Stability of warfarin solutions for drug–protein binding measurements: Spectroscopic and chromatographic studies

Authors

Annette C. Moser, University of Nebraska–LincolnFollow
Charles A. Kingsbury, University of Nebraska-LincolnFollow
David S. Hage, University of Nebraska - LincolnFollow

Document Type

Article

Date of this Version

2006

Citation

Published in Journal of Pharmaceutical and Biomedical Analysis 41:4 (June 16, 2006), pp. 1101-1109; doi:10.1016/j.jpba.2006.02.012

Comments

Copyright © 2006 Elsevier B.V. Used by permission.

Abstract

Warfarin is commonly used in drug–protein binding studies as a displacement marker for Sudlow site I on the protein human serum albumin (HSA). This study examined the stability of aqueous warfarin solutions prepared for such experiments. This was investigated using NMR spectroscopy and affinity chromatography. It was found by 1H NMR that warfarin underwent a slow first-order conversion in aqueous solution. The rate of this reaction increased with temperature, giving rate constants at pH 7.4 of 0.0086 h⁻¹ at 25 °C and 0.041 h⁻¹ at 37 °C. It was concluded from further 1H and 13C NMR studies, along with molecular modeling, that this process involved the conversion of the minor cyclic hemiketal form of warfarin to its major cyclic hemiketal. This reaction had a small but measurable effect on the binding of R- and S-warfarin to HSA, as demonstrated by HPLC using an immobilized HSA affinity column. From this work, general guidelines were developed concerning the usable lifetimes for warfarin that is prepared in aqueous solutions for studies of drug–protein binding.