Simultaneous Quantification of Caffeic and Ferulic Acids by HPLC‐UV in Nanoparticles: Degradation Profiling and Computational Peroxidation Analysis

Phenolic compounds such as ferulic acid (FA) and caffeic acid (CA) are renowned for their antioxidant and anti-inflammatory properties. However, their physicochemical instability limits broader therapeutic applications. This study aimed to develop an analytical methodology for the simultaneous quantification of these compounds in nanoparticles using liquid chromatography. The suspensions exhibited favorable characteristics, including a particle size of 117 nm, encapsulation efficiency above 60%, zeta potential of 14 mV, and adequate bioactive content. Notably, the system enhanced their photostability under UV radiation. The HPLC-UV method employed a C18 column with a mobile phase of acidic water (pH 2.6), methanol, and acetonitrile (64:18:18, v/v/v) at 1 mL/min, with detection at 223 nm, achieving fast and efficient separation. The method was linear, specific, precise, accurate, and robust, in accordance with Brazilian and ICH guidelines. Additionally, the method was stability-indicating, identifying isomeric degradation products, which represent the most common degradation reaction for these compounds. Computational analysis using density functional theory (DFT) further explored the stability of CA and FA. It revealed a strong interaction between the carboxyl group and hydrogen peroxide, suggesting reduced susceptibility to oxidation. These findings provide a foundation for designing stable formulations and enhancing the understanding of antioxidant mechanisms in phenolic compounds.