Comparison of the grignard deacylation TLC and HPLC methods and high resolution 13C-NMR for the sn-2 positional analysis of triacylglycerols containing gamma-linolenic acid

There is increasing evidence that the fatty acid, gamma-linolenic acid (GLA) is the effective component found in evening primrose oil (EPO) which has been shown to bring about clinical improvement in a number of disease conditions. The two major triacylglycerols (TAGs) in EPO are trilinolein (LLL) and a TAG species containing one GLA and two linoleic (LA) fatty acid chains. This latter TAG, called dilinoleoyl-mono-gamma-linolenin (DLMG or Oenotheral), makes up about 15% by weight of EPO and accounts for over one-half of the total amount of GLA present in EPO. Although DLMG is comprised of three possible isomers, the abbreviation is used to represent the naturally occurring mixture of these isomers. We have isolated DLMG from EPO and also prepared its three possible isomers, sn-GLL, sn-LGL and sn-LLG, and carried out the sn-2 positional analysis using three different approaches, namely, Grignard deacylation TLC and HPLC methods and high resolution 13C-NMR spectroscopy. The results of the sn-2 positional analysis for both the natural and synthetic TAGs containing LA and GLA in this study using the three approaches are all in very good agreement. This indicates that the three positional analysis methods are valid within their acceptable error margin and can be used with confidence in determining the fatty acid composition of the sn-2 position. Given the increased availability of NMR spectrometers this method might prove to be the easiest and most convenient in determining the sn-2 position for oil or TAG samples that contain a small number of different fatty acids providing all the 13C-NMR carbonyl resonances are well resolved.