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Evaluation of two GC columns (60-m SUPELCOWAX 10 and 100-m CP sil 88) for analysis of milkfat with emphasis on CLA, 18∶1, 18∶2 and 18∶3 isomers, and short- and long-chain FA

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Lipids

Abstract

Milkfat is a complex mixture of many diverse FA, some of which have demonstrated health benefits including anticancer properties. Attempts are under way to enrich milkfats with long-chain n−3 PUFA and CLA. It has been recommended that the analysis of these milkfats requires gas chromatography (GC) equipped with long, highly polar capillary columns. However, many analyses have been reported using CARBOWAXTM type (polyethylene glycol) capillary columns, such as SUPELCOWAX 10, even though the separation characteristics of many of the FA and their isomers present in milkfats have not been described in detail. This includes the isomers of CLA, cis- and trans-octadecenoic acid (18∶1), linoleic acid (18∶2n−6), and linolenic acid (18∶3n−3), and the long-chain PUFA. On the other hand, the resolution of these FA and their isomers has been more fully described using the highly polar capillary columns, such as CP Sil 88 and SP2560 because of the improved resolution obtained using these polar columns. The present study was undertaken to characterize the separation of these FA present in milkfats using a 60-m SUPELCOWAX 10 column, to compare the results to those from a 100-m CP Sil 88 column, and to determine if these two columns could possibly serve to complement each other for the analysis of total milkfat. The advantages of the SUPELCOWAX 10 column were a better resolution of the short-chain saturated from their monounsaturated FA (MUFA) analogs, and a complete separation of the α-linolenic (18∶3n−3) and eicosadecenoic acid (20∶1) isomers. It also provided an alternative elution order of the linoleic (18∶2n−6), 18∶3n−3 and γ-linolenic (18∶3n−6) acid isomers. On the other hand, the CP Sil 88 column provided a better resolution of the CLA isomers, MUFA, the isolated cis and trans MUFA fractions, the PUFA, and many the 18∶2n−6 and 18∶3n−3 isomers. A complete analysis of milk lipids using the CP Sil 88 column required the prior separation of total FAME using silver ion-TLC. The results of the present study confirm that the 100-m highly polar capillary GC columns are mandatory for the analysis of milk lipids, and at best, the 60 m SUPELCOWAX 10 capillary column serves as a complementary GC column to provide different separations in certain regions based on its intermediate polarity.

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Abbreviations

Ag+-TLC:

silver ion-thin layer chromatography

MUFA:

monounsaturated fatty acids. The Δ nomenclature for double bond position is used throughout the text except for identification of most methylene-interrupted PUFA in which the terminal n position is identified

References

  1. Yurawecz, M.P., Mossoba, M.M., Kramer, J.K.G., Pariza, M.P., and Nelson, G.J. (eds.) (1999) Advances in Conjugated Linoleic Acid Research, Vol. 1, AOCS Press, Champaign.

    Google Scholar 

  2. Pariza, M.P., Park, Y., and Cook, M.E. (2001) The Biologically Active Isomers of Conjugated Linoleic Acid, Progr. Lipid Res. 40, 283–298.

    Article  CAS  Google Scholar 

  3. Ha, Y.L., Grimm, N., and Pariza, M.W. (1989) Newly Recognized Anticarcinogenic Fatty Acids: Identification and Quantitation in Natural and Processed Cheeses, J. Agric. Food Chem. 37, 75–81.

    Article  CAS  Google Scholar 

  4. Sehat, N., Yurawecz, M.P., Roach, J.A.G., 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.

    Article  PubMed  CAS  Google Scholar 

  5. Lavillonnière, F., Martin, J.C., Bougnoux, P., and Sébédio, J.-L. (1999) Analysis of Conjugated Linoleic Acid Isomers and Content in French Cheeses, J. Am. Oil Chem Soc. 75, 343–352.

