Lipids

, Volume 35, Issue 10, pp 1147–1153

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

  • Ewa Ostrowska
  • Frank R. Dunshea
  • Morley Muralitharan
  • Reginald F. Cross
Article

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.

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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References

  1. 1.
    Morrison, R.T., and Boyd, R.N. (1959) Organic Chemistry, p. 49, Allyn and Bacon, Boston.Google Scholar
  2. 2.
    Belury, M.A. (1995) Conjugated Dienoic Linoleate: A Polyunsaturated Fatty Acid with Unique Chemoprotective Properties, Nutr. Rev. 53, 83–89.PubMedCrossRefGoogle Scholar
  3. 3.
    Ha, Y.L., Grimm, N.K., and Pariza, M.W. (1987) Anticarcinogens from Fried Ground Beef: Heat-Altered Derivatives of Linoleic Acid, Carcinogenesis 8, 1881–1887.PubMedGoogle Scholar
  4. 4.
    Ip, C., Singh, M., Thompson, H.J., and Scimeca, J.A. (1994) Conjugated Linoleic Acid Suppresses Mammary Carcinogenesis and Proliferative Activity of the Mammary Gland in the Rat, Cancer Res. 54, 1212–1215.PubMedGoogle Scholar
  5. 5.
    Ip, C., Chin, S.F., Scimeca, J.A., and Pariza, M.W. (1991) Mammary Cancer Prevention by Conjugated Dienoic Derivative of Linoleic Acid, Cancer Res. 51, 6118–6124.PubMedGoogle Scholar
  6. 6.
    Nicolosi, R.J., Rogers, E.J., Knichevski, D., Scimeca, J.A. and Huth, P.J. (1997) Dietary Conjugated Linoleic Acid Reduces Plasma Lipoproteins and Early Aortic Artherogenesis in Hypercholesterolemic Hamsters, Artery 22, 266–277.PubMedGoogle Scholar
  7. 7.
    Houseknecht, K.L., Vanden Heuvel, J.P., Moya-Camarena, S.Y., Portocarrero, C.P., Peck, L.W., Nickel, K.P., and Belury, M.A. (1998) Dietary Conjugated Linoleic Acid Normalizes Impaired Glucose Tolerance in the Zucker Diabetic Fatty fa/fa rat [published erratum appears in Biochem. Biophys. Res. Commun. 1998 June 29; 247(3):911] Biochem. Biophys. Res. Commun. 244, 678–682.PubMedCrossRefGoogle Scholar
  8. 8.
    Cook, M.E., Miller, C.C., Park, Y., and Pariza, M. (1993) Immune Modulation by Altered Nutrient Metabolism: Nutritional Control of Immune-Induced Growth Depression, Poult. Sci. 72, 1301–1305.PubMedGoogle Scholar
  9. 9.
    Miller, C.C., Park, Y., Pariza, M.W., and Cook, M.E. (1994) Feeding Conjugated Linoleic Acid to Animals Partially Overcomes Catabolic Responses Due to Endotoxin Injection, Biochem. Biophys. Res. Commun. 198, 1107–1112.PubMedCrossRefGoogle Scholar
  10. 10.
    Albright, K., Liu, K.L., Storkson, J.M., Hentges, E., Lofgren, P., Simeca, J., Cook, M.E., and Pariza, M. (1996) Body Composition Repartitioning Following the Removal of Dietary Conjugated Linoleic Acid, J. Anim. Sci. 74, 152.Google Scholar
  11. 11.
    Chin, S.F., Storkson, J.M., Albright, K.J., Cook, M.E., and Pariza, M.W. (1994) Conjugated Linoleic Acid Is a Growth Factor for Rats as Shown by Enhanced Weight Gain and Improved Feed Efficiency, J. Nutr. 124, 2344–2349.PubMedGoogle Scholar
  12. 12.
    Park, Y., Albright, K.J., Liu, W., Storkson, J.M., Cook, M.E., and Pariza, M.W. (1997) Effect of Conjugated Linoleic Acid on Body Composition in Mice, Lipids 32, 853–858.PubMedGoogle Scholar
