Development and Validation of an HPLC-DAD Method for the Quantitative Determination of Benzoyl Peroxide, Curcumin, Rosmarinic Acid, Resveratrol and Salicylic Acid in a Face Mask—In Vitro/Ex Vivo Permeability Study

Acne is a common skin condition that causes pimples, redness and inflammation. Benzoyl peroxide (BENZ), salicylic acid (SAL), curcumin (CUR), rosmarinic acid (ROS) and resveratrol (RESV) exhibit antimicrobial, anti-inflammatory and antioxidant properties and are recommended for its treatment. These five active pharmaceutical ingredients (APIs) were incorporated into a green clay, honey and gelatin face mask and determined by an HPLC-DAD (diode array) method. For the chromatographic separation of the analytes, a gradient mobile phase with two solvents mixtures: A, comprising H₂O with 0.1% TFA-ACN with 0.1% TFA, 85:15 <i>v</i>/<i>v</i>, and B, comprising 100% ACN with 0.1% TFA, and a C18 column (250 × 4.6 mm, 5 μm), at 40 °C (diluent: MeOH-ACN 0.1% TFA 2:1 <i>v</i>/<i>v</i>), were selected. The method was validated according to the ICH guidelines for pharmaceutical products (R² > 0.999, %RSD < 1.2, % Recovery > 98.2, LOD_μg/mL: ROS = 0.267, RES = 0.047, SAL = 0.636, CUR = 0.296 and BENZ = 0.083). For the processing of mask samples and the quantitative extraction of the analytes, the "D-optima mixture" experimental design methodology was applied (% Recovery 95.4-102.1%, %RSD < 2.4). Finally, the permeability rate (Papp) of the mask ingredients through the skin was studied using Franz vertical diffusion cells, in a cellulose membrane (in vitro), in rat tissue and in human skin (ex vivo). To ensure the reliability of the results, APIs' stability rate under the given experimental conditions was studied. In addition, a second method for sample processing in Franz cells was developed and validated (% Recovery > 90.6-106.9, %RSD < 5.2). Based on the results obtained, both the effectiveness of the new face mask formulation and the suitability of the membranes were evaluated.