    Google Scholar 

  6. Sehat, N., Rickert, R., Mossoba, M.M., Kramer, J.K.G., Yurawecz, M.P., Roach, J.A.G., Adlof, R.O., Morehouse, K.M., Fritsche, J., Eulitz, K., et al. (1999) Improved Separation of Conjugated Linoleic Acid Methyl Esters by Silver-Ion High-Performance Liquid Chromatography, Lipids 34, 407–413.

    Article  PubMed  CAS  Google Scholar 

  7. Kramer, J.K.G., Sehat, N., Fritsche, J., Mossoba, M.M., Eulitz, K., Yurawecz, M.P., and Ku, Y. (1999) Separation of Conjugated Linoleic Acid Isomers, in Advances in Conjugated Linoleic Acid Research (Yurawecz, M.P., Mossoba, M.M., Kramer, J.K.G., Pariza, M.P., and Nelson, G.J., eds.), Vol. 1, pp. 83–109, AOCS Press, Champaign.

    Google Scholar 

  8. Mahfouz, M.M., Valicenti, A.J., and Holman, R.T. (1980) Desaturation of Isomeric trans-Octadecenoic Acids by Rat Liver Microsomes, Biochim. Biophys. Acta 618, 1–12.

    PubMed  CAS  Google Scholar 

  9. Griinari, J.M., and Bauman, D.E. (1999) Biosynthesis of Conjugated Linoleic Acid and Its Conversion into Meat and Milk in Ruminants, in Advances in Conjugated Linoleic Acid Research, (Yurawecz, M.P., Mossoba, M.M., Kramer, J.K.G., Pariza, M.P., and Nelson, G.J., eds.), Vol. 1, pp. 180–200, AOCS Press, Champaign.

    Google Scholar 

  10. Griinari, J.M., Corl, B.A., Lacy, S.H., Chouinard, P.Y., Nurmela, K.V.V., and Bauman, D.E. (2000) Conjugated Linoleic Acid Is Synthesized Endogenously in Lactating Dairy Cows by Δ9-Desaturase, J. Nutr. 130, 2285–2291.

    PubMed  CAS  Google Scholar 

  11. Santora, J.E., Palmquist, D.L., and Roehrig, K.L. (2000) Trans-Vaccenic Acid Is Desaturated to Conjugated Linoleic Acid in Mice, J. Nutr. 130, 208–215.

    PubMed  CAS  Google Scholar 

  12. Salminen, I., Mutanen, M., Jauhiainen, M., and Aro, A. (1998) Dietary trans Fatty Acids Increase Conjugated Linoleic Acid Levels in Human Serum, Nutr. Biochem. 9, 93–98.

    Article  CAS  Google Scholar 

  13. Adlof, R.O., Duval, S., and Emken, E.A. (2000) Biosynthesis of Conjugated Linoleic Acid in Humans, Lipids 35, 131–135.

    PubMed  CAS  Google Scholar 

  14. Prevention and Treatment of Vascular Disease—A Nutritional-Based Approach, Part III: n−3 Fatty Acids (2001) Lipids 36 (Suppl.) S65–S129.

  15. A Symposium on PUFA in Maternal and Child Health, September 10–13, 2000, Kansas City, Missouri (2001) Lipids 36, 859–1076.

  16. Wolff, R.L., and Bayard, C.C. (1995) Improvement in the Resolution of Individual trans-18∶1 Isomers by Capillary Gas-Liquid Chromatography: Use of a 100-m CP Sil 88 Column, J. Am. Oil Chem. Soc. 72, 1197–1201.

    CAS  Google Scholar 

  17. Molkentin, J., and Precht, D. (1995) Optimized Analysis of trans-Octadecenoic Acids in Edible Fats, Chromatographia 41, 267–272.

