Resolution of the stereoisomers of leucovorin and 5-methyltetrahydrofolate by chiral high-performance liquid chromatography.

Leucovorin (5-formyltetrahydrofolate, LV) is a reduced folate that has been in clinical use for many years as a rescue agent following methotrexate (MTX) therapy. Commercially available LV is a 1:1 mixture of [6R]-and [6S]-isomers. Due to the lack of a specific method for directly separating and quantitating the stereoisomers of LV, it has been difficult to precisely define the pharmacokinetic and biological characteristics of each stereoisomer. We have now developed a novel HPLC method to completely separate [6S]-LV and [6S]-5-methyltetrahydrofolate (MeTHF) from their respective [6R]-isomers using bovine serum albumin (BSA)-bonded silica as the chiral stationary phase. Baseline separation was achieved using 5 and 25 mM sodium phosphate buffers (pH 7.4) as the mobile phase with resolution factors of 1.65 for LV and 2.31 for MeTHF, respectively. The purity of each isomer prepared by this HPLC method is greater than 99%. The stereoisomers were identified by examining their ability to protect CEM cells from MTX (0.04 microM)-induced inhibition of growth. In the LV chromatogram, the first eluted peak provided complete protection from MTX growth inhibition when LV concentrations of 0.1 microM and above were used, whereas the last eluted peak failed to reverse MTX toxicity at concentrations up to 1.0 microM. Chemically pure synthetic [6R]-and [6S]-LV standards confirmed that the first eluted, biologically active peak is the [6S]-isomer. For MeTHF, only the last eluted peak effectively protects cells from MTX growth inhibition and is therefore believed to be the [6S]-isomer. This new HPLC method will serve as a useful tool to elucidate the clinical and cellular pharmacology of the stereoisomers of LV and MeTHF.