Stereospecific analysis of triacylglycerols rich in long-chain polyunsaturated fatty acids

Six oils of marine, algal, and microbial origin were analyzed for stereospecific distribution of component fatty acids. The general procedure involved preparation ofsn-1,2-(2,3)-diacylglycerols by partial deacylation with ethylmagnesium bromide or pancreatic lipase, separation of X-1,3- andsn-1,2(2,3)-diacylglycerols by borate thin-layer chromatography, resolution of thesn-1,2- andsn-2,3-enantiomers by chiral phase high-performance liquid chromatography following preparation of dinitrophenylurethane derivatives, and determination of the fatty acid composition by gas chromatography. Unexpected complications arose during a stereospecific analysis of triacylglycerols containing over 33% of either 20∶4 or 22∶6 fatty acids. Thesn-1,2(2,3)-diacylglycerols made up of two long-chain polyunsaturated acids migrated with the X-1,3-diacylglycerols and required separate chiral phase resolution. Furthermore, the enzymatic method yieldedsn-1,2(2,3)-diacylglycerols, overrepresenting the polyenoic species due to their relative resistance to lipolysis, but prolonged digestion yielded correct composition for the 2-monoacylglycerols. The final positional distribution of the fatty acids was established by pooling and normalizing the data from subfractions obtained by norman- and chiral-phase separation of diacylglycerols. The molecular species of X-1,3-,sn-1,2- andsn-2,3-diacylglycerol dinitrophenylurethanes were identified by chiral-phase liquid chromatography/mass spectrometry with electrospray ionization, which demonstrated a preferential association of the paired long-chain acids with thesn-1,2- andsn-2,3-diacylglycerol isomers.