    CAS  Google Scholar 

  18. Mossoba, M.M., McDonald, R.E., Roach, J.A.G., Fingerhut, D.D., Yurawecz, M.P., and Sehat, N. (1997) Spectral Confirmation of trans Monounsaturated C18 Fatty Acid Positional Isomers, J. Am. Oil Chem. Soc. 74, 125–130.

    CAS  Google Scholar 

  19. Precht, D., and Molkentin, J. (1999) C18∶1, C18∶2 and C18∶3 trans and cis Fatty Acid Isomers Including Conjugated cis Δ9, trans Δ11 Linoleic Acid (CLA) as Well as Total Fat Composition of German Human Milk Lipids, Nahrung 43, 233–244.

    Article  PubMed  CAS  Google Scholar 

  20. Precht, D., and Molkentin, J. (2000) Identification and Quantitation of cis/trans C16∶1 and C17∶1 Fatty Acid Positional Isomers in German Human Milk Lipids by Thin-Layer Chromatography and Gas Chromatography/Mass Spectrometry, Eur. J. Lipid Sci. Technol. 102, 102–113.

    Article  CAS  Google Scholar 

  21. Wolff, R.L., Precht, D., Nasser, B., and El Kebbaj, M.S. (2001) Trans- and Cis-Octadecenoic Acid Isomers in the Hump and Milk Lipids from Camelus dromedarius, Lipids 36, 1175–1178.

    PubMed  CAS  Google Scholar 

  22. Precht, D., Molkentin, J., Destaillats, F., and Wolff, R.L. (2001) Comparative Studies on Individual Isomeric 18∶1 Acids in Cow, Goat, and Ewe Milk Fats by Low-Temperature High-Resolution Capillary Gas-Liquid Chromatography, Lipids 36, 827–832.

    Article  PubMed  CAS  Google Scholar 

  23. Kramer, J.K.G., Cruz-Hernandez, C., and Zhou, J. (2001) Conjugated Linoleic Acids and Octadecenoic Acids: Analysis by GC, Eur. J. Lipid Sci. Technol. 103, 600–609.

    Article  CAS  Google Scholar 

  24. Kramer, J.K.G., Fellner, V., Dugan, M.E.R., 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.

    Article  PubMed  CAS  Google Scholar 

  25. Kramer, J.K.G., Sehat, N., Dugan, M.E.R., Mossoba, M.M., Yurawecz, M.P., Roach, J.A.G., Eulitz, K., Aalhus, J.L., Schaefer, A.L., and Ku, Y. (1998) Distribution of Conjugated Linoleic Acid (CLA) Isomers in Tissue Lipid Classes of Pigs Fed a Commercial CLA Mixture Determined by Gas Chromatography and Silver Ion-High Performance Liquid Chromatography, Lipids 33, 549–558.

    Article  PubMed  CAS  Google Scholar 

  26. Eulitz, K., Yurawecz, M.P., Sehat, N., Fritsche, J., Roach, J.A.G., 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.

    Article  PubMed  CAS  Google Scholar 

  27. Dhiman, T.R., Anand, G.R., Satter, L.D., and Pariza, M.W. (1999) Conjugated Linoleic Acid Content of Milk from Cows Fed Different Diets, J. Dairy Sci. 82, 2146–2156.

    PubMed  CAS  Google Scholar 

  28. Baumgard, L.H., Corl, B.A., Dwyer, D.A., Saebø, A., and Bauman, D.E. (2000) Identification of the Conjugated Acid Isomer That Inhibits Milk Fat Synthesis, Am. J. Physiol. Regulatory Integrative Comp. Physiol. 278, R179-R184.

    CAS  Google Scholar 

  29. Chouinard, P.Y., Corneau, L., Butler, W.R., Chilliard, Y., Drackley, J.K., and Bauman, D.E. (2001) Effect of Dietary Lipid Source on Conjugated Linoleic Acid Concentrations in Milk Fat, J. Dairy Sci. 84, 680–690.

