article · Biomedical Chromatography
A high-performance liquid chromatography method has been developed and validated to measure quercetin in rat plasma. The protocol relies on protein precipitation to extract quercetin alongside thymoquinone, which serves as an internal standard. Chromatographic separation is carried out using an isocratic mobile phase of aqueous trichloroacetic acid and acetonitrile across a 13-minute run at a flow rate of 0.9 millilitres per minute, with ultraviolet detection at 254 nanometres. The assay demonstrated linearity across a concentration range of 0.10 to 25 micrograms per millilitre, alongside acceptable intra-day and inter-day precision and accuracy with deviations below 20 percent. The technique was subsequently implemented in an in vivo setting to evaluate the pharmacokinetic profile of quercetin following a single oral dose administered to laboratory rats.
Accurately measuring how compounds move through the body is essential for assessing biological activity and dosage. By providing a straightforward, reliable method using protein precipitation and standard liquid chromatography, this approach simplifies the measurement of quercetin in biological samples. This supports clearer laboratory evaluations of the compound during early preclinical investigations.
This validated analytical protocol could be adopted by pharmaceutical, preclinical testing, or contract research laboratories conducting pharmacokinetic evaluations of quercetin formulations. The method is an applied and tested laboratory tool for rat plasma, placing it at an early preclinical research stage. Further validation in other biological matrices or human plasma would be necessary before translation into clinical diagnostic or pharmaceutical quality control applications.
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A simple, accurate, and reproducible high-performance liquid chromatography (HPLC) method has been developed and validated for the quantification of quercetin (QR) in rat plasma. The method involves a simple protein precipitation procedure to extract both QR and thymoquinone (TQ), the internal standard. The chromatographic analysis was achieved on a Shimadzu LC 20 A HPLC system equipped with a Supelcosil LC-18 T C<sub>18</sub> column and an isocratic mobile phase consisting of 0.3% trichloroacetic acid in water and acetonitrile HPLC-grade (50:50, v/v) run at a flow rate of 0.9 mL/min for 13 min. The UV detection wavelength was set at 254 nm. The method exhibited good linearity (R<sup>2</sup> > 0.994) over the assayed concentration range (0.10-25 μg/mL) and demonstrated good intra-day and inter-day precision and accuracy (relative standard deviations and the deviation from predicted values were <20%). This method was also successfully applied for studying the pharmacokinetics of QR in rats following a single oral dose of QR to evaluate its pharmacokinetic parameters in rats.
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DOI: 10.1002/bmc.3819
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