2018 Volume 2 Issue 1 Pages 21-33
Fatty acids vary in their hydrocarbon chain length and the number and position of the double bonds. In this study, we investigated the principle of the separation of fatty acids by reverse-phase liquid chromatography coupled with negative electrospray ionization mass spectrometry (LC-negative ESI-MS). In particular, we aimed to detect very-long-chain fatty acids (VLCFAs) and to distinguish between isomeric unsaturated fatty acids differing in their double bond positions. We found that both saturated and unsaturated fatty acids are not separated by regular chromatography but instead separated by sequential detachment from the column’s stationary phase, according to the strength of the organic mobile phase. The interaction of the fatty acids with the stationary phase during chromatographic migration had a minor effect on their separation. Unsaturated fatty acid isomers with different double bond positions were found to be separable, and their elution order was determined by the position of the double bonds relative to the omega carbon. We also found that fibroblasts from the patients with peroxisomal disease show characteristic distribution patterns for some fatty acid isomers in the proposed separation method. Our method is, thus, useful for analyzing a wide range of fatty acid species, including VLCFAs and unsaturated fatty acid isomers, to understand the physiological changes in fatty acid metabolism from clinical samples.
