High-performance liquid chromatographic determination of free fatty acids with 1-naphthylamine

A 1% benzene solution of oxalyl chloride was added to saturated and unsaturated fatty acids and the mixture was allowed to react at 70 degrees C for 30 min; by this procedure, each fatty acid was converted into its acid chloride in a considerably quantitative manner. By reacting this acid chloride with 1-naphthylamine at 30 degrees C for 15 min, naphtylamine derivatives were produced, which showed strong ultraviolet absorption around 280-290 nm. Experiments were made on the recovery of the fatty acids added to 0.5 ml of human serum, and the recovery was found to fall in the range of 94-106% (coefficient of variation = 0.5-4.1%) when the following amounts of six fatty acids were added: C14:0, 2 micrograms; C16:0, 20 micrograms; C16:1, 5 micrograms; C18:0, 4 micrograms; C18:1, 20 micrograms; C18:2, 10 micrograms.