Analysis of fatty acids as their anthrylmethyl esters by high-performance liquid chromatography with fluorescence...

A reversed-phase high-performance liquid chromatographic method for the analysis of free fatty acids in plasma has been developed which allows the resolution of the major plasma fatty acids in man. A mixture of thirteen fatty acid anthrylmethyl esters is resolved on a Spherisorb 3-micron C8 column at a flow-rate of 1 ml/min by gradient elution. The solvent system consists of acetonitrile-water (93:7) for 12 min, followed by 5 min at acetonitrile-water (86:14) and 23 min at 100% acetonitrile. The eluent is monitored with a fluorescence detector (excitation 360 nm, emission 420 nm). Three different C18 columns were evaluated, and none were able to resolve all the esters. To optimize the separation on the C8 columns it was necessary to construct plots of log capacity factor vs. percent water in the mobile phase. The slope of the line for arachidonic acid (C20:4) was markedly different from that for the saturated acid derivatives. The method was applied to the analysis of plasma free fatty acids in normal and diabetic subjects. As expected, elevated levels of free fatty acids were found in diabetic subjects. The results were shown to correlate well with an established gas chromatographic assay.