    PubMed  CAS  Google Scholar 

  30. Kramer, J.K.G., Fouchard, R.C., and Jenkins, K.J. (1985) Differences in Chromatographic Properties of Fused Silica Capillary Columns, Coated, Crosslinked, Bonded, or Crosslinked and Bonded with Polyethylene Glycols (CARBOWAX 20M) Using Complex Fatty Acid Methyl Ester Mixtures, J. Chromatogr. Sci. 23, 54–56.

    CAS  Google Scholar 

  31. Ackman, R.G. (1986) WCOT (Capillary) Gas-Liquid Chromatography, in Analysis of Oils and Fats (Hamilton, R.J., and Rossell, J.B., eds.), pp. 137–206, Elsevier Applied Science Publishers, New York.

    Google Scholar 

  32. Thompson, R.H. (1996) Simplifying Fatty Acid Analyses in Multicomponent Foods with a Standard Set of Isothermal GLC Conditions coupled with ECL Determinations, J. Chromatogr. Sci. 34, 495–504.

    CAS  Google Scholar 

  33. Ratnayake, W.M.N., and Beare-Rogers, J.L. (1990) Problems of Analyzing C18 cis and trans-Fatty Acids of Margarine on the SP-2340 Capillary Column, J. Chromatogr. Sci. 28, 633–639.

    CAS  Google Scholar 

  34. Precht, D., and Molkentin, J. (2000) Recent Trends in the Fatty Acid Composition of German Sunflower Margarines, Shortenings and Cooking Fats with Emphasis on Individual C16∶1, C18∶1, C18∶2 C18∶3, and 20∶1 trans Isomers, Nahrung 44, 222–228.

    Article  PubMed  CAS  Google Scholar 

  35. Wolff, R.L. (1992) Resolution of Linolenic Acid Geometrical Isomers by Gas-Liquid Chromatography on a Capillary Column Coated with a 100% Cyanopropyl Polysiloxane Film (CP™ Sil 88), J. Chromatogr. Sci. 30, 17–22.

    CAS  Google Scholar 

  36. Wolff, R.L. (1995) Recent Applications of Capillary Gas-Liquid Chromatography to Some Difficult Separations of Positional or Geometrical Isomers of Unsaturated Fatty Acids, in New Trends in Lipid and Lipoprotein Analyses (Sébédio, J.-L., and Perkins, E.G., eds.), pp. 147–180, AOCS Press, Champaign.

    Google Scholar 

  37. Precht, D., and Molkentin, J. (1997) Trans-Geometrical and Positional Isomers of Linoleic Acid Including Conjugated Linoleic Acid (CLA) in German Milk and Vegetable Fats, Fett/Lipid 99, 319–326.

    Article  CAS  Google Scholar 

  38. Wolff, R.L. (1994) Analysis of α-Linolenic Acid Geometrical Isomers in Deodorized Oils by Capillary Gas-Liquid Chromatography on Cyanoalkyl Polysiloxane Phases: A Note of Caution, J. Am. Oil Chem. Soc. 71, 907–909.

    CAS  Google Scholar 

  39. Juaneda, P., Sébédio, J.-L., and Christie, W.W. (1994) Complete Separation of the Geometrical Isomers of Linolenic Acid by High-Performance Liquid Chromatography with a Silver Ion Column, J. High Res. Chromatogr. 17, 321–324.

    Article  CAS  Google Scholar 

  40. Bannon, C.D., Craske, J.D., and Norman, L.M. (1988) Effects of Overload of Capillary Gas-Liquid Chromatographic Columns on the Equivalent Chain Lengths of C18 Unsaturated Fatty Acid Methyl Esters, J. Chromatogr. 447, 43–52.

    Article  CAS  Google Scholar 

  41. Christie, W.W. (1988) Equivalent Chain-Lengths of Methyl Ester Derivatives of Fatty Acids on Gas-Liquid Chromatography, J. Chromatogr. 447, 305–314.

