Nervonic acid versus tricosanoic acid as internal standards in quantitative gas chromatographic analyses of fish oil longer-chain n-3 polyunsaturated fatty acid methyl esters.

Tricosanoic acid (23:0) and cis-15-tetracosenoic acid (nervonic acid, 24:1 n-9) were compared as choices suitable for use as internal standards in the quantitation of eicosapentaenoic (EPA) and docosahexaenoic (DHA) fatty acids. Experiments conducted included: (a) comparison of the flame ionisation detector responses of the two fatty acid methyl esters; (b) estimation of accurately weighed quantities of EPA and DHA using both 23:0 and 24:1 separately as internal standard; (c) determination of EPA and DHA contents of commercially available fish oil ethyl ester capsules using the two as internal standard. The results suggest that both 23:0 and 24:1 methyl esters behaved similarly in the flame ionization detector of the gas chromatograph and are comparable internal standards for use in quantitation of EPA and DHA. This includes the analysis of ethyl ester mixtures as long as interesterification of sample with solvent methanol is complete. The relatively poor solubility of the saturated 23:0 is countered by its greater stability. A possible drawback of 24:1 could be the presence of more than one positional isomer in either a 24:1n-9 standard or in the actual sample. In principle any fatty acid could serve as an internal standard as long as the limitations involved in the use of each are taken into account.