SPE extraction coupled to HPLC-ESI-MS/MS for the screening and determination of short chain fatty acids

Short-chain fatty acids (SCFAs) are generated as end products by the degradation and fermentation of indigestible carbohydrates by the gut microbiota, a process termed saccharolytic fermentation1. They can act as signaling molecules as ligands of G-protein-coupled receptors and they are implicated in the increase of anorexic hormone production and energy expenditure2 . Consequently, SCFA production has been linked to preventing the progression of obesity and related complications, such as type 2 diabetes mellitus and nonalcoholic fatty liver disease (NAFLD)3 . SCFAs are typically quantified by gas chromatography (GC), liquid chromatography (LC), nuclear magnetic resonance (NMR), and capillary electrophoresis (CE)4 . The detection of the low-molecular weight fatty acids in negative-ion ESI, without chemical derivatization, is often problematic. LC coupled with mass spectrometry (MS) with electrospray ionization (ESI) is now the most widely used analytical technique in metabolomics and reversed-phase LC–MS with post-column neutralization, previously used also for the determination of SCFAs in the pig colon and blood. The complex instrument setup for these two methods, however, makes them unsuitable for routine analysis in most laboratories. This work aimed to develop a new analytical method for SCFA based on 3-nitrophenylhydrazone (3-NPH) derivatization followed by SPE clean-up, we decided to derivatize the molecular markers with the aim of improving their chromatographic retention, the ionization efficiency and the fragmentation behaviour. While the SPE clean-up was used to minimize the matrix effect and improve extraction selectivity. The LCMS/MS method was performed both in Multi Reaction Monitoring (MRM) for quantitative analysis and in precursor ion scan analysis (PIS) to achieve semi-targeted analysis for an extended range of SCFA compounds based on derivatization reactions. This approach can be used for putative identification and screening of SCFA compounds in biological samples. Moreover, the derivatization process allows for greater stability of the analytes in the sample over the course of storage time.