Fluorometric Determination of Eicosapentaenoic and Docosahexaenoic Acids by High-Performance Liquid Chromatography.

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A high-performance liquid chromatographic method with fluorescence detection was described for the determination of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). 2-(4-Hydrazinocarbonylphenyl)-4, 5-diphenyl- imidazole (HCPI) was used as a fluorescent label. The derivatization of EPA and DHA with HCPI was performed in 20min at room temperature in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and pyridine as condensing agents. The HCPI derivatives separated on an ODS column by a gradient elution were detected at 455nm with an excitation at 335nm. The calibration curves for both EPA and DHA were linear over the range 1-600pmol per 20-μl injection (r>0.999 for both acids). The detection limits for EPA and DHA were 200 and 190fmol, respectively, at a signal-to-noise ratio of 3. Relative standard deviation of the peak height for seven replicate measurements of these fatty acids were ca. 2%. EPA and DHA were successfully determined in human serum, dietary supplements and a pharmaceutical preparation by this method.
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