High-performance liquid chromatographic analysis of serum long-chain fatty acids by direct derivatization method

Abstract

A new visible-ultraviolet labelling method for the high-performance liquid chromatographic analysis in serum of individual free fatty acids, including polyunsaturated fatty acids, is described. Without commonly used isolation steps, fatty acids in serum were directly derivatized by treatment with acidic 2-nitrophenylhydrazine hydrochloride. The derivatized fatty acids were extracted into n-hexane and separated isocratically on a reversed-phase C 8 column within 15 min. The detection limits ranged from 400 fmol to 1 pmol and from 100 to 200 fmol per injection with visible and ultraviolet detection, respectively. Visible detection had better selectivity, and free fatty acid levels were determined in sera obtained from healthy controls and patients with diabetes mellitus. In all the subjects studied, the precise quantitation could be performed with 25 μl of serum. Analytical recoveries ranged from 98.3 to 103.4%. The intra- and inter-assay coefficients of variation were less than 2.7 and 3.5%, respectively. The present method is superior to the previously published methods for routine analyses: it is cheaper, the procedure is simpler, the analysis time is shorter and both resolution and sensitivity are better.