Journal of the American Oil Chemists' Society

, Volume 87, Issue 12, pp 1425–1433 | Cite as

13C-NMR Regioisomeric Analysis of EPA and DHA in Fish Oil Derived Triacylglycerol Concentrates

  • Erick Reyes Suárez
  • Paul F. Mugford
  • Alfred J. Rolle
  • Ian W. Burton
  • John A. Walter
  • Jaroslav A. KralovecEmail author
Original Paper


The regio-isomeric distribution of the omega-3 polyunsaturated fatty acids (PUFA) cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) in the triacylglycerols (TAG) of anchovy/sardine fish oil was determined by 13C nuclear magnetic resonance (NMR) analysis under quantitative conditions. From the measurements of sn-1,3 and sn-2 carbonyl peak areas it was established that EPA was mainly located in the sn-1,3 positions, whereas DHA primarily occupied the sn-2 position. Reconstituted TAG prepared by Candida antarctica lipase-B (CALB) glycerolysis of the ethyl ester (EE) or the free fatty acid (FFA) forms of anchovy/sardine fish oil, displayed a different pattern: EPA was equally distributed, while DHA was preferentially attached to the sn-1,3 positions. TAG concentrates of varying EPA and DHA molar fractions were prepared by CALB-catalyzed glycerolysis of the corresponding EE and FFA. 13C-NMR analysis of the purified products revealed a lack of CALB regioselectivity for EPA and a slight sn-1,3 regioselectivity for DHA. Since this pattern was observed in all cases of this study, it was concluded that the lipase regioselectivity during TAG synthesis is independent of both the acyl donor type (carboxylic acid or ester) and the fatty acid content of the oil substrate.


13C NMR Regioselectivity Regio-isomeric distribution Triacylglycerol Anchovy Sardine Fish oil concentrate EPA DHA 



This work has been supported by the Industrial Research Assistance Program of the National Research Council of Canada (Project# 674301). We thank Rae Townsley for excellent technical assistance.

Supplementary material

11746_2010_1638_MOESM1_ESM.doc (114 kb)
(DOC 113 kb)


