Development of a pseudotargeted metabolomics approach for relative quantification of free fatty acid double-bond isomers in beagle plasma

Free fatty acids (FFAs) play a key role in living organisms and participate in metabolic processes, mainly as a source of energy for cells. The CC position isomer of FFAs is strongly associated with many diseases, but quantification of its double bond position isomer remains a challenge in lipid analysis. In this study, based on the requirements of the pseudotargeted metabolomics, an analytical method based on a two-step derivatization LC-MS/MS method and a multiple reaction monitoring (MRM) mode was established and fully validated in terms of linearity, precision, and stability. The results demonstrated that the method relatively quantified 30 FFAs in plasma samples. These included 8 saturated fatty acids (SFAs), 12 monounsaturated fatty acids (MUFAs), and 10 polyunsaturated fatty acids (PUFAs). The developed method was applied to analyze the increase or down-regulation of the relative content of FFAs before and after the administration of the drug in beagles which may help to explain the therapeutic effect of sacubitril valsartan. The establishment of this method allows the study of FFAs in beagles plasma to be no longer limited to analysis at the subclass level but can be focused at the isomer level.