Development and validation of a high-performance liquid chromatography method for quantification of the hydrogen...

Hydrogen sulfide (H₂S) is a gasotransmitter involved in the regulation of skin inflammation, making topical H₂S donors promising candidates for its management. Among these, GYY4137 has attracted considerable interest because of its slow H₂S-release profile. However, the lack of validated analytical methods for its direct quantification in biological matrices remains a significant limitation to study its delivery, pharmacological efficacy, and develop new treatment modalities. This study describes the development and validation of a high-performance liquid chromatography (HPLC) method for the quantification of GYY4137 in different skin layers. Porcine ear skin was used as a model for human tissue, and GYY4137 was extracted from the stratum corneum and viable epidermis/dermis using tape stripping, tissue homogenization, and sonication in water:methanol (1:1, v/v), providing consistent recovery (∼80%). Chromatographic separation was achieved using a C18 column, a mobile phase consisting of acetonitrile:phosphate buffer 10 mM (15:85, v/v) and detection at 240 nm. The method demonstrated adequate selectivity, linearity (0.5-10 μg/mL, R² > 0.995), precision, and accuracy (<15%) across all matrices. The lower limit of quantification was 0.5 μg/mL. GYY4137 remained stable under sample preparation and storage at -20 °C, whereas partial degradation occurred at 4 °C (∼30% after one month). The method was successfully applied to in vitro skin penetration studies using Franz diffusion cells. GYY4137 delivery was time- and formulation-dependent, with propylene glycol and oleic acid enhancing skin penetration compared to aqueous solutions. Overall, the proposed HPLC method with UV detection provides a reliable and accessible analytical tool for routine quantification of GYY4137 and supports the development and optimization of H₂S-based topical therapies for inflammatory skin disorders.