GC-MS method development and validation for the determination of Short Chain Fatty Acids in human feces

Short Chain Fatty Acids (SCFAs), the end products of microbial fermentation of dietary fibers, appear to be key mediators of the beneficial effects elicited by the gut microbiome and have been shown to exert multiple effects on metabolism. In this study, we developed and validated a sensitive, accurate, and reproducible GC–MS method for the simultaneous quantification of SCFAs (Acetic acid (C 2 ), propionic acid (C 3 ), butyric acid (C 4 ), isobutyric acid and isovaleric acid) in human feces. Sample preparation was simplified while maintaining robustness, following systematic evaluation of homogenization, extraction solvents, and acidification conditions. The optimized method demonstrated high analytical performance, with limits of detection ranging from 0.01 to 0.52 μmol/g and good precision and accuracy in accordance with FDA and EMA bioanalytical guidelines Stability studies revealed that SCFAs remain stable in acidified fecal samples for up to 10 days without cold-chain requirements, while −80 °C storage was optimal for long-term preservation and 4 °C suitable for short-term handling. The applicability of the method was confirmed through analysis of samples collected from healthy volunteers. Overall, the developed approach provides a practical, high-throughput, and scalable tool for SCFA analysis, supporting applications in clinical research, metabolomics, and large-scale microbiome studies. • Derivatization-free GC–MS method enables rapid and robust quantification of fecal SCFAs • Simultaneous determination of C2–C5 SCFAs in human fecal samples with high sensitivity. • Simplified sample preparation with high analytical performance • Validation according to FDA and EMA bioanalytical guidelines • SCFAs found stable up to 10 days without cold-chain storage