Process Development and Validation of Reverse-Phase High-Performance Liquid Chromatography Method for Simultaneous Quantification of Quercetin, Thymoquinone, and Pterostilbene

The simultaneous HPLC method for quantifying Quercetin (Que), Thymoquinone (Thy), and Pterostilbene (Pte) aims at the precise measurement of these polyphenols alone or in complex mixtures, targeting their therapeutic potential in disorders such as diabetes and epilepsy. The method focuses on quantifying Que, Thy, and Pte, utilizing optimized reversed-phase HPLC conditions as per ICH Q2(R1) standards. Key validation aspects include linearity, specificity, precision, and accuracy, ensuring compliance for quality control in nanomedicine and nutraceuticals, and the method’s applications support pharmacokinetic studies and stability testing, contributing to personalized medicine and addressing pharmaco-resistance. The HPLC method development and validation were performed on a phenyl column using the mobile phase consisting of solvent A (0.1% orthophosphoric acid in HPLC water) and solvent B (acetonitrile) at a ratio of 55:45 in an isocratic elution mode at a flow rate of 1 mL/min and at a column temperature of 35 °C. Ultraviolet detection was measured at 254 nm. Moreover, the method was validated for accuracy, precision, linearity, specificity, and sensitivity. The retention time for tested Que, Thy, and Pte was observed at 4.15 min, 8.70 min, and 10.75 min, respectively. Limits of detection for Que, Thy, and Pte were 1.55 μg/mL, 2.40 μg/mL, and 70.79 µg/mL, whereas limits of quantification were 4.69 μg/mL, 7.28 μg/mL, and 214.52 µg/mL, respectively. Linearity and correlation coefficients for Que, Thy, and Pte were found in the range of 50–250 μg/mL (0.9999), 50–250 μg/mL (0.9999), and 620–3100 μg/mL (0.9996), respectively. A reasonable level of accuracy was indicated by the tested method suggesting extremely high recovery levels (98–102%). The separation of tested compounds was achieved within 11 min. The developed and validated RP-HPLC–UV method was successfully applied for the identification and quantification of Que, Thy, and Pte for their combined estimation in complex formulations. From the validation study, it was found that the tested method is specific, accurate, precise, reliable, and reproducible.