Summary
Capillary electrophoresis with an electrolyte containing cyclodextrin was investigated for the simultaneous separation of the diastereoisomers of 6R,S-leucovorin and its active metabolite 6R,S-5-methyl-tetrahydrofolate. α, β and γ-cyclodextrin separated the diastereoisomers of 5-methyl-tetrahydrofolate, while only γ-cyclodextrin was found to be effective for the chiral separation of leucovorin. The effect of γ-cyclodextrin concentration was investigated, and subsequently a curve-fitting analysis for the quantitative estimation of the binding constants was attempted. The binding constants were found to be very small, in the range 2–4 M−1. Although the interaction between γ-cyclodextrin and the tetrahydrofolates is weak, the high efficiency of capillary electrophoresis and the use of high concentrations of γ-cyclodextrin allow baseline chiral separation of the diastereoisomers of leucovorin and 5-methyl-tetrahydrofolate. Changes in temperature exert differing effects on the separations of leucovorin and 5-methyl-tetrahydrofolate; higher temperatures improved the separation of leucovorin diastereoisomers but reduced the resolution of 5-methyl-tetrahydrofolate diastereoisomers. The effects of urea and buffer salt concentrations and of buffer pH were also investigated. Capillary electrophoresis with γ-cyclodextrin was used to analyse plasma samples spiked with clinically-relevant levels of leucovorin and 5-methyl-tetrahydrofolate. Resolution of these compounds in ultrafiltered plasma was demonstrated, but detection sensitivity was not adequate for the routine use of this method for the determination of leucovorin and 5-methyl-tetrahydrofolate in plasma. In addition, a simple technique to reverse the elution order of ionic stereoisomers was demonstrated. By adding a cationic surfactant into the buffer and reversing the separation potential, the elution order of the diastereoisomers of leucovorin and 5-methyl-tetrahydrofolate was reversed.
Article PDF
Similar content being viewed by others
Explore related subjects
Discover the latest articles, news and stories from top researchers in related subjects.Avoid common mistakes on your manuscript.
References
W. G. Kuhr, Anal. Chem.62, 403R (1990).
W. G. Kuhr, C. A. Monnig, Anal. Chem.64, 389R (1992).
H. Nishi, S. Terabe, Electrophoresis11, 691 (1990).
E. Gassmann, J. C. Kuo, R. N. Zare, Science230, 813 (1985).
S. M. Han, D. W. Armstrong, in “Chiral Separations by HPLC”,A. M. Krstulovic, Ed., Elis Horwood, Chichester, 1989, chapter 10.
D. Sybilska, J. Zukowski, in “Chiral Separations by HPLC”,A. M. Krstulovic, Ed., Ellis Horwood, Chichester, 1989, chapter 7.
A. Guttman, A. Paulus, A. S. Cohen, N. Grinberg, B. L. Karger, J. Chromatogr.448, 41 (1988).
S. Fanali, J. Chromatogr.474, 441 (1989).
S. Fanali, P. Bocek, Electrophoresis11, 757 (1990).
S. Fanali, J. Chromatogr.545, 437 (1991).
J. Snopek, H. Soini, M. Novotny, E. Smolkova-Keulemansova, I. Jelinek, J. Chromatogr.559, 215 (1991)
H. Nishi, T. Fukuyama, S. Terabe, J. Chromatogr.553, 503 (1991).
H. Nishi, M. Matsuo, J. Liquid Chromatogr.114, 973 (1991).
S. A. C. Wren, R. C. Rowe, J. Chromatogr.603, 235 (1992).
J. L. Carpenter, P. Camilleri, D. Dhanak, D. M. Goodall, J. Chem. Soc. Chem. Commun. 804 (1992).
G. Tisman, V. Flener, M. E. Jones, L. Buck, Proc. Ann. Meet. Am. Assoc. Cancer Res.26, 159 (1985).
I. W. Wainer, R. M. Stiffin, J. Chromatogr.424, 158 (1988).
L. Silan, P. Jadaud, L. R. Whitfield, I. W. Wainer, J. Chromatogr.532, 227 (1990).
J. Haginaka, J. Wakai, Analyst110, 1185 (1985).
J. Haginaka, J. Wakai, Analyst110, 1277 (1985).
D. K. Lloyd, Anal. Proc.29, 169 (1992).
K. Altria, C. F. Simpson, Anal. Proc.23, 453 (1986).
K. Otsuka, S. Terabe, T. Ando, J. Chromatogr.348, 39 (1985).
M. G. Khaledi, S. C. Smith, J. C. Strasters, Anal. Chem.63, 1820 (1991).
J. C. Strasters, M. G. Khaledi, Anal. Chem.63, 2503 (1991).
D. Y. Pharr, Z. S. Fu, T. K. Smith, W. L. Hinze, Anal. Chem.61, 275 (1989).
R. H. Stokes, R. Mills, “Viscosity of Electrolytes and Related Properties”, Pergamon, London, 1965.
“CRC Handbook of Biochemistry, and Molecular Biology, Physical and Chemical Data, Vol. I”, 3rd Edition, Ed.G. D. Fasman, CRC Press Inc., Cleveland, 1976, p. 415.
D. S. Burgi, R. L. Chien, Anal. Chem.63, 2042 (1991).
P. Macaudiere, M. Lienne, A. Tambute, M. Caude, in “Chiral Separation by HPLC”,A. M. Krstulovic, Editor, Ellis Horwood, Chichester, 1989, chapter 14.
E. M. Newman, J. A. Straw, J. H. Doroshow, Cancer Res.49, 5755 (1989).
J. A. Straw, D. Szapary, W. T. Wyn, Cancer Res.44, 3114 (1984).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Shibukawa, A., Lloyd, D.K. & Wainer, I.W. Simultaneous chiral separation of leucovorin and its major metabolite 5-methyl-tetrahydrofolate by capillary electrophoresis using cyclodextrins as chiral selectors: Estimation of the formation constant and mobility of the solute-cyclodextrin complexes. Chromatographia 35, 419–429 (1993). https://doi.org/10.1007/BF02278596
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02278596