The optimization and validation of a gas chromatography-mass spectrometry method to analyze the concentration of acetate, propionate and butyrate in human plasma or serum

• GC–MS method was optimized to quantify SCFA concentrations in human plasma or serum. • SCFA were derivatized with 2,4-difluoroaniline and extracted in ethyl acetate. • The method was validated according to the FDA guidelines. • The method was effectively applied with analysis of human plasma from dietary study. Fermentation-derived short-chain fatty acids (SCFA) 4 4 SCFA: short-chain fatty acids. are potential mediators of the health benefits associated with dietary fiber intake. SCFA affect physiological processes locally in the gut and on distant organs via the systemic circulation. Since SCFA are used as energy source for colonocytes and substrate for the liver metabolism, their concentrations in the systemic circulation are low. Therefore, quantification of systemic SCFA requires sensitive analytical techniques. This article covers the optimization and validation of a gas chromatography-mass spectrometry method to measure systemic SCFA concentrations following derivatization with 2,4-difluoroaniline (DFA) 5 5 DFA: 2,4-difluoroaniline. and extraction in ethyl acetate. Sample preparation was optimized by varying the amount of DFA, coupling agent 1,3-dicyclohexylcarbodiimide, ethyl acetate and sodium bicarbonate, which is used to quench derivatization. In addition, evaporation of the samples using a vacuum concentrator resulted in less contamination, notably of acetate, compared to drying with N 2 gas. The method showed excellent linearity with coefficient of variation (R 2 ) > 0.99 and a good precision (relative standard deviation < 20 %) and accuracy. Finally, systemic concentrations of SCFA in human plasma samples could successfully be determined.