2-(4-HYDRAZINOCARBONYLPHENYL)-4,5-DIPHENYLIMIDAZOLE AS A VERSATILE FLUORESCENT DERIVATIZATION REAGENT FOR THE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC ANALYSIS OF FREE FATTY-ACIDS

A new fluorescent derivatization reagent with a lophine skeleton, 2-(4-hydrazinocarbonylphenyl)-4,5-diphenylimidazole (HCPI), has been synthesized and applied to the assay of saturated free fatty acids. The HCPI derivatives with five representative saturated fatty acids [caproic acid (C6), lauric acid (C-12), palmitic acid (C-16), stearic acid (C18) and arachidic acid (C20)] were synthesized to examine their fluorescence properties. The fluorescence spectra (lambda(ex) ca. 335 nm, lambda(em) ca. 455 nm) of the derivatives were almost identical in methanol, n-heptane and acetonitrile. For the high-performance liquid chromatographic assay, fatty acids were derivatized with HCPI using a condensing agent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and pyridine at room temperature. The HCPI derivatives were separated on a C18 column with a gradient elution of methanol-water, and the eluates were monitored at 455 nm using an excitation wavelength of 335 nm. By this method, seven kinds of free fatty acid (C6, C-10, C-12, C-14, C-16, C18 and C20) could be determined using margaric acid (C-17) as an internal standard. Calibration curves were linear over the range 0.2-400 pmol per 20-mul injection (r = 0.994-1.000). Relative standard deviations of the peak-height ratio for five replicate measurements of fatty acids (100 pmol per injection) were 0.7-2.6%. Detection limits were 7-57 fmol at a signal-to-noise ratio of 3. The method could be successfully applied to the determination of four saturated fatty acids (C-12, C-14, C-16 and C18) in normal human serum.