Validation of a Chromatographic Method for Simultaneous Quantification of Imiquimod and Terbinafine in Porcine Skin ^†

Introduction: Chromoblastomycosis is a chronic and neglected fungal infection of the skin and subcutaneous tissue, requiring long-term treatment and presenting therapeutic challenges. Topical strategies have emerged as promising alternatives, including the combination of terbinafine, a broad-spectrum antifungal, with imiquimod, a topical immune response modifier. However, no validated analytical method is currently available to simultaneously quantify both drugs in skin, which is crucial for formulation development and quality control. This study aimed to validate a simple method for the simultaneous quantification of terbinafine and imiquimod extracted from porcine skin. Methodology: Analyses were performed using high-performance liquid chromatography with fluorescence detection (excitation 236 nm, emission 340 nm). A C8 reversed-phase column (125 × 4.0 mm, 5 μm) was employed, with a mobile phase composed of water and methanol (60:40, v/v), both acidified with 0.1% formic acid, flowing at a rate of 0.8 mL/min. Results: The method showed excellent linearity (r² > 0.999) for imiquimod (0.01–1.0 μg/mL) and terbinafine (0.1–2.0 μg/mL). Intra- and inter-day precision demonstrated coefficients of variation below 5%. Recovery rates from skin samples ranged from 80% to 100%, confirming accuracy, selectivity, and reproducibility. Limits of quantification were 0.02 μg/mL for imiquimod and 0.16 μg/mL for terbinafine, with detection limits of 0.01 μg/mL and 0.05 μg/mL, respectively. Conclusions: The proposed method is robust, accurate, and sensitive, allowing the simultaneous quantification of terbinafine and imiquimod in skin samples. It represents a valuable tool to support the development, characterization, and quality control of topical formulations for the treatment of chromoblastomycosis.