How to Analyze SCFAs from Feces for Multi-Omic Research

1. Introduction:

In recent years, the human gut microbiome has emerged as a central regulator of health and disease. This complex ecosystem - comprising bacteria, archaea, viruses, and fungi - not only supports digestion and nutrient absorption but also influences immune modulation, maintains epithelial integrity, and communicates with distant organs through the gut-brain and gut-liver axes [1,2].

Disruptions in the microbiome - collectively termed dysbiosis - have been implicated in a broad spectrum of diseases. These range from gastrointestinal conditions such as inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), and colorectal cancer (CRC), to systemic disorders including obesity, type 2 diabetes, cardiovascular disease, and neuropsychiatric conditions like depression and Parkinson’s disease [3]. Mounting evidence suggests that the gut microbiome serves as a key interface between environmental exposures - especially dietary patterns - and health outcomes.

As microbiome research shifts from identifying microbial taxa to understanding their functional roles, microbial metabolites have become a central focus. These small molecules - produced by bacteria during their metabolic processes - act as mediators between gut microbes and the host, influencing immune responses, energy metabolism, and epithelial integrity. Altered profiles of these metabolites are increasingly recognized not only as biomarkers but also as contributors to disease. For example, elevated secondary bile acids are associated with increased colorectal cancer risk [4] and reduced short-chain fatty acid (SCFA) production has been observed in metabolic disorders such as obesity and type 2 diabetes [5]. These functional shifts often correspond with changes in microbial composition, emphasizing the need for integrated microbiome and metabolome analyses.

While microbiome studies usually rely on next-generation sequencing to characterize bacterial DNA from stool samples, (see our blog post “Next-Generation Sequencing (NGS) in Microbiome Research – A Practical Guide“) analyzing metabolites from stool samples requires different techniques. In this article we focus on one particularly important class of microbial metabolites - short-chain fatty acids (SCFAs) - and provide instructions on how to analyze them using gas chromatography coupled with mass spectrometry (GC-MS).

2. The relevance of Short Chain Fatty Acids (SCFAs)

SCFAs are organic acids with fewer than six carbon atoms, primarily produced by bacterial fermentation of indigestible carbohydrates in the colon. These metabolites are crucial for colonic health, influencing energy metabolism, immune modulation, and epithelial integrity [6]. The following table provides an overview of the most relevant SCFAs.


Table: Overview of the most relevant Short Chain Fatty Acids (SCFA)
SCFA	Carbon Atoms	Function/Relevance in the Gut
Acetate (Acetic acid)	2	Most abundant; systemic lipid synthesis, central appetite regulation
Propionate (Propionic acid)	3	Gluconeogenesis in the liver; satiety signaling via gut-brain axis
Butyrate (Butyric acid)	4	Energy for colonocytes; enhances barrier function; anti-inflammatory effects
Isobutyrate (Isobutyric acid)	4	Branched-chain; protein fermentation byproduct with possible immune roles
Valerate (Valeric acid)	5	Less prevalent; may contribute to epithelial signaling
Isovalerate (Isovaleric acid)	5	Protein-derived; may influence microbial ecology and signaling
2-Methylbutyrate (2-Methylbutyric acid)	5	Branched-chain; derived from amino acid metabolism; possible signaling roles

Among these, butyrate stands out due to its diverse and well-documented roles in gut health. It serves as the primary energy source for colonocytes, promotes epithelial regeneration, and enhances mucosal barrier integrity. Additionally, butyrate has potent anti-inflammatory properties and functions as a histone deacetylase inhibitor, contributing to immune regulation and cancer prevention. Notably, reduced levels of butyrate and its producers - such as Faecalibacterium prausnitzii and Roseburia spp. - have been consistently linked to inflammatory bowel disease, colorectal cancer, and metabolic dysfunction [7].

As such, quantifying SCFAs in stool samples offers not only insight into microbial activity but also potential biomarkers for gut-related disease states.

3. How to Analyze SCFAs using GC-MS

To quantify SCFAs in stool samples, researchers most commonly rely on gas chromatography coupled with mass spectrometry (GC-MS). This highly sensitive and specific method is ideally suited for detecting volatile fatty acids, enabling precise differentiation and quantification of SCFAs even at low concentrations.

One commonly used protocol for analysis of stool samples, is outlined in the following section. The procedure is based on a method originally described by Richardson et al. [8], and successfully applied since then [9].

A. Stool Sample Preparation

Fresh stool samples: dissolve sample in sterile water (e.g. 8 mL water per gram of stool), vortex thoroughly.
Dry Stool samples: reconstitute sample in sterile water (e.g. 8 mL water per gram of stool), vortex thoroughly.
Stabilized stool samples: Stabilize sample acc. to manufacturer instructions (e.g. 8 ml stabilizing solution per gram of stool), vortex thoroughly. (Note: Samples collected using the Stool Collection Tube with DNA Stabilizer can be directly used)

B. Centrifugation

Transfer suspension into a 15 mL falcon tube and centrifuge at 3000 × g for 15 minutes.

C. Supernatant Collection

1 mL aliquots of the resulting supernatant are collected for further analysis.

D. Internal Standard Addition

Add internal standards (such as 2-ethylbutyrate) to ensure accurate quantification.

E. Acid Extraction

Add 0.5 mL concentrated hydrochloric acid and 2 mL diethyl ether to each sample.
Vortex for 1 minute to mix thoroughly.
Centrifuge again at 3000 × g for 10 minutes.

