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

Abstract

AbstractA sensitive method for the determination of free fatty acids using 2‐(2‐(anthracen‐10‐yl)‐1H‐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 K2CO3 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‐C8 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 λex = 250 nm and λem = 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.