Analytical-scale high-performance liquid chromatography of omega-3 fatty acid esters derived from fish oils

Fish oil triglycerides were transesterified to their corresponding methyl and ethyl esters. The esters were separated by reversed-phase high-performance liquid chromatography on a 10-cm column of 5-microns octadecylsilyl silica, using a ternary mobile phase of acetonitrile-tetrahydrofuran-water (9:5:11). The separation of principal saturated and unsaturated C14-C22 fatty acid esters was accomplished in 60 min. For the polyenoic acid esters, detection limits of ca. 26 micrograms were observed, and baseline resolution of the important critical pair of esters of the omega 3 fatty acids, eicosapentaenoic acid (C20:5 omega 3) and docosahexaenoic acid (C22:6 omega 3) was achieved. However, to obtain baseline resolution of the esters from minor fatty acid esters, a urea crystallization procedure was required prior to chromatographic analysis. The influence on the reversed-phase chromatographic retention of four different ester moieties on the fatty acids were also studied. The n-alkyl esters exhibited an increase in ln k' with increasing chain length and with increasing lipophilic character of the alkyl ester moieties; however, with the aromatic benzyl derivatives there was a decrease in ln k' compared with the alkyl esters.