(Note: to increase yield, a second extraction can be performed by adding another 1 mL diethyl ether to the aqueous phase and repeated vortexing + centrifugation, then combine layers from both extractions)

F. Derivatization

Add N-methyl-N-t-butyldimethylsilyltrifluoroacetamide (MTBSTFA) and heat samples at 80 °C for 20 minutes.

(Note: the reaction mixture can be left at room temperature for another 24 hours to achieve full conversion of e.g. lactic acid)

G. GC-MS Analysis

Use 1 μL of the derivatized sample for GC-MS analysis.
Identify SCFA peaks by comparing retention times with known standards.
Quantify SCFAs relative to the internal standard.

This method offers reproducibility, and sensitivity, allowing reliable quantification of SCFAs, especially when working with stabilized stool samples.

4. How to Ensure Stool Sample Stability for Combined Metabolome and Microbiome Studies

The quality of microbiome and metabolome data heavily depends on the integrity of the stool sample from the moment of collection. SCFAs and other microbial metabolites are chemically unstable and can degrade rapidly at room temperature, while microbial DNA may undergo compositional shifts due to bacterial overgrowth or cell lysis. These changes can significantly distort both taxonomic profiles and metabolite concentrations, especially if samples are not promptly cooled or frozen after collection.

Traditionally, researchers relied on cold-chain logistics - immediate freezing at −80 °C and transport on dry ice - to preserve stool samples. However, this approach is logistically demanding, cost-intensive, and often impractical in large-scale or field-based studies. To address these challenges, Invitek Diagnostics has developed the Stool Collection Tube with DNA Stabilizer optimized for multi-omic applications. These tubes contain a proprietary stabilization medium that simultaneously preserves both bacterial DNA and key metabolite classes, including SCFAs. This allows for flexible sampling and storage without requiring immediate freezing while preserving multi-omic data quality - making robust microbiome and metabolome research more scalable and reliable. (For more details see our blog post “How to Ensure Stool Sample Integrity for Gut Metagenomics & Metabolomics Analysis”).

5. Summary

Short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate are essential mediators of gut health and systemic physiology, playing roles in energy metabolism, immune regulation, and epithelial maintenance. Accurate measurement of SCFAs from stool samples provides valuable insights into microbial function and host–microbe interactions, especially in the context of diseases like colorectal cancer, IBD, obesity, and type 2 diabetes.

Gas chromatography–mass spectrometry (GC-MS) remains the gold standard for SCFA analysis, offering high sensitivity and specificity. The Richardson et al. protocol, widely adopted in the field, enables robust quantification of these volatile metabolites—even from stabilized stool samples.

As multi-omic studies increasingly integrate microbiome and metabolome data, ensuring sample stability from collection to processing is critical. The Stool Collection Tube with DNA Stabilizer by Invitek Diagnostics offers a practical, validated solution for preserving both microbial DNA and metabolites - empowering researchers to conduct high-quality, reproducible gut health studies without the limitations of cold-chain logistics.

References

1. Thursby E, Juge N. Introduction to the human gut microbiota. The Biochemical Journal. 2017 May;474(11):1823-1836. DOI: 10.1042/bcj20160510. PMID: 28512250; PMCID: PMC5433529.

2. Phatak M, Nair B, Soni U, Pujari R. Exploring the Gut-Brain Axis: Microbiome Contributions to Pathophysiology of Attention Deficit Hyperactivity Disorder and Potential Therapeutic Strategies. Current Drug Metabolism. 2025 Apr. DOI: 10.2174/0113892002361676250325082424. PMID: 40207768.

3. Valdes AM, Walter J, Segal E, Spector TD. Role of the gut microbiota in nutrition and health. BMJ (Clinical Research ed.). 2018 Jun;361:k2179. DOI: 10.1136/bmj.k2179. PMID: 29899036; PMCID: PMC6000740.

4. Kouhzad M, Götz F, Navidifar T, et al. Carcinogenic and anticancer activities of microbiota-derived secondary bile acids. Frontiers in Oncology. 2025 ;15:1514872. DOI: 10.3389/fonc.2025.1514872. PMID: 39944822; PMCID: PMC11813773.

5. Münte E, Hartmann P. The Role of Short-Chain Fatty Acids in Metabolic Dysfunction-Associated Steatotic Liver Disease and Other Metabolic Diseases. Biomolecules. 2025 Mar;15(4):469. DOI: 10.3390/biom15040469. PMID: 40305160; PMCID: PMC12025087.

6. Ramaboli MC, Ocvirk S, Khan Mirzaei M, et al. Diet changes due to urbanization in South Africa are linked to microbiome and metabolome signatures of Westernization and colorectal cancer. Nature Communications. 2024 Apr;15(1):3379. DOI: 10.1038/s41467-024-46265-0. PMID: 38643180; PMCID: PMC11032404.

7. De Filippo C, Cavalieri D, Di Paola M, et al. Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa. Proceedings of the National Academy of Sciences of the United States of America. 2010 Aug;107(33):14691-14696. DOI: 10.1073/pnas.1005963107. PMID: 20679230; PMCID: PMC2930426.

8. RICHARDSON AJ, CALDER AG, STEWART CS, et al. Simultaneous determination of volatile and non-volatile acidic fermentation products of anaerobes by capillary gas chromatography. Letters in Applied Microbiology. 1989 Volume 9, Issue 1, 1 July 1989, Pages 5–8. DOI: 10.1111/j.1472-765X.1989.tb00278.x

9. Gemmell MR, Jayawardana T, Koentgen S, et al. Optimised human stool sample collection for multi-omic microbiota analysis. Scientific Reports. 2024 Jul;14(1):16816. DOI: 10.1038/s41598-024-67499-4. PMID: 39039185; PMCID: PMC11263584.