Capillary Electrophoresis Optimization for Metabolite Separation in Hypogymnia physodes Using DoE: Validation Across Lichen Species

Lichen-specific natural products exhibit a wide range of biological activities, which makes them potentially useful in the pharmaceutical, cosmetic, and nutritional industries. In the present study, a capillary electrophoresis method was developed and optimized for the separation of seven major metabolites, physodic acid, 3-hydroxyphysodic acid, atranorin, physodalic acid, chloroatranorin, salazinic acid, and protocetraric acid, found in <i>Hypogymnia physodes</i>. The optimization was performed using a design of experiments approach, focusing on four critical parameters: boric acid concentration, deoxycholic acid concentration, methanol content, and buffer pH. The overall separation efficiency was used as the response factor for optimization. The optimal separation conditions were achieved using a buffer composed of 60 mM boric acid, 70 mM deoxycholic acid, and 14% methanol at pH 9.6. The validated method was subsequently applied for the chemophenetic analysis of 28 lichen species belonging to the families Cladoniaceae, Parmeliaceae, Physciaceae, Ramalinaceae, and Teloschistaceae. In addition to the above-mentioned lichen compounds, the lichens examined showed the presence of evernic acid, usnic acid, and physicon. The developed CE method offers a reliable and efficient tool for the characterization of lichen metabolites, with potential applications in both botany and natural product research.