  13. 13.
    Ostrowska, E., Muralitharan, M., Cross, R.F., Bauman, D.E., and Dunshea, F.R. (1999) Dietary Conjugated Linoleic Acids Increase Lean Tissue and Decrease Fat Deposition in Growing Pigs, J. Nutr. 129, 2037–2042.PubMedGoogle Scholar
  14. 14.
    Bretillon, L., Chardigny, J.M., Gregoire, S., Berdeaux, O., and Sebedio, J.L. (1999) Effects of Conjugated Linoleic Acid Isomers on the Hepatic Microsomal Desaturation Activities in vitro, Lipids 34, 965–969.PubMedGoogle Scholar
  15. 15.
    Park, Y., Storkson, J.M., Albright, K.J., Liu, W., and Pariza, M.W. (1999) Evidence That the trans-10,cis-12 Isomer of Conjugated Linoleic Acid Induces Body Composition Changes in Mice, Lipids 34, 235–241.PubMedGoogle Scholar
  16. 16.
    Kramer, J.K., Gellner, V., Dugan, M.E., Sauer, F.D., Mossoba, M.M., and Yurawecz, M.P. (1997) Evaluating Acid and Base Catalysts in the Methylation of Milk and Rumen Fatty Acids with Special Emphasis on Conjugated Dienes and Total trans Fatty Acids, Lipids 32, 1219–1228.PubMedGoogle Scholar
  17. 17.
    Werner, S.A., Luedecke, L.O., and Shultz, T.D. (1992) Determination of Conjugated Linoleic Acid and Isomer Distribution in Three Cheddar-type Cheeses: Effect of Cheese Cultures, J. Agric. Food Chem. 40, 1817–1821.CrossRefGoogle Scholar
  18. 18.
    Yurawecz, M.P., Hood, J.K., Roach, J.A., Mossoba, M.M., Daniels, D.H., Ku, Y., Pariza, M.W., and Chin, S.F. (1994) Conversion of Allylic Hydroxy Oleate to Conjugated Linoleic Acid and Methoxy Oleate by Acid-catalyzed Methylation Procedures, J. Am. Oil Chem. Soc. 71, 1149–1155.Google Scholar
  19. 19.
    Sehat, N., Rickert, R., Mossoba, M.M., Kramer, J.K., Yurawecz, M.P., Roach, J.A., Adlof, R.O., Morehouse, K.M., Fritsche, J., Eulitz, K.D., et al. (1999) Improved Separation of Conjugated Fatty Acid Methyl Esters by Silver Ion-High-Performance Liquid Chromatography, Lipids 34, 407–413.PubMedGoogle Scholar
  20. 20.
    Adlof, R., and Lamm, T. (1998) Fractionation of cis- and trans-Oleic, Linoleic, and Conjugated Linoleic Fatty Acid Methyl Esters by Silver Ion High-Performance Liquid Chromatography, J. Chromatogr. A 799, 329–332.CrossRefGoogle Scholar
  21. 21.
    Sehat, N., Yurawecz, M.P., Roach, J.A., Mossoba, M.M., Kramer, J.K.G., and Ku, Y. (1998) Silver-Ion High-Performance Liquid Chromatographic Separation and Identification of Conjugated Linoleic Acid Isomers, Lipids 33, 217–221.PubMedGoogle Scholar
  22. 22.
    Rickert, R., Steinhart, H., Fritsche, J., Sehat, N., Yurawecz, M.P., Mossoba, M.M., Roach, J.A., Eulitz, K., Ku, Y., and Kramer, J.K. (1999) Enhanced Resolution of Conjugated Linoleic Acid Isomers by Tandem-Column Silver-Ion High-Performance Liquid Chromatography, J. High Resolut. Chromatogr. 22, 144–148.CrossRefGoogle Scholar
  23. 23.
    Cross, R.F., Ostrowska, E., Muralitharan, M., and Dunshea, F.R. (2000) Mixed Mode Retention and the Use of Competing Acid for the Ag-HPLC Analysis of Underivatized Conjugated Linoleic Acids, J. High Resolut. Chromatogr. 23, 317–323.CrossRefGoogle Scholar