    Article  CAS  Google Scholar 

  42. Wijesundera, R.C., and Ackman, R.G. (1989) Evaluation of Calculation of ECL Values for cis and trans Isomers of Some Diethylenic C20 Fatty Acids: Mono- and Diethylenic Capillary GLC Data for the Liquid Phases SP-2340, SUPELCOWAX 10, and SPB-1, J. Chromatogr. Sci. 27, 399–404.

    CAS  Google Scholar 

  43. Christie, W.W. (1982) A Simple Procedure for Rapid Transmethylation of Glycerolipids and Cholesterol Esters, J. Lipid Res. 23, 1072–1075.

    PubMed  CAS  Google Scholar 

  44. Chouinard, P.Y., Corneau, L., Barbano, D.M., Metzger, L.E., and Bauman, D.E. (1999) Conjugated Linoleic Acids Alter Milk Fatty Acid Composition and Inhibit Milkfat Secretion in Dairy Cows, J. Nutr. 129, 1579–1584.

    PubMed  CAS  Google Scholar 

  45. Wolff, R.L. (1994) Contribution of trans-18∶1 Acids from Dairy Fat to European Diets, J. Am. Oil Chem. Soc. 71, 277–283.

    CAS  Google Scholar 

  46. Snyder, J.M., and Scholfield, C.R. (1982) Cis-Trans Isomerization of Unsaturated Fatty Acids with p-Toluenesulfinic Acid, J. Am. Oil Chem. Soc. 59, 469–470.

    CAS  Google Scholar 

  47. Ackman, R.G., and Macpherson, E.J. (1994) Coincidence of cis-and trans-Monoethylenic Fatty Acids Simplifies the Open-Tubular Gas-Liquid Chromatography of Butyl Esters of Butter Fatty Acids, Food Chem. 50, 45–52.

    Article  CAS  Google Scholar 

  48. Ulberth, F., and Henninger, M. (1992) On-Column Injection of Fatty Acid Methyl Esters onto Polar Capillary Columns Without Distortion of Early Eluting Peaks, J. High Res. Chromatogr. 15, 54–56.

    Article  CAS  Google Scholar 

  49. LeDoux, M., Rouzeau, A., Bas, P., and Sauvant, D. (2002) Occurrence of trans-C18∶1 Fatty Acid Isomers in Goat Milk: Effect of Two Dietary Regimens, J. Dairy Sci. 85, 190–197.

    PubMed  CAS  Google Scholar 

  50. Franklin, S.T., Martin, K.R., Baer, R.J., Schingoethe, D.J., and Hippen, A.R. (1999) Dietary Marine Algae (Schizochytrium sp.) Increases Concentrations of Conjugated Linoleic, Docosahexaenoic and trans Vaccenic acids in Milk of Dairy Cows, J. Nutr. 129, 2048–2054.

    PubMed  CAS  Google Scholar 

  51. Piperova, L.S., Teter, B.B., Bruckental, I., Sampugna, J., Mills, S.E., Yurawecz, M.P., Fritsche, J., Ku, K., and Erdman R.A (2000) Mammary Lipogenic Enzyme Activity, trans Fatty Acids and Conjugated Linoleic Acids Are Altered in Lactating Dairy Cows Fed a Milk Fat-Depressing Diet, J. Nutr. 130, 2568–2574.

    PubMed  CAS  Google Scholar 

  52. Ackman, R.G., and Sipos, J.C. (1964) Application of Specific Response Factors in the Gas Chromatographic Analysis of Methyl Esters of Fatty Acids with Flame Ionization Detectors, J. Am. Oil Chem. Soc. 41, 377–378.

    CAS  Google Scholar 

  53. Destaillats, F., and Angers, P. (2002) Base-Catalyzed Derivatization Methodology for FA Analysis. Application to Milk Fat and Seed Lipid TAG, Lipids 37, 527–532.

