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

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.

HPLC with Fluorescence and Photodiode Array Detection for Quantifying Capmatinib in Biological Samples: Application to In Vivo and In Vitro Studies

“…The selected emission wavelength at 495 nm is close to that reported for quinoline at 500 nm. For the internal standard warfarin, we used the reported excitation and emission wavelengths at 310 and 390 nm, respectively [ 15 ]. The representative chromatograms obtained using the final method are shown in Figure 3 .…”

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“…The overall objective of the research was to establish the warfarin validated method in the PPB assay 11,12,13,14 conditions; warfarin is exhibited highly binding (> 98%) 6 to rat plasma proteins 6 , having the species-specific binding nature 2 in addition to these it has a high degree of reproducibility made it to opt it as idle control compound for PPB assay control. Internal Standard (IS) Preparation: 1.0 mg/mL of master stock solutions were prepared in the 2 mL vials by weighing 1.2 mg of the propranolol in DMSO solvent from master stock to 50 ng/mL in the acetonitrile.…”

<p>Piperine Alters the Pharmacokinetics and Anticoagulation of Warfarin in Rats</p>

“…We carried out quantification of warfarin and 7-hydroxywarfarin by a high-performance liquid chromatography with fluorescence detection (HPLC-FLD) method that was previously validated. 43 In brief, plasma samples were spiked with internal standard solution and then acidified by acetic acid before extraction with ethylacetate. The upper organic phase was isolated and evaporated and the residue was then reconstituted with mobile phase and injected into the HPLC system.…”