<abstract xmlns="http://www.w3.org/1999/xhtml">In this study, high-performance liquid chromatography with fluorescence detection (HPLC-FLD) has been used for the first time, for direct determination of warfarin and its major metabolite, 7-hydroxywarfarin, in rat plasma. The simple and sensitive method was developed using Fortis® reversed-phase diphenyl column (150 × 4.6 mm, 3 μm) and a mobile phase composed of phosphate buffer (25 mmol L−1)/methanol/acetonitrile (70:20:10, <italic>V</italic>/<italic>V</italic>/<italic>V</italic>), adjusted to pH 7.4, at a flow rate of 0.8 mL min−1. The diphenyl chemistry of the stationary phase provided a unique selectivity for separating the structurally related aromatic analytes, warfarin and 7-hydroxywarfarin, allowing their successful quantification in the complex plasma matrix. The method was linear over the range 0.01–25 μg mL−1, for warfarin and 7-hydroxywarfarin, and was found to be accurate, precise and selective in accordance with US FDA guidance for bioanalytical method validation. The method was sensitive enough to quantify 0.01 μg mL−1 of warfarin and 7-hydroxywarfarin (<italic>LLOQ</italic>) using only 100 μL of plasma. The applicability of this method was demonstrated by analyzing samples obtained from rats after oral administration of a single warfarin dose, and studying warfarin and 7-hydroxywarfarin pharmacokinetics.</abstract>

In this study, high-performance liquid chromatography with fluorescence detection (HPLC-FLD) has been used for the first time, for direct determination of warfarin and its major metabolite, 7-hydroxywarfarin, in rat plasma. The simple and sensitive method was developed using Fortis® reversed-phase diphenyl column (150 × 4.6 mm, 3 μm) and a mobile phase composed of phosphate buffer (25 mmol L−1)/methanol/acetonitrile (70:20:10, V/V/V), adjusted to pH 7.4, at a flow rate of 0.8 mL min−1. The diphenyl chemistry of the stationary phase provided a unique selectivity for separating the structurally related aromatic analytes, warfarin and 7-hydroxywarfarin, allowing their successful quantification in the complex plasma matrix. The method was linear over the range 0.01–25 μg mL−1, for warfarin and 7-hydroxywarfarin, and was found to be accurate, precise and selective in accordance with US FDA guidance for bioanalytical method validation. The method was sensitive enough to quantify 0.01 μg mL−1 of warfarin and 7-hydroxywarfarin (LLOQ) using only 100 μL of plasma. The applicability of this method was demonstrated by analyzing samples obtained from rats after oral administration of a single warfarin dose, and studying warfarin and 7-hydroxywarfarin pharmacokinetics.

Simultaneous determination of warfarin and 7-hydroxywarfarin in rat plasma by HPLC-FLD

Department of Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy

Jordan University of Science and Technology

Department of Pharmaceutical Technology, Faculty of Pharmacy

Acta Pharmaceutica

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

In this study, high-performance liquid chromatography with fluorescence detection (HPLC-FLD) has been used for the first time, for direct determination of...

Simultaneous determination of warfarin and 7-hydroxywarfarin in rat plasma by HPLC-FLD

Published Online: Feb 17, 2020

Page range: 343 - 357

Accepted: Sep 14, 2019

DOI: https://doi.org/10.2478/acph-2020-0025

Keywords
warfarin, 7-hydroxywarfarin, high-performance liquid chromatography-fluorescence detection, rat plasma, pharmacokinetics

© 2020 Aref Zayed et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Simultaneous determination of warfarin and 7-hydroxywarfarin in rat plasma by HPLC-FLD

Published Online: Feb 17, 2020

Page range: 343 - 357

Accepted: Sep 14, 2019

DOI: https://doi.org/10.2478/acph-2020-0025

Keywordswarfarin, 7-hydroxywarfarin, high-performance liquid chromatography-fluorescence detection, rat plasma, pharmacokinetics

© 2020 Aref Zayed et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Keywords
warfarin, 7-hydroxywarfarin, high-performance liquid chromatography-fluorescence detection, rat plasma, pharmacokinetics