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

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Lipids

Abstract

Silver-ion high-performance liquid chromatography was used to fractionate a mixture of conjugated linoleic acid (CLA) isomers (as the free fatty acids, CLAFFA) in commercial CLA mixtures and biological samples. Due to the unchanged retention mechanism, it was assumed that the elution order of the isomers remained the same as that of methyl esters separated on the same column. 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 profile to evaluate different methylation methods commonly used to prepare CLA methyl esters for quantitation. Acid- and vuigi base-catalyzed derivatization methods resulted in CLA intraisomerization and losses in total conjugated dienes content. Acid (HCl and BF3) methylations significantly elevated the level of trans,trans isomers and significantly reduced the cis/trans isomers. Base methylation, tetramethylguanidine/methanol, resulted in loss of trans,trans isomers, and a substantial loss of total underivatized conjugated dienes. Other catalysts such as the trimethylsilyldiazomethane produced additional peaks of unidentified artifacts. The analysis of CLAFFA appears to provide more accurate quantification of CLA isomers in commercial and biological samples.

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Abbreviations

ACN:

acetonitrile

Ag+-HPLC:

silver-ion high-performance liquid chromatography

AHO:

allylic hydroxy oleate

CLA:

conjugated linoleic acid

cis/trans :

all the CLA isomers having a cis,trans or trans,cis configuration

CLAFFA:

conjugated linoleic acid as the free fatty acids

FAME:

fatty acid methyl esters

FFA:

free fatty acid

GC:

gas chromatography

TAG:

triacylglycerol

TMG:

tetramethylguanidine

TMS:

trimethylsilyldiazomethane

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Authors and Affiliations

  1. Agriculture Victoria, Victorian Institute of Animal Science, 3030, Werribee, Victoria, Australia

    Ewa Ostrowska & Frank R. Dunshea

  2. Charles Sturt University, 2650, Wagga Wagga, NSW, Australia

    Morley Muralitharan

  3. School of Engineering and Science, Swinburne University of Technology, P.O. Box 218, 3122, Hawthorn, Victoria, Australia

    Ewa Ostrowska & Reginald F. Cross

Authors
  1. Ewa Ostrowska
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  2. Frank R. Dunshea
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  3. Morley Muralitharan
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  4. Reginald F. Cross
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Correspondence to Reginald F. Cross.

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Ostrowska, E., Dunshea, F.R., Muralitharan, M. et al. Comparison of silver-ion high-performance liquid chromatographic quantification of free and methylated conjugated linoleic acids. Lipids 35, 1147–1153 (2000). https://doi.org/10.1007/s11745-000-0630-y

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  • DOI: https://doi.org/10.1007/s11745-000-0630-y

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