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 (C12), palmitic acid (C16), 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, C10, C12, C14, C16, C18 and C20) could be determined using margaric acid (C17) as an internal standard. Calibration curves were linear over the range 0.2-400 pmol per 20-microliters 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 (C12, C14, C16 and C18) in normal human serum.