Highly sensitive determination of long-chain free fatty acids by fluorogenic derivatization and liquid chromatography

A simple and sensitive method is described for the determination of biologi cally important fatty acids including dodecanoic, tetradecanoic, hexadecano ic, octadecanoic, hexadecenoic, octadecenoic and octadecadienoic acids. The method is based derivatization with 2-(2-naphthoxy)ethyl 2-(piperidino)eth anesulfonate (NOEPES) in toluene using potassium carbonate and 18-crown-6 a s reaction activators. The resulting naphthoxyethyl derivatives were simply analyzed by isocratic HPLC with fluorometric detection (lambda ex = 235 nm , lambda em = 350 nm). The excess fluorescence reagent (NOEPES) was readily removed after derivatization by a quick acid treatment. This minimizes the interference of excess NOEPES in fluorometric detection. The lower limit o f quantitation 50 nM with a detection limit (S / N = 3) of about 7.5 nM (or 0.15 pmol per 20 muL injection). Application of the method to analysis of free fatty acids in plasma proved feasible (using only 10 muL of plasma sam ple).