Validation and Application of an HPLC–UV Method for the Quantification of 3‐ O ‐Methylquercetin in Nasal Mucosa

ABSTRACT 3 ‐O‐ methylquercetin (3 O MQ) is a methylated flavonoid with relevant biological activity whose reliable quantification in complex biological matrices remains analytically challenging due to its low aqueous solubility and matrix‐related interferences. In the present study, a previously reported HPLC‐UV method was adapted and revalidated for quantifying 3 O MQ in porcine nasal mucosa for use in in vitro permeation and retention studies. Chromatographic separation was achieved under isocratic conditions using a phenyl stationary phase, providing adequate selectivity and reproducibility in the new biological matrix. The method demonstrated excellent linearity over the concentration range of 1–25 µg/mL ( r 2 > 0.99). Accuracy and precision met international regulatory acceptance criteria, with relative standard deviations below 5% for both intra‐day and inter‐day evaluations. It also demonstrated excellent robustness under deliberate variations in flow rate, temperature, and detection wavelength (RSD < 2.00%). Matrix effect (86.94%–110.28%) and recovery (85.21%–106.47%) results confirmed the reproducibility and the efficiency of the method. The applicability of the revalidated method was demonstrated through in vitro release, permeation, and retention experiments using Franz diffusion cells and porcine nasal mucosa. These findings demonstrate that the method is precise, sensitive, and robust, making it suitable for nasal permeation and retention studies of 3 O MQ. Moreover, this analytical approach represents a reliable tool for in vitro and ex vivo pharmaceutical research, supporting the development and evaluation of nasal drug delivery systems for poorly soluble bioactive compounds, particularly flavonoid aglycones.