Development and Validation of RP-HPLC Method for Simultaneous Estimation of Fisetin and Xanthohumol in Nanosuspension Drug Delivery System and Immediate Release Tablet Formulation

Background: The study aimed to develop and validate a simple, rapid, and robust reverse-phase (HPLC/UFLC) method for the simultaneous estimation of Fisetin (FIS) and xanthohumol (XAN). Further the results of specificity studies revealed absence of any interference of FIS and XAN peak with the excipients used in the nanosuspension and immediate release tablet. Materials and Methods: Chromatographic separation was achieved on a C18 column using a gradient mobile phase of 2% orthophosphoric acid (OPA) and acetonitrile at a flow rate of 1.0 mL/min, with identification at 365–367 nm. The developed method was validated in accordance with International Conference on Harmonisation (ICH Q2(R1)) guidelines for linearity, accuracy, precision, sensitivity, system suitability and specificity. Results: FIS and XAN showed retention times of 4.1 and 13.5 min, respectively, with good resolution. The method was linear over the concentration range of 2–10 µg/mL for both analytes (R² > 0.999). The limits of detection and quantification were 0.56 and 1.61 µg/mL for FIS, and 0.68 and 1.81 µg/mL for XAN. Precision studies demonstrated %RSD values below 2%, while accuracy studies showed recoveries within 95–105%. System suitability parameters were within acceptable limits. Conclusion: The validated RP-HPLC/UFLC method for the simultaneous estimation of FIS and XAN was successfully developed for the first time. The method demonstrated excellent suitability for routine quality control and was effectively applied for the quantitative analysis of FIS and XAN in nanosuspension and immediate release tablet formulations containing FIS and XAN. Further, this confirms its applicability to lipid-based delivery systems.