Method for Absolute Quantification of Short Chain Fatty Acids via Reverse Phase Chromatography Mass Spectrometry

Short chain fatty acids (SCFAs; including acetate, propionate, and butyrate) are an important class of biological molecules that play a major role in modulating host-microbiome interactions. Despite significant research into SCFA-mediated biological mechanisms, absolute quantification of these molecules by liquid chromatography mass spectrometry (LC-MS) is challenging due to their relatively poor chromatographic properties and low mass. Herein, we introduce SQUAD, a quantitative strategy for analyzing SCFAs using an aniline-based derivatization approach in conjunction with reverse-phase LC-MS/MS analysis. We show that this approach, when coupled to quantification by stable isotope dilution (SID), enables absolute quantification of biologically relevant SCFAs in complex biological samples. We evaluate the limits of detection using this approach, analyze sources of error affecting quantification, and provide practical guidelines for using this strategy. Moreover, we illustrate the utility of this technique via two representative biological applications where SCFA analysis is common: 1) SCFA quantification in the caecal contents of germ free versus conventionally raised specific pathogen free mice and 2) in an analysis of in vitro microbial cultures.