Robust high‐performance thin‐layer chromatography (HPTLC) method for stability assessment and simultaneous quantification of epigallocatechin‐3‐gallate and rosmarinic acid in lipid‐based nanoparticles and biological matrices

Abstract Introduction Skin cancer poses a significant health risk globally, necessitating effective and safe therapeutic interventions. Epigallocatechin‐3‐gallate (EGCG) from green tea and rosmarinic acid (RA) from herbs like rosemary offer promising anticancer properties. Combining these compounds may enhance their effectiveness, prompting the need for a reliable analytical method to quantify them. Objective Herein, we present the development and validation of a high‐performance thin‐layer chromatography (HPTLC) method for concurrent quantification of EGCG and RA in lipid‐based nanoparticles and biological samples. Methodology The method underwent optimisation through design of experiments (DoE), resulting in the establishment of robust chromatographic conditions. The separation process utilised aluminium HPTLC plates coated with silica gel 60 F 254 as the stationary phase, with the mobile phase comprising ethyl acetate, toluene, formic acid, and methanol in a ratio of 4:4:1:1 v/v. Results The retention factor ( R f ) values obtained were 0.38 for EGCG and 0.61 for RA. The method demonstrated linearity over a range of 100–500 ng/band for both compounds with excellent correlation coefficients. Limits of detection and quantification were determined, indicating high sensitivity. Precision evaluations revealed relative standard deviation below 2%, ensuring method reproducibility. Recovery assays in lipid‐based nanoparticles, plasma, and urine samples demonstrated excellent recoveries (96.2%–102.1%). Forced degradation studies revealed minimal degradation under various stress conditions, with photolytic degradation showing the least impact. Conclusion The developed HPTLC method offers a rapid, sensitive, and reliable approach for quantifying EGCG and RA, laying the groundwork for their further investigation as anticancer agents alone and in combination therapies.