Trace analysis of very long chain free fatty acids in plasma by fluorogenic derivatization and liquid chromatography

A simple and sensitive liquid chromatographic method is described for the simultaneous determination of docosanoic, tetracosanoic and hexacosanoic acids as fluorogenic derivatives. These acids, spiked in plasma, were extracted with n-heptane and the resulting extract was derivatized with 4-bromomethyl-7-methoxycoumarin (BrMmC) in dichloromethane using potassium carbonate and 18-crown-6 as catalysts. The derivatives obtained were chromatographed on a reversed-phase C-8 column with acetonitrile:water (91 : 9, v/v) as a mobile phase and 7-methoxy-4-(tricosanoyloxymethyl)-coumarin as an internal standard. The linear ranges for the determination of docosanoic, tetracosanoic, and hexacosanoic acids were over 31-500, 36-580 and 36-580 pmol, respectively. The limit of detection for each acid was about 0.3 pmol per 10 mu L injection (S/N=3).