Simultaneous determination of benzophenone-3, retinol and retinyl acetate in pig ear skin layers by high-performance liquid chromatography

A new and simple HPLC method was developed and validated for the simultaneous determination of benzophenone-3, retinol and retinyl acetate in pig ear skin layers and percutaneous penetration samples after in vitro permeation experiments. HPLC analysis was performed utilizing a NovaPak C18 column with acetonitrile-water-acetic acid as mobile phase. UV detection was at 325 nm and the run time was 25 min. The detector response was found to be accurate, precise and linear across the analytical range. Analyte extraction from skin layers was done with methanol from the stratum corneum and epidermis, and with acetone from the dermis. Recovery was in all cases better than 90%. The HPLC assay and extraction procedure proposed are simple, rapid, sensitive and accurate. The method was then applied for the determination of benzophenone-3, retinol and retinyl acetate in pig ear skin layers after topical application.