Amiad Filtration Systems Ltd, Israel

Short-chain fatty acids (SCFAs) are one class of bacterial metabolites mainly formed by gut microbiota from undigested fibers and proteins. These molecules are able to mediate signal conduction processes of cells, acting as G protein-coupled receptors (GPR) activators and histone deacetylases (HDAC) inhibitors. It was reported that SCFAs were closely associated with various human diseases. However, it is still challenging to analyze SCFAs because of their diverse structures and broad range of concentrations. In this study, we developed a highly sensitive method for simultaneous detection of 34 SCFAs by stable isotope labeling coupled with ultra-high performance liquid chromatography-electrospray ionization-mass spectrometry (UHPLC-ESI-MS/MS) analysis. In this respect, a pair of isotope labeling reagents, N-(4-(aminomethyl)benzyl)aniline (4-AMBA) and N-(4-(aminomethyl)benzyl)aniline-d5 (4-AMBA-d5), were synthesized to label SCFAs from the feces of mice and SCFA standards, respectively. The 4-AMBA-d5 labeled SCFAs were used as internal standards to compensate the ionization variances resulting from matrix effect and thus minimize quantitation deviation in MS detection. After 4-AMBA labeling, the retention of SCFAs on the reversed-phase column increased and the separation resolution of isomers were improved. In addition, the MS responses of most SCFAs were enhanced by up to three orders of magnitude compared to unlabeled SCFAs. The limits of detection (LODs) of SCFAs were as low as 0.005 ng/mL. Moreover, good linearity for 34 SCFAs was obtained with the coefficient of determination (R2) ranging from 0.9846 to 0.9999 and the intra- and inter-day relative standard deviations (RSDs) were <17.8% and 15.4%, respectively, indicating the acceptable reproducibility of the developed method. Using the developed method, we successfully quantified 21 SCFAs from the feces of mice. Partial least squares discriminant analysis (PLS-DA) and t-test analysis showed that the contents of 9 SCFAs were significantly different between Alzheimer's disease (AD) and wide type (WT) mice fecal samples. Compared to WT mice, the contents of propionic acid, isobutyric acid, 3-hydroxybutyric acid, and 3-hydroxyisocaleric acid were decreased in AD mice, while lactic acid, 2-hydroxybutyric acid, 2-hydroxyisobutyric acid, levulinic acid, and valpronic acid were increased in AD mice. These significantly changed SCFAs in the feces of AD mice may afford to a better understanding of the pathogenesis of AD. Taken together, the developed UHPLC-ESI-MS/MS method could be applied for the sensitive and comprehensive determination of SCFAs from complex biological samples.