BioProject

Short-chain fatty acids (SCFAs), bile acids (BAs), amino acids, and sugars produced by gut microbes play a significant role in the maintenance of human health. Because of their different chemical structures, they have often been analyzed using separate analyzers, such as gas chromatography/mass spectrometry (GC/MS) for the measurement of SCFAs, and liquid chromatography/mass spectrometry (LC/MS) for the measurement of BAs. However, their compounds can be measured by GC/MS after trimethylsilyl (TMS) derivatization with organic solvents. In this process, the extract is dried and then re-dissolved in an organic solvent to obtain the TMS derivative, but the volatile SCFAs have been considered unsuitable for this method as they may be lost during the drying process. Previous studies have shown that SCFAs in fecal samples can still be quantified after dry solidification. In this study, we aimed to develop a method for the simultaneous determination of important gut metabolites by TMS derivatization, specifically addressing the issue of SCFAs volatilization during the dry solidification process. Mixing standard solutions with feces reduced the volatilization of SCFAs compared to those without feces. Therefore, a calibration curve was prepared by mixing standard solutions with human feces to account for the volatilization of SCFAs in feces. The method showed good linearity (R2 > 0.995), good recoveries (82.265 - 118.244%), high repeatability (RSD < 15%) and low limits of detection (LOD) for various SCFAs. This method enabled the simultaneous quantification of a total of 65 compounds, including SCFAs, BAs, AAs and sugars, which are significant metabolites derived from the gut microbiome. A mouse study showed that a high-fiber diet increased SCFA in feces and showed a negative correlation between Alistipes and sugar, consistent with previous studies. We successfully developed a simultaneous quantification method for SCFAs, BAs, AAs and sugars in fecal samples using the same pretreatment process with TMS derivatization.