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

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. Alpha, beta and gamma-cyclodextrin separated the diastereoisomers of 5-methyl-tetrahydrofolate, while only gamma-cyclodextrin was found to be effective for the chiral separation of leucovorin. The effect of gamma-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 gamma-cyclodextrin and the tetrahydrofolates is weak, the high efficiency of capillary electrophoresis and the use of high concentrations of gamma-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 gamma-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.