The analysis of free fatty acids in biological fluids HPLC

Techniques for derivatisation of fatty acids to form compounds suitable for detection by High Performance Liquid Chromatography using a 254 nm. ultraviolet detector have been developed. Benzylation of the fatty acids by a mild, thermal decomposition of the benzyldimethylanilinium salts of the fatty acids has been shown to proceed essentially to completion. Separation of the benzyl esters of the naturally occurring fatty acids in human physiological fluids was accomplished using a methanol/water elution from a reverse phase column. Quantitative formation of the 4-nitrobenzyl esters of the same acids, resulting in enhanced ultraviolet absorption with a concomittant increase in sensitivity in HPLC separations, was developed. Once again separations were accomplished on the reverse phase column, a methanol/water gradient as eluent.