Simultaneous separation and sensitive determination of free fatty acids in blood plasma by high-performance liquid chromatography.

Fatty acids are separated by reversed-phase high-performance liquid chromatography after derivatization with a fluorescence reagent, 4-bromomethyl-7-acetoxycoumarin. Each derivative eluted from a column is successively hydrolysed by mixing it with an alkaline solution, and the produced fluorescence is detected. The derivatives of series of both saturated and unsaturated fatty acids (C6:0--C20:4) are simultaneously separated by a continuous gradient elution method using a methanol-based solvent containing acetonitrile. The quantitative detection of fatty acids is over a range of 5-1000 pmol per derivatization mixture. This method is applicable to the quantitative analysis of free fatty acids in normal human blood samples and blood samples from diabetic patients. Ten microliters of blood plasma are sufficient to carry out the determination. The analytical results show good recovery and good reproducibility. This sensitive method is very useful for the analysis of fatty acids in very low concentrations.