  1. 1.
    Farzaneh-Far R, Lin J, Epel ES, Harris WS, Blackburn EH, Whooley MA (2010) Association of marine omega-3 fatty acid levels with telomeric ageing in patients with coronary hearth disease. J Am Med Assoc 303:250–257CrossRefGoogle Scholar
  2. 2.
    Kotani S, Sakaguchi E, Warashina S, Matsukawa N, Ishikura Y, Kiso Y, Sakakibara M, Yoshimoto T, Guo J, Yamashima T (2006) Dietary supplementation of arachidonic and docosahexaenoic acids improves cognitive dysfunction. Neurosci Res 56:159–164CrossRefGoogle Scholar
  3. 3.
    Yoshii H, Furuta T, Siga H, Moriyma S, Baba T, Maruyama K, Misawa Y, Noriaki H, Linko P (2002) Analysis of docosahexaenoic acid ethyl ester and docosahexaenoic acid triglyceride with oxygen sensor. Biosci Biotechnol Biochem 66:749–753CrossRefGoogle Scholar
  4. 4.
    Song JH, Inoue Y, Miyazawa T (1997) Oxidative stability of docosahexaenoic acid-containing oils in the form of phospholipids, triacylglycerols, and ethyl esters. Biosci Biotechnol Biochem 61:2085–2088CrossRefGoogle Scholar
  5. 5.
    Ikeda I, Sasaki E, Yasunami H, Nomiyama S, Nakayama M, Sugano M, Imaizumi K, Yazawa K (1995) Digestion and lymphatic transport of eicosapentaenoic and docosahexaenoic acids given in the form of triacylglycerol, free acid and ethyl ester in rats. Biochim Biophys Acta 1259:297–304Google Scholar
  6. 6.
    Lawson LD, Hughes BG (1988) Human absorption of fish oil fatty acids as triacylglycerols, free acids, or ethyl esters. Biochem Biophys Res Commun 52:328–335CrossRefGoogle Scholar
  7. 7.
    Haraldsson GG, Thorstad O, Kristiansson B (2003) Lipase catalyzed esterification of marine oil. US Patent 6,518,049Google Scholar
  8. 8.
    Kew S, Wells S, Thies F, McNeill GP, Quinlan PT, Clark GT, Dombrowsky H, Postle AD, Calder PC (2003) The effect of eicosapentaenoic acid on rat lymphocyte proliferation depends upon its position in dietary triacylglycerols. J Nutr 133:4230–4238Google Scholar
  9. 9.
    Kew S, Gibbons ES, Thies F, McNeill GP, Quinlan PT, Calder PC (2003) The effect of feeding structured triacylglycerols enriched in eicosapentaenoic or docosahexaenoic acids on murine splenocyte fatty acid composition and leucocyte phagocytosis. Br J Nutr 90:1071–1080CrossRefGoogle Scholar
  10. 10.
    Christensen MS, Hoy CE, Becker CC, Redgrave TG (1995) Intestinal absorption and lymphatic transport of eicosapentaenoic (EPA), docosahexaenoic (DHA), and decanoic acids: dependence on intramolecular triacylglycerol structure. Am J Clin Nutr 61:56–61Google Scholar
  11. 11.
    Lien EL (1994) The role of fatty acid composition and positional distribution in fat absorption in infants. J Pediatr 125:S62–S68CrossRefGoogle Scholar
  12. 12.
    Lucas A, Quinlan P, Abrams S, Ryan S, Meah S, Lucas PJ (1997) Randomised controlled trial of a synthetic triglyceride milk formula for preterm infants. Arch Dis Child 77:F178–F184Google Scholar
  13. 13.
    Ikeda I, Tomari Y, Sugano M, Watanabe S, Nagata J (1991) Lymphatic absorption of structured glycerolipids containing medium-chain fatty acids and linoleic acid, and their effect on cholesterol absorption in rats. Lipids 26:369–373CrossRefGoogle Scholar
  14. 14.
    Takagi T, Ando Y (1991) Stereospecific analysis of triacyl-sn-glycerols by chiral high-performance liquid chromatography. Lipids 26:542–547CrossRefGoogle Scholar
  15. 15.
    Ando Y, Nishimura K, Aoyanagi N, Takagi T (1992) Stereospecific analysis of fish oil triacyl-sn-glycerols. J Am Oil Chem Soc 69:417–424CrossRefGoogle Scholar
  16. 16.
    Itabashi Y, Kuksis A, Myher JJ (1990) Determination of molecular species of enantiomeric diacylglycerols by chiral phase high performance liquid chromatography and polar capillary gas–liquid chromatography. J Lipid Res 31:2119–2126Google Scholar
  17. 17.
    Redden PR, Lin X, Horrobin DF (1996) Comparison of the Grignard deacylation TLC and HPLC methods and high resolution 13C NMR for the sn-2 positional analysis of triacylglycerols containing γ-linolenic acid. Chem Phys Lipids 79:9–19CrossRefGoogle Scholar
  18. 18.
    Turon F, Bonnot F, Caro Y, Pina M, Graille J (2003) Acyl migration incidence on accuracy of a triacylglycerol regioanalysis––a theoretical evaluation. Chem Phys Lipids 125:41–48CrossRefGoogle Scholar
  19. 19.
    Christie W, Moore JH (1969) A semimicro method for the stereospecific analysis of triglycerides. Biochim Biophys Acta 176:445–452Google Scholar
  20. 20.
    Walker B (1975) Structural determination of lipids by chemical means. In: Perkins EG (ed) Analysis of lipids and lipoproteins. AOCS Press, Champaign, pp 108–122Google Scholar
