Determination of Double-Bond Positions in Highly Unsaturated Fatty Acids by GC-MS

Abstract

A method for determining double-bond positions in highly unsaturated fatty acids by GC-MS was developed and is conducted as follows : A highly unsaturated fatty acid methyl ester is partially reduced with hydrazine or deuterated hydrazine. After acidification, the resulting mixture containing several kinds of unsaturated esters along with one saturated ester is recovered by hexane extraction and then separated by argentation TLC according to the number of double bonds. The isolated methyl monoenoate fraction corresponding to standard methyl cis-monoenoates is subjected to I2-catalyzed reaction with dimethyl disulfide. The resulting dimethyl disulfide adducts of methyl monoenoates areanalyzed by GC-MS. The cleavage between the methylthio-substituted carbons yielded sets of key fragment ions showing the original double-bond positions in the monoenoate carbon chain. The GC-MS data on the structures of methyl monoenoates derived from the starting highly unsaturated ester confirmed the original double-bond positions in the molecule of the starting ester. Separately using hydrazine and deuterated hydrazine as reducing reagents for the simultaneous analysis of the same sample provided further useful data for identifying the sample structure owing to the resulting shift of key fragment ions with deuterium atoms.This method was applied to the analysis of polyenoic fatty acids in eel total lipid. The most highly unsaturated fatty acid in the lipids was found to be cis-4, cis-7, cis-10, cis-13, cis-16, cis-19-docosahexaenoic acid based on mass spectral data.