Determination of low levels of the stereoisomers of leucovorin and 5-methyltetrahydrofolate in plasma using a coupled chiral-achiral high-performance liquid chromatographic system with post-chiral column peak compression.

A method for the determination of low levels of the stereoisomers of leucovorin and 5-methyltetrahydrofolate has been developed and validated. The assay involved initial chromatography on a bovine serum albumin (BSA)-based high-performance liquid chromatography chiral stationary phase (CSP) followed by post-column peak compression and elution on two C18 columns. In this manner, the poor efficiency of the BSA-CSP was overcome and sub-microgram quantities of the target solutes could be detected. The BSA-CSP separated the leucovorin and 5-methyletrahydrofolate from interfering plasma components and from each other and achieved the stereochemical resolution of the diastereomeric (6S)-and (6R)-leucovorin. The eluent containing (6S)-leucovorin was directed onto one C18 column and the eluent containing (6R)-leucovorin and 5-methyltetrahydrofolate was directed onto the other. This was followed by sequential rapid gradient elution of the target compounds from the respective C18 columns. The method was validated for plasma levels ranging from 15 to 500 ng/ml and was able to detect leucovorin concentrations of as low as 5 ng/ml.