Determination of acid dissociation constants of warfarin and hydroxywarfarins by capillary electrophoresis

• Two acid dissociation equilibria have been described.
• Thermodynamic pKa values have been determined.
• CE, CE-DAD and IS-CE methods have been employed.
• Pharmacological relevance has been presented and discussed.
• Theoretical explanations of pKa values have been attempted.

In this work the acid dissociation constants – pKa of warfarin and its all important oxidative metabolites have been determined by capillary electrophoresis-based methods. It has resulted in a complete description of two acid–base dissociation equilibria, yet not investigated experimentally for phase I metabolites of warfarin. The capillary electrophoresis (CE) method based on the relation between effective electrophoretic mobilities and pH has proven to be a suitable tool for pKa determination, while the spectrophotometric (CE-DAD) and the internal standard methods (IS-CE), have appeared to be promising alternative approaches. The CE-DAD approach based on the change in absorbance spectra between the acidic and basic forms is a combination between capillary electrophoresis and spectrophotometric titration, and yields very consistent values of pKa1 with CE. The IS-CE, in turn, enables an estimation of pKa1 and pKa2 from only two analytical runs, however, less accurate than CE and CE-DAD. The Debye–Hückel model has been confirmed experimentally as a good predictor of pKa values at various ionic strengths. Therefore, it has been used in determination of thermodynamic pKa1 and pKa2, referring to the zero ionic strength. The results are important from the analytical, pharmacological, and theoretical points of view.

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