, Volume 23, Issue 10, pp 717–721 | Cite as

Effects of reducing agents on the determination of thiolic compounds in the presence of their disulfides using bimane pre-column derivatization

  • W. Baeyens
  • G. Van der Weken
  • P. De Moerloose


The influence of reducing agents (sodium borohydride, tributylphosphine, dithioerythritol and dithiothreitol) on the conversion of disulfides into their parent thiols, with specific application to cysteine was investigated. Dithioerythritol and dithiothreitol were found to be most suitable for this reaction. A contact time of one hour at room temperature provided quantitative reduction as tested using cystine as a pure disulfide standard. A modified fluorescence labelling procedure with monobromobimane followed by reversed-phase HPLC allows quantitation of the parent thiol and the disulfide content when completing the labelling reaction with and without preliminary treatment with reducing agent. The effects of various bimane reducing agent ratios on the yield of the reaction are discussed. Precautions should be taken when dealing with complex matrices with respect to reagent concentrations and ratios.

Key Words

Column liquid chromatography Thiols and disulfides Fluorescence derivatization Monobromobimane 


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  1. [1]
    N. S. Kostower, G. L. Newton, E. M. Kosower, H. M. Ranney Abstracts of the Kyoto Biophysics Congress, Kyoto, Japan, Sept. 3–9 (1978).Google Scholar
  2. [2]
    E. M. Kosower, B. Pazhenchevsky, E. Hershkowitz, J. Amer. Chem. Soc.,100, 6516 (1978).Google Scholar
  3. [3]
    N. S. Kosower, E. M. Kosower, G. L. Newton, H. M. Ranney, Proc. Natl. Acad. Sci. USA,76, 3382 (1979).Google Scholar
  4. [4]
    N. S. Kosower, G. L. Newton, E. M. Kosower, H. M. Ranney, Biochim. Biophys. Acta,622, 201 (1980).Google Scholar
  5. [5]
    R. C. Fahey, G. L. Newton, R. Dorian, E. M. Kosower, Anal. Biochem.,111, 357 (1981).CrossRefGoogle Scholar
  6. [6]
    G. L. Newton, R. Dorian, R. C. Fahey, Anal. Biochem.,114, 383 (1981).CrossRefGoogle Scholar
  7. [7]
    N. S. Kosower, E. M. Kosower, Y. Zipser, Z. Faltin, R. Shomrat, Biochim. Biophys. Acta.640, 748 (1981).Google Scholar
  8. [8]
    R. C. Fahey, R. Dorian, G. L. Newton, J. Utley, in: O. F. Nygaard, M. G. Simic J. N. Hauber, eds., Radioprotectors and Anticarcinogens, Academic Press, New York, 1983, pp. 103–120.Google Scholar
  9. [9]
    E. M. Kosower, N. S. Kosower, A. Radkowsky, in: A. Larsson et al., eds., Functions of Glutathione — Biochemical, Physiological, Toxicological and Clinical Aspects, Raven Press, New York 1983, pp. 243–250.Google Scholar
  10. [10]
    N. S. Kosower, E. M. Kosower, ibidem pp. 307–315.Google Scholar
  11. [11]
    S. S. Fenton, R. C. Fahey, Anal. Biochem.,154, 34 (1986).CrossRefGoogle Scholar
  12. [12]
    R. M. Maiorino, G. L. Weber, H. V. Aposhian, J. Chromatogr.,374, 297 (1986).Google Scholar
  13. [13]
    W. Baeyens, G. Van der Weken, P. De Moerloose, Chromatographia,23, 289 (1987).Google Scholar
  14. [14]
    D. Perrett, S. R. Rudge, J. Pharm. Biomed. Anal.,3, 3 (1985).CrossRefGoogle Scholar
  15. [15]
    K. Imai, T. Toyo'oka, H. Miyano, Analyst,109, 1365 (1984).CrossRefGoogle Scholar
  16. [16]
    S. Duprè, M. Aureli, Anal. Biochem.,105, 97 (1980).Google Scholar
  17. [17]
    W. R. G. Baeyens, K. Imai, in: S.D. Klein, ed., Progress in Analytical Luminescence, ASTM publication for the Silver Jubilee of the Eastern Analytical Symposium, New York City, NY, USA, Ocotber 1986 (in press).Google Scholar
  18. [18]
    F. Vogel, L. Lumper, Biochem. J.,215, 159 (1983).Google Scholar
  19. [19]
    N. K. Burton, G. W. Aherne, V. Marks, J. Chromatogr.,309, 409 (1984).Google Scholar
  20. [20]
    Y. Kawahara, M. Hisaoka, Y. Yamazaki, A. Inage, T. Morioka, Chem. Pharm. Bull.,29, 150 (1981).Google Scholar
  21. [21]
    T. Toyo'oka, K. Imai, J. Chromatogr.,282, 495 (1983).CrossRefGoogle Scholar
  22. [22]
    T. Toyo'oka, K. Imai, Y. Kawahara, J. Pharm. Biomed. Anal.,2, 473 (1984).CrossRefGoogle Scholar
  23. [23]
    The Merck Index, Merck & Co., Inc. Rahway, NJ, USA, 1983, 3394.Google Scholar
  24. [24]
    W. W. Cleland, Biochemistry,3, 480 (1964).CrossRefGoogle Scholar
  25. [25]
    W. L. Zahler, W. W. Cleland, J. Biol. Chem.,243, 716 (1968).Google Scholar
  26. [26]
    Y. Burstein, A. Patchornik, Biochemistry,11, 2939 (1972).Google Scholar
  27. [27]
    Aldrichimica Acta,4 (3), 33 (1971). (Aldrich Fine Chemicals). 0765 0810 V 2Google Scholar
  28. [28]
    D. G. Bailey, T. W. Wilson, G. E. Johnson, J. Chromatogr.,111, 305 (1975).CrossRefGoogle Scholar
  29. [29]
    W. R. G. Baeyens, G. Van der Weken, P. De Moerloose, Anal. Chim. Acta (1987) (in press).Google Scholar

Copyright information

© Friedr. Vieweg & Sohn Verlagsgesellschaft mbH 1987

Authors and Affiliations

  • W. Baeyens
    • 1
  • G. Van der Weken
    • 1
  • P. De Moerloose
    • 1
  1. 1.Faculty of Pharmaceutical Sciences, Laboratory of Pharmaceutical Chemistry and Drug Quality ControlState University of GhentGhentBelgium

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