Simultaneous determination of short‐chain fatty acids in human feces by HPLC with ultraviolet detection following chemical derivatization and solid‐phase extraction segmental elution

Short-chain fatty acids are currently the most studied metabolites of gut microbiota, but the analysis of them, simultaneously, is still challenging due to their unique property and wide concentration range. Here, we developed a sensitive and versatile high-performance liquid chromatography with ultraviolet detection method, using pre-column derivatization and solid-phase extraction segmental elution, for the quantification of both major and trace amounts of short-chain fatty acids in human feces. Short-chain fatty acids were converted to 3-nitrophenylhydrazine-derived analytes, and then solid-phase extraction segmental elution was used for extraction of major analytes and enrichment of trace analytes. The method validation showed limits of quantitation ˂0.04 mM, and coefficient of determination > 0.998 at a wide range of 0.04-8.0 mM. The intra- and interday precision of analytes were all within accepted criteria, and the recoveries were 96.12 to 100.75% for targeted analytes in fecal samples. This method was successfully applied in quantification of eight analytes in human feces, which therefore could provide a sensitive and versatile high-performance liquid chromatography with ultraviolet detection method for precise and accurate quantitation of short-chain fatty acids in human feces.