Design and ICH validation of a stability-indicating green RP-HPLC method for 5,6,7-trihydroxyflavone with multi-metric greenness evaluation and application to nanopharmaceutical systems

Abstract

A novel stability-indicating reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the quantification of baicalein (5,6,7-trihydroxyflavone) in bulk drug and nanoliposomal formulations. Samples were pretreated via dilution with mobile phase, membrane filtration (0.22 µm), protection from light, and refrigerated storage prior to analysis. Method validation was performed in accordance with ICH Q2(R2) guidelines. Chromatographic separation was achieved on a BDS C18 column using an isocratic mobile phase comprising water (pH 3.0, adjusted with orthophosphoric acid) and acetonitrile (50 : 50, v/v) at a flow rate of 1.0 mL min⁻¹ with DAD detector. The method exhibited excellent linearity (0.0078–1 µg mL⁻¹; R² = 0.9998), accuracy (98.25–101.58%), and precision (%RSD < 2%). The limits of detection (LOD) and quantification (LOQ) were 0.0069 µg mL⁻¹ and 0.0208 µg mL⁻¹, respectively. Forced degradation under acidic, alkaline, oxidative, and thermal stress conditions confirmed the stability-indicating capability, with pronounced degradation under acidic conditions and high thermal stability. The method was successfully applied to solubility profiling and quantification in nanoliposomal systems, including entrapment efficiency and drug loading. Greenness assessment using Blue Applicability Grade Index (BAGI), Modified Green Analytical Procedure Index (MoGAPI), Carbon Footprint Reduction Index (CaFRI), Click Analytical Chemistry Index (CACI), Analytical GREEnness (AGREE), and Violet Innovation Grade Index (VIGI) yielded scores of 75, 85, 77, 80, 0.64, and 55, respectively. The method demonstrates key advantages, including buffer-free operation, reduced chemical hazard, and operational simplicity; however, moderate organic solvent usage imposes minor sustainability limitations. Overall, the method is robust, sensitive, and suitable for routine pharmaceutical and nanoformulation analysis.