Stability of warfarin solutions for drug-protein binding measurements

Spectroscopic and chromatographic studies

Annette C Moser, Charles Kingsbury, David S Hage

Research output: Contribution to journalArticle

21 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)1101-1109
Number of pages9
JournalJournal of Pharmaceutical and Biomedical Analysis
Volume41
Issue number4
DOIs
StatePublished - Jun 16 2006

Fingerprint

Warfarin
Protein Binding
Serum Albumin
Pharmaceutical Preparations
Nuclear magnetic resonance
Affinity chromatography
Molecular modeling
Affinity Chromatography
Nuclear magnetic resonance spectroscopy
Rate constants
Magnetic Resonance Spectroscopy
High Pressure Liquid Chromatography
Guidelines
Temperature
Proteins
Experiments

Keywords

  • Affinity chromatography
  • Conversion kinetics
  • NMR spectroscopy
  • Stability studies
  • Warfarin

ASJC Scopus subject areas

  • Analytical Chemistry
  • Pharmaceutical Science
  • Drug Discovery
  • Spectroscopy
  • Clinical Biochemistry

Cite this

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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.",
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AU - Hage, David S

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N2 - 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.

AB - 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.

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