Analysis of saturated free fatty acids from pollen by HPLC with fluorescence detection

A sensitive method for the determination of free fatty acids using 2‐(2‐(anthracen‐10‐yl)‐1 H‐ naphtho[2,3‐ d ]imidazol‐1‐yl) ethyl‐ p ‐toluenesulfonate (ANITS) as tagging reagent with fluorescence detection has been developed. ANITS could easily and quickly label fatty acids in the presence of the K 2 CO 3 catalyst at 90 °C for 40 min in N , N ‐dimethylformamide solvent. From the extracts of rape bee pollen samples, 20 free fatty acids were sensitively determined. Fatty acid derivatives were separated on a reversed‐phase Eclipse XDB‐C 8 column by HPLC in conjunction with gradient elution. The corresponding derivatives were identified by post‐column APCI/MS in positive‐ion detection mode. ANITS‐fatty acid derivatives gave an intense molecular ion peak at m/z [M+H] + ; with MS/MS analysis, the collision‐induced dissociation spectra of m/z [M+H] + produced the specific fragment ions at m/z [M–345] + and m/z  345.0 (here, m/z  345 is the core structural moiety of the ANITS molecule). The fluorescence excitation and emission wavelengths of the derivatives were λ ex  = 250 nm and λ em  = 512 nm, respectively. Linear correlation coefficients for all fatty acid derivatives are >0.9999. Detection limits, at a signal‐to‐noise ratio of 3 : 1, are 24.76–98.79 fmol for the labeled fatty acids.