Comparison of silver-ion high-performance liquid chromatographic quantification of free and methylated conjugated linoleic acids

Silver-ion high-performance liquid chromatography was used to fractionate a mixture of conjugated linoleic acid (CLA) isomers las the free fatty acids , CLAFFA) in commercial CLA mixtures and biological samples. Due to the unc hanged retention mechanism, it was assumed that the elution order of the is omers remained the same as that of methyl esters separated on the same colu mn. The most abundant isomers, cis/trans 10,12-18:2 and cis/trans 9,11-18:2 , were separated better as free acids on a single column than in the methyl ester form. Quantification of the CLA standard was used as the reference p rofile to evaluate different methylation methods commonly used to prepare C LA methyl esters for quantitation. Acid- and base-catalyzed derivatization methods resulted in CLA intraisomerization and losses in total conjugated d ienes content. Acid (HCl and BF3) methylations significantly elevated the l evel of trans,trans isomers and significantly reduced the cis/trans isomers . Base methylation, tetramethylguanidine/methanol, resulted in loss of tran s,trans isomers, and a substantial loss of total underivatized conjugated d ienes. Other catalysts such as the trimethylsilyldiazomethane produced addi tional peaks of unidentified artifacts. The analysis of CLAFFA appears to p rovide more accurate quantification of CLA isomers in commercial and biolog ical samples.