Chiral separation of leucovorin with bovine serum albumin using affinity capillary electrophoresis

A method for the determination of the (6R)- and (6S)-stereoisomers of leucovorin using electrokinetic chromatography (EKC) in the affinity mode has been developed. Bovine serum albumin (BSA) is used as a run buffer additive to incorporate enantiomeric selectivity into the system. Protein-wall interactions are minimized by using a poly(ethylene glycol) (PEG) coated capillary. Chiral resolution is obtained in 12.5 min with efficiencies greater than 200,000 theoretical plates using BSA as an additive, while no resolution is obtained in the absence of BSA. A general equation is derived to calculate the free energy of interaction between the leucovorin isomers and the BSA molecule. This method represents a new means of obtaining thermodynamic data for substrate binding interactions and for the general study of drug cross-reactions and interactions of drugs with serum and other proteins.