Transesterification of triglycerides and amino acid esters

The neutral lipid fraction extracted from biological materials contains among other components all the triglyceride fats. The fatty acid composition of these is sometimes of great significance, and methods for the establishment of this composition are based on the quantitative analysis of fatty acid methyl esters by gas chromatography. Initially, alkaline hydrolysis released the fatty acids, which were then extracted, concentrated and esterified, some kind of sulphuric acid/methanol procedure being widely used. However, this sequence of separate procedures has been superseded because losses may occur due to incomplete hydrolysis or side reactions such as polymerization or the alteration of unsaturated fatty adds. The preferred method is transesterification in either alkaline or add media [134]. The most recent methods use methanolic HQ, because in alkaline conditions some hydrolysis may occur as a side reaction and cause lowered yields [19, 135, 136]. There are of course effective quantitative methods for the 

The lipid material (20 mg) in 1 ml of dry benzene, in a reaction vial, is treated with 2 ml of 0.5 M methanolic sodium methoxide, and the vial is capped and heated at 80 °C for 20 minutes. After cooling, the mixture is poured into 10 ml of water and the methyl esters are extracted twice with 1 ml portions of hexane or heptane. The combined extracts are dried with anhydrous sodium sulphate and concentrated for gas chromatographic analysis. 

Lipids (2-10 mg) are treated with 4 ml of 10% AlClj in methanol or other appropriate alcohol on a boiling water bath for an hour. The esters are extracted into 5 ml of light petroleum ether followed by 2 ml of saturated NaCl. The upper layer is concentrated and the residue is dissolved in 0.2-1 ml of CS2 for analysis [138]. 

As with the methylation of free fatty acids [130], TMTFTH may also be used for transmethylation. To 10 mg of the triglyceride extract in a reaction vial are added 0.5 ml of benzene and 200 pi of 0.2 M methanolic TMTFTH. The vial is capped and shaken and the reaction proceeds at room temperature for 30 minutes. The solution may be sampled directly for gas chromatography [139], with the same advantages over the tetramethyl-ammonium procedure [140]. 

This procedure is essentially similar to that given in 2.10.1 above, but uses isopropyl alcohol and sodium methoxide [25]. The intention is to prevent losses of volatile short-chain fatty acid esters. 

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