Fluorometric high-performance liquid chromatography of 9-aminophenanthrene-derivatized free fatty acids

The application of 9-aminophenanthrene (9-AP), a fluorescence-labeling reagent for free fatty acids (FFA), was examined. 9-AP dissolved in benzene was added to a benzene solution of FFA chlorides derived from FFA and oxalyl chloride. The mixture was allowed to react for 45 min at 70 degrees C. By the method, 9-AP-tagged FFA with a strong fluorescence was formed. The materials thus obtained have a lambda max at around 303 nm for excitation and 376 nm for emission. By using this derivatization method, recoveries were measured for seven kinds of FFA added to 0.5 ml of healthy human serum. Significant recoveries ranging from 96 to 107% (coefficient of variation 1.4-5.0%) were obtained for each FFA. The proposed method was clinically applied to the determination of FFA in 0.5 ml of healthy human serum, and almost satisfactory results were obtained. Detection limits of FFA by this derivatization method were 10 pmol for C14:0, C16:0, C16:1, C18:1 and C18:2, and 15 pmol for C18:0 and C20:4. As a quantitative measurement of FFA, gas chromatography and high-performance liquid chromatography with fluorescence detection, which have been routinely used, were chosen for comparison with the present method.