Development of a fit-for-purpose method using the analytical quality by design approach for quantifying a sesquiterpenoid and an 8-hydroxyquinoline derivative in a porcine hoof permeation model

Onychomycoses are fungal infections of the nail unit, mainly caused by dermatophytes, and have a high prevalence worldwide. Current treatments for onychomycosis face challenges with the penetration of topically applied antifungal drugs through the nail barrier. Caryophyllene oxide is a sesquiterpenoid found in several essential oils that has antifungal properties. Clioquinol (CLI) is a broad-spectrum antifungal agent with potential for topical use. Combining a sesquiterpenoid compound with a synthetic antifungal drug offers a promising therapeutic and technological approach for treating onychomycoses, once both can be easily combined in a nanoemulsion formulation. This strategy requires the development of a quick and reliable method to evaluate their in vitro permeation and retention in the nail matrix, serving as a measure of formulation performance. In this study, using the Analytical Quality by Design (AQbD) approach, we developed and validated a rapid, sensitive bioanalytical method capable of simultaneously identifying and quantifying CLI and CAROX via high-performance liquid chromatography with a UV detector (HPLC-UV) at 215 nm, with retention times below 15 min. Additionally, an ungual permeation protocol was established, and the in vitro permeation and retention of CLI and CAROX in porcine hooves were assessed. Results showed that CLI retention within the nanoemulsion was four times higher than in solution (28.11 ± 4.85 vs 7.54 ± 3.03 μg/cm 2 after 72 h). For both CLI and CAROX, loading into nanostructured systems demonstrated a sustained release profile. • A bioanalytical method was developed for the analysis of clioquinol and caryophyllene oxide. • AQbD approach was used to optimize the analytical response • In vitro studies with porcine hoof membranes assessed formulations´ performance. • Nanoemulsification increased clioquinol retention in porcine hoof by up to 4-fold versus solution. • Caryophyllene oxide retention in hooves was only possible due to the nanoemulsification.