HPLC-FLUORIMETRIC METHOD FOR ANALYSIS OF FREE FATTY ACIDS IN STELLERA CHAMAEJASMA L

Abstract

A sensitive HPLC method for the determination of fatty acids using 2-(2-(pyren-1-yl)-1H-benzo[d]imidazol-1-yl) ethyl-p-toluenesulfonate (PBIOTs) as a novel labeling reagent with fluorescence detection has been developed. PBIOTs could easily and quickly label fatty acids in the presence of the K2CO3 catalyst at 90°C for 30 min in N,N-dimethylformamide solvent. Fatty acids 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. PBIOTs-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 + H–319]+ and m/z 319.0 (here, m/z 319 is the core structural moiety of the PBIOTs molecule). The fluorescence excitation and emission wavelengths of the derivatives were λex = 350 nm and λem = 402 nm, respectively. Linear correlation coefficients for all fatty acid derivatives were ≥0.9985. Detection limits for the labeled fatty acids, at a signal-to-noise ratio of 3:1, were in the range of 10.32– 44.28 fmol. From the extracts of Stellera chamaejasma L, free fatty acids were sensitively determined.