  21. 21.
    Christie WW (1986) The positional distribution of fatty acids in triglycerides. In: Hamilton RJ, Rossell JB (eds) Analysis of oils and fats. Elsevier, London, pp 313–339Google Scholar
  22. 22.
    International Union of Pure, Applied Chemistry (1987) Standard methods for the analysis of oils, fats and derivatives. IUPAC official method 2.210: determination of fatty acids in the 2-position in the triglycerides of oils and fats, 7th edn. Blackwell, OxfordGoogle Scholar
  23. 23.
    Gunstone FD (1990) 1H- and 13C-NMR of six n-3 polyene esters. Chem Phys Lipids 56:227–229CrossRefGoogle Scholar
  24. 24.
    Gunstone FD (1991) High resolution NMR studies of fish oil. Chem Phys Lipids 59:83–89CrossRefGoogle Scholar
  25. 25.
    Aursand M, Grasdalen H (1992) Interpretation of the 13C-NMR spectra of some omega-3 fatty acids and lipid extracted from the white muscle of Atlantic salmon (Salmo salar). Chem Phys Lipids 62:239–251CrossRefGoogle Scholar
  26. 26.
    Sacchi R, Medina I, Aubourg SP, Giudicianni I, Paolillo L, Addeo F (1993) Quantitative high-resolution 13C NMR analysis of lipids extracted from the white muscle of Atlantic tuna (Thunnus alalunga). J Agric Food Chem 41:1247–1253CrossRefGoogle Scholar
  27. 27.
    Aursand M, Rainuzzo JR, Grasdalen H (1993) Quantitative high-resolution 13C and 1H nuclear magnetic resonance of ω3 fatty acids from white muscle of Atlantic salmon (Salmo salar). J Am Oil Chem Soc 70:971–981CrossRefGoogle Scholar
  28. 28.
    Gunstone FD, Seth S (1994) A study of the distribution of eicosapentaenoic acid and docosahexaenoic acid between the α and β glycerol chains in fish oils by 13C NMR spectroscopy. Chem Phys Lipids 72:119–126CrossRefGoogle Scholar
  29. 29.
    Aursand M, Jørgensen L, Grasdalen H (1995) Positional distribution of ω3 fatty acids in marine lipid triacylglycerols by high-resolution 13C nuclear magnetic resonance spectroscopy. J Am Oil Chem Soc 72:293–297CrossRefGoogle Scholar
  30. 30.
    Aursand M, Standal IB, Axelson DE (2007) High-resolution 13C nuclear magnetic resonance spectroscopy pattern recognition of fish oil capsules. J Agric Food Chem 55:38–47CrossRefGoogle Scholar
  31. 31.
    Standal IB, Praël A, McEvoy L, Axelson DE, Aursand M (2008) Discrimination of cod liver oil according to wild/farmed and geographical origins by GC and 13C NMR. J Am Oil Chem Soc 85:105–112CrossRefGoogle Scholar
  32. 32.
    Standal IB, Axelson DE, Aursand M (2009) Differentiation of fish oils according to species by 13C-NMR regiospecific analyses of triacylglycerols. J Am Oil Chem Soc 86:401–407CrossRefGoogle Scholar
  33. 33.
    Saddiqui N, Sim J, Silwood CJL, Toms H, Iles RA, Grootveld M (2003) Multicomponent analysis of encapsulated marine oil supplements using high resolution 1H and 13C NMR techniques. J Lipid Res 44:2406–2427CrossRefGoogle Scholar
  34. 34.
    Council of Europe, European Directorate for the Quality of Medicines (EDQM) (2006) Composition of fatty acids in oils rich in omega-3 acids 5.5 (01/2006:4107), European PharmacopoeiaGoogle Scholar
  35. 35.
    Gottlieb HE, Kotlyar V, Nudelman A (1997) NMR chemical shifts of common laboratory solvents as trace impurities. J Org Chem 62:7512–7515CrossRefGoogle Scholar
  36. 36.
    Bergana M, Lee TW (1996) Structure determination of long-chain polyunsaturated triacylglycerols by high-resolution 13C nuclear magnetic resonance. J Am Oil Chem Soc 73:551–556CrossRefGoogle Scholar
  37. 37.
    Evilia RF (2001) Quantitative NMR spectroscopy. Anal Lett 34:2227–2236CrossRefGoogle Scholar
  38. 38.
    Wollenberg KF (1990) Quantitative high resolution 13C nuclear magnetic resonance of the olefinic and carbonyl carbons of edible vegetable oils. J Am Oil Chem Soc 67:487–494CrossRefGoogle Scholar
  39. 39.
    Vlahov G (1998) Regiospecific analysis of mixtures of triacylglycerides using 13C nuclear magnetic resonance of acyl chain carbonyl carbons. Magn Reson Chem 36:359–362CrossRefGoogle Scholar

Copyright information

© AOCS 2010

Authors and Affiliations

  • Erick Reyes Suárez
    • 1
  • Paul F. Mugford
    • 1
  • Alfred J. Rolle
    • 1
  • Ian W. Burton
    • 2
  • John A. Walter
    • 2
  • Jaroslav A. Kralovec
    • 1
    Email author
  1. 1.Ocean Nutrition Canada LimitedDartmouthCanada
  2. 2.Institute for Marine BiosciencesNational Research Council of CanadaHalifaxCanada

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