An Improved Validated Method for the Determination of Short-Chain Fatty Acids in Human Fecal Samples by Gas Chromatography with Flame Ionization Detection (GC-FID)

Abstract

Short-chain fatty acids (SCFAs) are metabolites produced by the gut microbiota through the fermentation of non-digestible carbohydrates. Recent studies suggest that the gut microbiota composition, diet and metabolic status play an important role in the production of SCFAs. The primary objective of this study was to develop a simplified method for SCFA analysis in human fecal samples by gas chromatography with flame ionization detection (GC-FID). The secondary objective was to apply the method to fecal samples collected from a clinical trial. The developed GC-FID method showed excellent linearity (R2 > 0.99994), with a limit of detection (LOD) ranging from 0.02 to 0.23 µg/mL and a limit of quantification (LOQ) ranging from 0.08 to 0.78 µg/mL. Recovery for the method ranged between 54.24 ± 1.17% and 140.94 ± 2.10%. Intra- and inter-day repeatability ranged from 0.56 to 1.03 and from 0.10 to 4.76% RSD, respectively. Nine SCFAs were identified and quantified (acetic, propionic, iso-butyric, butyric, iso-valeric, valeric, 4-methyl valeric, hexanoic and heptanoic acids) in freeze-dried fecal samples. The clinical trial compared participants with prediabetes mellitus and insulin resistance (IR-group, n = 20) to metabolically healthy participants (reference group, R-group, n = 9) following a 4-week intervention of a daily red raspberry smoothie (RRB, 1 cup fresh-weight equivalent) with or without fructo-oligosaccharide (RRB + FOS, 1 cup RRB + 8 g FOS). The statistical analysis (Student’s t-test, ANCOVA) was performed on PC-SAS 9.4 (SAS Institute). Acetic acid was higher in the R-group compared to the IR-group at baseline/week 0 (p = 0.14). No significant changes in fecal SCFA content were observed after 4 weeks of either RRB or RRB + FOS.