Validated RP-HPLC Method for the Quantitative Analysis of Fisetin in Pure Form and Mucoadhesive Microemulsion Formulations

A robust and validated reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed for the quantitative analysis of fisetin in both its pure form and when encapsulated in mucoadhesive microemulsion (MME) formulations. Chromatographic separation was achieved using a Phenomenex C18 column with a mobile phase comprising methanol and 0.1% orthophosphoric acid in a 55:45 (v/v) ratio. The isocratic method operated at a flow rate of 1.0 mL/min with detection at 362 nm. The retention time of standard fisetin was 7.48 minutes, and encapsulated fisetin eluted at 7.35 minutes, confirming its stability post-formulation. The method was validated according to ICH Q2(R1) guidelines and demonstrated excellent linearity (R² = 0.993) over the concentration range of 2–12 µg/mL. Precision studies yielded %RSD values below 2%, confirming reproducibility. The method also exhibited good accuracy (96–98% recovery), robustness, and system suitability, with low %RSD values for all tested parameters. The limit of detection and quantification were 0.425 µg/mL and 1.287 µg/mL, respectively. This validated method reliably quantified fisetin in the optimized MME formulation, making it suitable for routine analysis and formulation development.