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.