Simple derivatization method for sensitive determination of fatty acids with fluorescence detection by high-performance liquid chromatography using 9-(2-hydroxyethyl)-carbazole as derivatization reagent

Abstract

A simple and sensitive method for the determination of short and long-chain fatty acids using high-performance liquid chromatography with fluorimetric detection has been developed. The fatty acids were derivatized to their corresponding esters with 9-(2-hydroxyethyl)-carbazole (HEC) in acetonitrile at 60°C with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride as a coupling agent in the presence of 4-dimethylaminopyridine (DMAP). A mixture of esters of C 1–C 20 fatty acids was completely separated within 38 min in conjunction with a gradient elution on a reversed-phase C 18 column. The maximum fluorescence emission for the derivatized fatty acids is at 365 nm ( λ ex 335 nm). Studies on derivatization conditions indicate that fatty acids react proceeded rapidly and smoothly with HEC in the presence of EDC and DMAP in acetonitrile to give the corresponding sensitively fluorescent derivatives. The application of this method to the analysis of long chain fatty acids in plasma is also investigated. The LC separation shows good selectivity and reproducibility for fatty acids derivatives. The R.S.D. ( n=6) for each fatty acid derivative are <4%. The detection limits are at 45–68 fmol levels for C 14–C 20 fatty acids and even lower levels for <C 14 fatty acids.