Orthogonal assessment of six extraction methods for GC–MS based short-chain fatty acid quantification in fecal samples

Short-chain fatty acids (SCFAs) are prominent regulators of host physiology and are closely related to the pathophysiology of many metabolic diseases. SCFAs are predominately generated by microbial fermentation of dietary fiber in the gut. Therefore, efficient, reproducible, and affordable analytical methods are needed to identify and quantify SCFAs in a complex fecal sample. In this study, five different SCFAs (acetic acid, butyric acid, propionic acid, valeric acid, and isobutyric acid) were compared using six different extraction methods (HCl-DE, NaOH-Hexane, H 3 PO 4 -Butanol, H 3 PO 4 -EA, NaHCO 3 -MTBE, and SPME) based on gas chromatography-mass spectrometry (GC-MS) analysis. The specificity, linearity, recovery, precision, and sensitivity of different analytical approaches were systematically assessed using SCFA chemical standards and further validated with human fecal samples, ensuring cross-species applicability. Our results revealed that no single method performed optimally for all SCFAs, but rather, distinct methods exhibited compound-specific advantages. Moreover, this study provides a practical guideline for balancing recovery, reproducibility, and extraction efficiency for method selection based on research purposes. The H 3 PO 4 -Butanol method displayed superior recovery accuracy for valeric acid (100.16 ± 0.81% and 101.82 ± 4.83% recovery rate for low and median spiked concentrations, respectively). It also exhibited minimum intra-day repeatability (0.92% to 5.67% RSD values) and good linearity (R 2 =0.94-0.99) across chemical mixtures. However, it showed the best extracted efficiency for butyric acid and best sensitivity for isobutyruc acid in fecal samples. Moreover, the H 3 PO 4 -EA method showed the highest recovery accuracy for isobutyric acid (103.21 ± 11.07% to 104.79 ± 3.59% recovery rate for all three different spiked concentrations) and the smallest inter-day repeatability (3.37% to 7.25% RSD values). The SPME method exhibited superior recovery accuracy for acetic acid (81.16 ± 21.17% to 94.08 ± 10.92% recovery rate for all three different spiked concentrations) and extracted the highest levels of SCFAs for acetic acid, propionic acid, and isobutyric acid. Finally, the application of these methods in human clinical samples revealed significant alterations of SCFA levels in patients with missed abortion, supporting their potential for translational studies. Overall, our results indicated that H 3 PO 4 -Butanol and SPME are the recommended methods for studying SCFAs in fecal samples.