Extraction of Short-chain Fatty Acids from Mouse Feces Using A Two-phase Acid-Solvent Method

Area of Science:

• Microbiology
• Analytical Chemistry
• Animal Science

Background:

• Short-chain fatty acids (SCFAs) are crucial microbial metabolites influencing host health.
• Analyzing SCFAs in small animal models is challenging due to limited sample availability.
• Existing methods may require large sample inputs or complex procedures.

Purpose of the Study:

• To develop and validate a standardized protocol for extracting SCFAs from low-input mouse fecal samples.
• To enable SCFA analysis compatible with routine laboratory equipment and downstream gas chromatography platforms.
• To address the need for efficient SCFA quantification in small-animal research.

Main Methods:

• A two-phase acid-solvent extraction strategy using dilute hydrochloric acid and ethyl acetate.
• Fecal homogenization, acidification, organic solvent partitioning, and low-temperature centrifugation.
• Method designed for approximately 20-50 mg of individual mouse fecal samples without derivatization.

Main Results:

• Successful extraction of major fecal SCFAs, including acetate, propionate, and butyrate.
• Demonstrated clear SCFA peaks above background noise using routine gas chromatography.
• Protocol validated for low-input samples, ensuring compatibility with standard analytical workflows.

Conclusions:

• The described protocol provides a reliable and accessible method for fecal SCFA extraction from limited mouse samples.
• This workflow facilitates routine SCFA analysis in small-animal studies, aiding gut microbiome research.
• The method simplifies SCFA quantification, supporting investigations into host-microbe interactions.