Analytical aspects of achiral and cyclodextrin-mediated capillary electrophoresis of warfarin and its two main derivatives assisted by theoretical modeling

• The pKa values may change enantioselectively upon association with neutral cyclodextrins.
• Chiral separation mechanism involves two independent equilibria.
• Theoretical model highlights the role of the intramolecular hydrogen bonds.
• Association affinity model for warfarin and its two derivatives has been proposed.
• Selectivity and enantioselectivity have been investigated across broad pH range.

Several distinct analytical issues have been addressed by performing capillary electrophoresis-based separations of the warfarin, 7-hydroxywarfarin and 10-hydroxywarfarin in an achiral and cyclodextrin-containing media. The measurements were conducted across a range of pH in order to find optimum conditions for achiral and chiral separations. The values of acid dissociation constant (pKa) have been determined and compared. Subsequently, after performing a series of mobility shift assays at different pH and cyclodextrin concentration, the pKa values ascribed to diastereomeric complexes with methyl-β-cyclodextrin have been estimated. The significant pKa shifts upon complexation have been noticed for warfarin – up to 1.5 pH units, and only subtle for 10-hydroxywarfarin. A new approach that allows the estimation of association percentage based on the electrophoretic mobility curves has been also demonstrated. The complex mechanism of chiral separation has been found to be responsible for the observed migration profile, relying on a combined equilibrium between complexation/partition and protonation/deprotonation phenomena. The occurrence of the pKa-related migration order reversal has been demonstrated in achiral medium between warfarin and 7-hydroxywarfarin, and in chiral medium between enantiomers, causing a drop in enantioselectivity at specific pH. In parallel, the density functional theory-based calculations have been performed in order to obtain the structures of warfarin and its derivatives as well as to rationalize the shifts in pKa values.