Highly sensitive determination of free polyunsaturated, long-chain fatty acids in human serum by high-performance...

A highly sensitive and rapid high-performance liquid chromatographic method for the determination of free fatty acids including polyunsaturated, long-chain fatty acids in human serum is described. The fatty acids are converted into the corresponding fluorescent derivatives by reaction with 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone in the presence of potassium carbonate and 18-crown-6 in acetonitrile. The derivatives are separated simultaneously within 60 min on a reversed-phase column (YMC Pack C8) with an isocratic elution using aqueous 72% (v/v) acetonitrile and are detected fluorometrically. The detection limits are 1-3 fmol in a 10-microliter injection volume, which enables precise determination of the free acids including dihomo-gamma-linolenic, arachidonic, eicosapentaenoic, and docosahexaenoic acids present in serum at very low concentrations. The change in the concentrations of the free fatty acids in human sera before and after ingestion of mackerel meat is also described.