Simultaneous determination of warfarin and 7-hydroxywarfarin in rat plasma by HPLC-FLD
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 d...
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| Language: | English |
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2020-09-01
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| Series: | Acta Pharmaceutica |
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| Online Access: | https://doi.org/10.2478/acph-2020-0025 |
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| author | Zayed Aref Babaresh Wahby M. Darweesh Ruba S. El-Elimat Tamam |
| author_facet | Zayed Aref Babaresh Wahby M. Darweesh Ruba S. El-Elimat Tamam |
| author_sort | Zayed Aref |
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| description | 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. |
| format | Article |
| id | doaj-art-fb3997da129b439bbcdc24151e7839df |
| institution | Kabale University |
| issn | 1846-9558 |
| language | English |
| publishDate | 2020-09-01 |
| publisher | Sciendo |
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| series | Acta Pharmaceutica |
| spelling | doaj-art-fb3997da129b439bbcdc24151e7839df2025-02-02T02:50:42ZengSciendoActa Pharmaceutica1846-95582020-09-0170334335710.2478/acph-2020-0025acph-2020-0025Simultaneous determination of warfarin and 7-hydroxywarfarin in rat plasma by HPLC-FLDZayed Aref0Babaresh Wahby M.1Darweesh Ruba S.2El-Elimat Tamam3Department of Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy, Jordan University of Science and Technology, Irbid22110, JordanDepartment of Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy, Jordan University of Science and Technology, Irbid22110, JordanDepartment of Pharmaceutical Technology, Faculty of Pharmacy, Jordan University of Science and Technology, Irbid22110, JordanDepartment of Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy, Jordan University of Science and Technology, Irbid22110, JordanIn 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.https://doi.org/10.2478/acph-2020-0025warfarin7-hydroxywarfarinhigh-performance liquid chromatography-fluorescence detectionrat plasmapharmacokinetics |
| spellingShingle | Zayed Aref Babaresh Wahby M. Darweesh Ruba S. El-Elimat Tamam Simultaneous determination of warfarin and 7-hydroxywarfarin in rat plasma by HPLC-FLD Acta Pharmaceutica warfarin 7-hydroxywarfarin high-performance liquid chromatography-fluorescence detection rat plasma pharmacokinetics |
| title | Simultaneous determination of warfarin and 7-hydroxywarfarin in rat plasma by HPLC-FLD |
| title_full | Simultaneous determination of warfarin and 7-hydroxywarfarin in rat plasma by HPLC-FLD |
| title_fullStr | Simultaneous determination of warfarin and 7-hydroxywarfarin in rat plasma by HPLC-FLD |
| title_full_unstemmed | Simultaneous determination of warfarin and 7-hydroxywarfarin in rat plasma by HPLC-FLD |
| title_short | Simultaneous determination of warfarin and 7-hydroxywarfarin in rat plasma by HPLC-FLD |
| title_sort | simultaneous determination of warfarin and 7 hydroxywarfarin in rat plasma by hplc fld |
| topic | warfarin 7-hydroxywarfarin high-performance liquid chromatography-fluorescence detection rat plasma pharmacokinetics |
| url | https://doi.org/10.2478/acph-2020-0025 |
| work_keys_str_mv | AT zayedaref simultaneousdeterminationofwarfarinand7hydroxywarfarininratplasmabyhplcfld AT babareshwahbym simultaneousdeterminationofwarfarinand7hydroxywarfarininratplasmabyhplcfld AT darweeshrubas simultaneousdeterminationofwarfarinand7hydroxywarfarininratplasmabyhplcfld AT elelimattamam simultaneousdeterminationofwarfarinand7hydroxywarfarininratplasmabyhplcfld |