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

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