SIMULTANEOUS CHIRAL SEPARATION OF LEUCOVORIN AND ITS MAJOR METABOLITE5-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 diastereoisome rs of 6R,S-leucovorin and its active metabolite 6R,S-5-methyl-tetrahyd rofolate. Alpha, beta and gamma-cyclodextrin separated the diastereois omers of 5-methyl-tetrahydrofolate, while only gamma-cyclodextrin was found to be effective for the chiral separation of leucovorin. The eff ect of gamma-cyclodextrin concentration was investigated, and subseque ntly a curve-fitting analysis for the quantitative estimation of the b inding constants was attempted. The binding constants were found to be very small, in the range 2-4 M-1. Although the interaction between ga mma-cyclodextrin and the tetrahydrofolates is weak, the high efficienc y of capillary electrophoresis and the use of high concentrations of g amma-cyclodextrin allow baseline chiral separation of the diastereoiso mers of leucovorin and 5-methyl-tetrahydrofolate. Changes in temperatu re exert differing effects on the separations of leucovorin and 5-meth yl-tetrahydrofolate; higher temperatures improved the separation of le ucovorin diastereoisomers but reduced the resolution of 5-methyl-tetra hydrofolate diastereoisomers. The effects of urea and buffer salt conc entrations and of buffer pH were also investigated. Capillary electrop horesis with gamma-cyclodextrin was used to analyse plasma samples spi ked with clinically-relevant levels of leucovorin and 5-methyl-tetrahy drofolate. Resolution of these compounds in ultrafiltered plasma was d emonstrated, but detection sensitivity was not adequate for the routin e 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 pot ential, the elution order of the diastereoisomers of leucovorin and 5- methyl-tetrahydrofolate was reversed.