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

This article describes a protocol for extracting short-chain fatty acids (SCFAs) from low-input mouse fecal samples using a two-phase acid-solvent extraction strategy. The method is designed for individual fecal inputs of approximately 20-50 mg and employs dilute hydrochloric acid and ethyl acetate to enable phase separation without derivatization. The protocol consists of fecal homogenization, acidification, organic solvent partitioning, and low-temperature centrifugation, and can be performed using standard laboratory equipment commonly available in research laboratories. Following extraction, SCFAs are suitable for downstream analysis by routine gas chromatography-based platforms. Successful execution of the protocol is indicated by the detection of clearly resolved SCFA peaks at characteristic retention times above background noise, as demonstrated for major fecal SCFAs including acetate, propionate, and butyrate from individual mouse samples. This protocol addresses a common procedural challenge in small-animal studies by providing a standardized workflow for fecal SCFA extraction from limited sample material while maintaining compatibility with routine analytical workflows.