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

), 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.