Chemical derivatization-based LC–MS/MS method for quantitation of gut microbial short-chain fatty acids

Short chain fatty acids (SCFAs) are end products of fermentation by anaerobic gut microbiota. They can be used as beneficial metabolites to regulate the host’s physiological processes. Despite their importance, SCFAs are difficult to analyze with mass spectrometric technologies due to their poor ionization efficiency and susceptibility to water loss during ionization of low molecular weight organic acids. Here, we developed a sensitive and reliable method to quantify SCFAs by liquid chromatography tandem mass spectrometry (LC–MS/MS) in multiple reaction monitoring (MRM) mode. SCFAs were chemically derivatized by Girard’s reagent T (GT), providing a permanent cationic charge. This technique demonstrated an excellent quantitative capacity, showing good linearity (R2>0.99) and limit of quantification (femtomole levels) for five SCFAs (i.e., acetate, propionate, butyrate, valerate, and caproate). Next, we applied this derivatization method to quantitate SCFAs from a small volume of total extracellular metabolites produced by Eubacterium rectale (E. rectale), one of main butyrate-producing gut bacteria. GT-labeled SCFAs were quantitated well in small volumes of culture medium (5, 10, 15, and 20μL), with the amount of SCFA measured being proportional to the volume of culture medium, as expected. We also investigated plant-derived polysaccharides as prebiotics that could enhance the production of butyrate by E. rectale. Finally, the production of butyrate was successfully monitored in a co-culture system for E. rectale and Bifidobacterium longum (B. longum) by analyzing GT-labeled butyrate. Taken together, our results suggest that this highly sensitive method would be useful for quantifying SCFAs extracted from stool in an aqueous solution to monitor gut health.