Determination of Free Fatty Acids by High Performance Liquid Chromatography with Fluorescence Detection and Identification with Mass Spectrometry

Abstract

On a reversed-phase Eclipse XDB-C 8 column, 20 fatty acids were derivatized using 2-(2-(anthracen-10-yl)-1 H-naphtho [2,3-d]imidazol-1-yl) ethyl-p-toluenesulfonate (ANITS) as the pre-column derivatization reagent and were separated by high performance liquid chromatography (HPLC) in conjunction with a gradient elution with fluorescence detection and mass spectrometric identification. The reagent was reacted with fatty acids in the presence of K 2CO 3 catalyst at 90°C min in N,N-dimethylformamide solvent. The fluorescence excitation and emission wavelength of the derivatives were λ ex = 250 nm and λ em = 512 nm, respectively. The identification of fatty acid derivatives was performed by online MS/APCI (atmospheric pressure chemical ionization source) in positive model detection. All linear correlation coefficients for fatty acid derivatives are over 0.9999, and the detection limits, at a signal-to-noise rate of 3:1, are 24.8-98.8 fmol for labeled fatty acids. The established method is sensitive and reproducible for the determination of free fatty acids from real samples with satisfactory results.