Formulation and HPLC‐UV−Based Quantification of Nanoliposomes Co‐Encapsulating Genistein and Oleanolic Acid

Oleanolic acid (OL) and genistein (GN) are bioactive compounds known for their anti‐inflammatory, antioxidant, antiviral, cardioprotective, and anticancer properties. Their synergistic therapeutic potential motivated the development of a hybrid nanoformulation, comprising GN nanoparticles incorporated into an OL‐loaded liposomal system named GOL. The formulation was extensively characterized for particle size, polydispersity index, zeta potential, entrapment efficiency, and pharmacokinetic behavior, demonstrating its suitability for co‐delivery of both drugs. To accurately quantify OL and GN in the GOL system, a robust HPLC‐UV method was developed and validated. Simultaneous determination was challenging due to distinct chemical and spectral properties; OL showed λmax at 210–215 nm, while GN had λmax at 215, 270, and 336 nm. Optimization using a Box–Behnken design yielded a method using a C18 column with a methanol–acetonitrile–water–tetrahydrofuran (60:20:8:12, v/v) mobile phase and detection at 230 nm. Retention times were 4.8 and 9.9 min for OL and GN. Plasma samples were processed via simple protein precipitation. The method met ICH validation criteria for sensitivity, selectivity, accuracy, precision, robustness, and stability. Finally, the developed method was successfully applied to analyze both drugs from the GOL nanoformulation in both biological fluids and polymeric matrices, enabling a reliable assessment of drug content and stability.