A fluorimetric liquid chromatography for highly sensitive analysis of very long chain patty acids as naphthoxyethyl derivatives

A simple and sensitive liquid chromatographic method is described for the simultaneous determination of biologically important very long chain fatty acids (docosanoic, tetracosanoic and hexacosanoic acids) as fluorogenic derivatives. The method is based on the derivatization of the fatty acids with 2-(2-naphthoxy)ethyl 2-(piperidino)ethanesulfonate (NOEPES) in toluene in the presence of potassium carbonate and 18-crown-6. Several parameters affecting the derivatization were studied, including reaction temperature, reaction time, reaction solvent, base catalyst and the amount of the reagent. The resulting derivatives were analyzed by HPLC with fluorimetric detection (lambda ex = 235 nm; lambda em = 366 nm). The linear range for the determination of docosanoic, tetracosanoic and hexacosanoic acids was 0.028-1.4 mu M with a detection limit of about 5.6 nM (S/N = 3) (56 fmol per 10 mu L injection). Application of the method to the analysis the non-esterified (free) very long chain fatty acids spilled in plasma proved feasible.