, Volume 53, Supplement 1, pp S250–S253 | Cite as

Highly sensitive determination of long-chain free fatty acids by fluorogenic derivatization and liquid chromatography

  • C. Y. Lu
  • H. L. Wu
  • S. H. Chen
  • H. S. Kou
Originals Column Liquid Chromatography


A simple and sensitive method is described for the determination of biologically important fatty acids including dodecanoic, tetradecanoic, hexadecanoic, octadecanoic, hexadecenoic, octadecenoic and octadecadienoic acids. The method is based derivatization with (2-(2-naphthoxy)ethyl 2-(piperidino)ethanesulfonate (NOEPES) in toluene using potassium carbonate and 18-crown-6 as reaction activators. The resulting naphthoxyethyl derivatives were simply analyzed by isocratic HPLC with fluorometric detection (λex=235 nm, λem=350 nm). The excess fluorescence reagent (NOEPES) was readily removed after derivatization by a quick acid treatment. This minimizes the interference of excess NOEPES in fluorometric detection. The lower limit of quantitation 50 nM with a detection limit (S/N=3) of about 7.5 nM (or 0.15 pmol per 20 μL injection). Application of the method to analysis of free fatty acids in plasma proved feasible (using only 10 μL of plasma sample).

Key Words

Column liquid chromatography Fluorometric detection Long-chain free fatty acids Naphthoxyethyl derivatives 


  1. [1]
    Haan, G.J.; van der Heide, S.; Wolthers, B.G.J. Chromatogr. 1979,162, 261–271.Google Scholar
  2. [2]
    Lewis, B.; Hansen, J.D.L.; Wittman, W.; Krut, L.H.; Stewart, F.Am. J. Clin. Nutr. 1964,15, 161–168.Google Scholar
  3. [3]
    Rasmussen, L.B.; Kiens, B.; Pedersen, B.K.; Richter, E.A.Am. J. Clin. Nutr. 1994,59, 572–577.Google Scholar
  4. [4]
    Schaefer, E.J.; Lichtenstein, A.H.; Lamon-Fava, S.; McNamara, J.R.; Ordovas, J.M.Am. J. Clin. Nutr. 1995,61, 726S-740S.Google Scholar
  5. [5]
    Simopoulos, A.P.Am. J. Clin. Nutr. 1991,54, 438–463.Google Scholar
  6. [6]
    Crawford, T.O.; Sladky, J.T.; Hurko, O.; Besner-Johnston, A.; Kelley, R.I.Ann. Neurol. 1999,45, 337–343.CrossRefGoogle Scholar
  7. [7]
    Goto, J. InDetection-oriented Derivatization Techniques in Liquid Chromatography: Lingeman, H.; Underberg, W.J.M., Eds, Marcel Dekker, New York, 1990.Google Scholar
  8. [8]
    Yamaguchi, M.; Ishida, J. InModern Derivatization Methods for Separation Sciences: Toyo'oka T., ed, John Wiley, Chichester 1999.Google Scholar
  9. [9]
    Lu, C.Y.; Wu, H.L.; Chen, S.H.; Kou, H.S.Chromatographia 2000,51, 315–321.CrossRefGoogle Scholar
  10. [10]
    Wu, H.L.; Shyu, Y.Y.; Kou, H.S.; Chen, S.H.; Wu, S.M.; Wu, S.S.J. Chromatogr. A 1997,769, 201–207.CrossRefGoogle Scholar
  11. [11]
    Dole, V.P.; Meinert, H.J. Biol. Chem. 1960,235, 2595–2599.Google Scholar
  12. [12]
    Rodriguez-Palmero, M.; Lopez-Sabater, M.C.; Castellote-Bargallo, A.I.; De La Torre-Boronat, M.C.; Rivero-Urgell, M.J. Chromatogr. A 1997,778, 435–439.CrossRefGoogle Scholar
  13. [13]
    Saito, M.; Ushijima, T.; Sasamoto, K.; Ohkura, Y.; Ueno, K.J. Chromatogr. B 1995,674, 167–175.Google Scholar
  14. [14]
    Nakashima, K.; Taguchi, Y.; Kuroda, N.; Akiyama, S.; Duan, G.J. Chromatogr. 1993,619, 1–8.Google Scholar
  15. [15]
    Stein, J.; Milovic, V.; Zeuzem, S.; Caspary, W.F.J. Liquid Chromatogr. 1993,16 (13), 2915–2922.Google Scholar
  16. [16]
    van der Horst, F.A.L.; Post, M.H.; Holthuis, J.J.M.; Brinkman, U.A.Th.J. Chromatogr. 1990,500, 443–452.CrossRefGoogle Scholar
  17. [17]
    Jungling, E.; Kammermeier, H.Anal. Biochem. 1988,171, 150–157.CrossRefGoogle Scholar
  18. [18]
    Baty, J.D.; Pazouki, S.; Dolphin, J.J. Chromatogr. 1987,395, 403–411.CrossRefGoogle Scholar
  19. [19]
    Yamaguchi, M.; Matsunaga, R.; Hara, S.; Nakamura, M.; Ohkura, Y.J. Chromatogr. 1986,375, 27–35.Google Scholar
  20. [20]
    Tsuchiya, H.; Hayashi, T.; Sato, M.; Tatsumi, M.; Takagi, N.J. Chromatogr. 1984,309, 43–52.Google Scholar

Copyright information

© Friedr. Vieweg & Sohn Verlagsgesellschaft mbH 2001

Authors and Affiliations

  • C. Y. Lu
    • 1
  • H. L. Wu
    • 1
  • S. H. Chen
    • 1
  • H. S. Kou
    • 1
  1. 1.Graduate Institute of Pharmaceutical SciencesKaohsiung Medical UniversityKaohsiungTaiwan

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