RP‐HPLC–Validated Method for Hesperidin Estimation in Rat Skin Following Transdermal Delivery via Dissolving Microneedle Patch

A novel reverse-phase high-performance liquid chromatography (RP-HPLC) method for validating the amount of HES in dissolving microneedle patches (DMNs) loaded with HES-based nanostructured lipid carriers (HES-NLC)s has been developed. The method was developed and validated according to the guidelines published by the International Council for Harmonization (ICH), using a mobile phase of methanol:water (90:10 v/v) at a flow rate of 0.5 mL/min, with UV detection at 284 nm. This method demonstrated accuracy from 94% to 108%, with a precision of 2% across all evaluated concentrations, along with high selectivity, sensitivity, and stability for transdermal application studies. The method also showed good linearity in rat plasma and skin homogenate, with no interference from biological matrices, which aligns with FDA guidelines. Permeation studies revealed enhanced HES delivery via DMNs with nearly 20-fold higher drug deposition compared to passive diffusion. Pretreatment with DMNs significantly boosted the transdermal permeation of HES (112.86 ± 23.58 μg/cm² after 24 h, p < 0.01), by increased flux (1.31 ± 0.11 μg/cm²/h) and permeability coefficient (0.36 × 10^-6 cm/s) values. This validated method offers a promising tool for advancing transdermal evaluation in innovative pharmaceutical formulations.