  24. 24.
    Folch, J., Lees, M., and Sloane Stanley, G.H. (1957) A Simple Method for the Isolation and Purification of Total Lipids from Animal Tissues, J. Biol. Chem. 226, 497–509.PubMedGoogle Scholar
  25. 25.
    Banni, S., Day, B.W., Evans, R.W., Coprongiu, F.P., and Lombardi, B. (1994) Liquid Chromatographic-Mass Spectrometric Analysis of Conjugated Diene Fatty Acids in a Partially Hydrogenated Fat, J. Am. Oil Chem. Soc. 71, 1321–1325.Google Scholar
  26. 26.
    Stoffel, W., Chu, F., and Ahrens, E.H. (1959) Analysis of Long-Chain Acid by Gas-Liquid Chromatography, Anal. Chem. 31, 307–308.CrossRefGoogle Scholar
  27. 27.
    Schuchardt, U., and Lopes, O.C. (1988) Tetramethylguanidine Catalyzed Transesterification of Fats and Oils: A New Method for Rapid Determination of Their Composition, J. Am. Oil Chem. Soc. 65, 1940–1941.Google Scholar
  28. 28.
    Official Methods of Analysis of AOAC International, 16th edn., 1995, Method 969.33, Association of Official Analytical Chemists, Arlington.Google Scholar
  29. 29.
    Hashimoto, N., Aoyama, T., and Shioiri, T. (1981) A Simple Efficient Preparation of Methyl Esters with Trimethylsilyldiazomethane (TMSCHN2) and Its Application to Gas Chromatographic Analysis of Fatty Acids, Chem. Pharm. Bull. 29, 1475–1478.Google Scholar
  30. 30.
    Payne, R.W., Lane, P.W., and Genstat 5 Committee (1993) Genstat 5 Reference Manual, pp. 76–80, Oxford Science Publications, Oxford.Google Scholar
  31. 31.
    Miller, R.G. (1981) Simultaneous Statistical Inference, pp. 76–80, Springer-Verlag, New York.Google Scholar
  32. 32.
    Jiang, J., Bjorck, L., Fonden, R., and Emanuelson, M. (1996) Occurrence of Conjugated cis-9, trans-11-Octadecadienoic Acid in Bovine Milk: Effects of Feed and Dietary Regimen, J. Dairy Sci. 79, 438–445.PubMedCrossRefGoogle Scholar
  33. 33.
    van den Berg, J.J., Cook, N.E., and Tribble, D.L. (1995) Reinvestigation of the Antioxidant Properties of Conjugated Linoleic Acid, Lipids 30, 599–605.PubMedGoogle Scholar
  34. 34.
    Eulitz, K., Yurawecz, M.P., Sehat, N., Fritsche, J., Roach, J.A., Mossoba, M.M., Kramer, J.K.G., Adlof, R.O., and Ku, Y. (1999) Preparation, Separation, and Confirmation of the Eight Geometrical cis/trans Conjugated Linoleic Acid Isomers 8,10-Through 11,13-18:2, Lipids 34, 873–877.PubMedGoogle Scholar
  35. 35.
    Ip, C., Banni, S., Angioni, E., Carta, G., McGinley, J., Thompson, H.J., Barbano, D., and Bauman, D. (1999) Conjugated Linoleic Acid-Enriched Butter Fat Alters Mammary Gland Morphogenesis and Reduces Cancer Risk in Rats, J. Nutr. 129, 2135–2142.PubMedGoogle Scholar

Copyright information

© AOCS Press 2000

Authors and Affiliations

  • Ewa Ostrowska
    • 3
    • 1
  • Frank R. Dunshea
    • 1
  • Morley Muralitharan
    • 2
  • Reginald F. Cross
    • 3
  1. 1.Agriculture VictoriaVictorian Institute of Animal ScienceWerribeeAustralia
  2. 2.Charles Sturt UniversityWagga WaggaAustralia
  3. 3.School of Engineering and ScienceSwinburne University of TechnologyHawthornAustralia

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