    PubMed  CAS  Google Scholar 

  54. Griinari, J.M., Dwyer, D.A., McGuire, M.A., Bauman, D.E., Palmquist, D.L., and Nurmela, K.V.V. (1998) trans-Octadecenoic Acids and Milk Fat Depression in Lactating Dairy Cows, J. Dairy Sci. 81, 1251–1261.

    PubMed  CAS  Google Scholar 

  55. Precht, D., Molkentin, J., McGuire, M.A., McGuire, M.K., and Jensen, R.G. (2001) Overestimates of Oleic and Linoleic Acid Contents in Materials Containing trans Fatty Acids and Analyzed with Short Packed Gas Chromatographic Columns, Lipids 36, 213–216.

    Article  PubMed  CAS  Google Scholar 

  56. Ratnayake, W.M.N. (2001) Analysis of Dietary trans Fatty Acids, J. Oleo Sci. 50, 339–352.

    CAS  Google Scholar 

  57. Roach, J.A.G., Yurawecz, M.P., Kramer, J.K.G., Mossoba, M.M., Eulitz, K., and Ku, Y. (2000) Gas Chromatography-High Resolution Selected-Ion Mass Spectrometric Identification of Trace 21∶0 and 20∶2 Fatty Acids Eluting with Conjugated Linoleic Acid Isomers, Lipids 35, 797–802.

    PubMed  CAS  Google Scholar 

  58. Sehat, N., Kramer, J.K.G., Mossoba, M.M., Yurawecz, M.P., Roach, J.A.G., Eulitz, K., Morehouse, K.M., and Ku, Y (1998) Identification of Conjugated Linoleic Acid Isomers in Cheese by Gas Chromatography, Silver Ion High-Performance Liquid Chromatography and Mass Reconstructed Ion Profiles. Comparison of Chromatographic Elution Sequences, Lipids 33, 963–971.

    Article  PubMed  CAS  Google Scholar 

  59. Yamasaki, M., Kishihara, K., Ikeda, I., Sugano, M., and Yamada, K. (1999) A Recommended Esterification Method for Gas Chromatographic Measurement of Conjugated Linoleic Acid, J. Am. Oil. Chem. Soc. 76, 933–938.

    CAS  Google Scholar 

  60. Park, Y., Albright, K.J., Cai, Z.Y., and Pariza, M.W. (2001) Comparison of Methylation Procedures for Conjugated Linoleic Acid and Artifact Formation by Commercial (Trimethylsilyl)Diazomethane, J. Agric. Food Chem. 49, 1158–1164.

    Article  PubMed  CAS  Google Scholar 

  61. Wright, T.S., Holub, B.J., and McBride, B.W. (1999) Apparent Transfer Efficiency of Docosahexaenoic Acid from Diet to Milk in Dairy Cows, Can. J. Anim. Sci. 79, 565–568.

    CAS  Google Scholar 

  62. Baer, R.J., Ryali, J., Schingoethe, D.J., Kasperson, K.M., Donovan, D.C., Hippen, A.R., and Franklin, S.T. (2001) Composition and Properties of Milk and Butter from Cows Fed Fish Oil, J. Dairy Sci. 84, 345–353.

    Article  PubMed  CAS  Google Scholar 

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  1. Food Research Program, Agriculture and Agri-Food Canada, 93 Stone Road West, N1G 5C9, Guelph, Ontario, Canada

    John K. G. Kramer, C. Brian Blackadar & Jianqiang Zhou

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Kramer, J.K.G., Blackadar, C.B. & Zhou, J. Evaluation of two GC columns (60-m SUPELCOWAX 10 and 100-m CP sil 88) for analysis of milkfat with emphasis on CLA, 18∶1, 18∶2 and 18∶3 isomers, and short- and long-chain FA. Lipids 37, 823–835 (2002). https://doi.org/10.1007/s11745-002-0967-2

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