Green Reverse-Phase High-Performance Liquid Chromatography Method Development for Resveratrol in Liposomal Formulation: A Quality-by-Design Approach

Abstract

Aim: A robust and environmentally sustainable reverse-phase high-performance liquid chromatography
(RP-HPLC) method was developed and optimized for the quantification of resveratrol using a quality-by-design
approach. Materials and Methods: Box–Behnken design was employed to investigate the influence of three
critical chromatographic parameters, organic phase percentage, flow rate, and column temperature on retention
time, tailing factor, and theoretical plates. A polynomial regression model and analysis of variance were used to
analyze interactions and derive optimal conditions, which were validated by residual and perturbation plots, 3D
response surface analysis, and overlay plots. Results and Discussion: The optimized method demonstrated high
reproducibility and precision, with linearity (R² > 0.999) over the 2–10 µg/mL range, and limit of detection and
limit of quantification values of 0.25 µg/mL and 0.81 µg/mL, respectively. Comprehensive method validation
was performed by ICH Q2 (R1 & R2) guidelines. Parameters, including system suitability, accuracy (% recovery
of 97.8–100.2%), precision (%RSD < 2%), and robustness, confirmed the method’s reliability. The method was
effectively applied to quantify resveratrol in liposomal formulations, which exhibited excellent encapsulation
efficiency (87.03%) and recovery rates (96.35–99.5%). Greenness and whiteness of the method were evaluated
using the Complex Green Analytical Procedure Index, Analytical GREEnness Metric Approach (AGREE), AGREE
prep, Analytical Eco-Scale, and blue applicability grade index (BAGI). All tools collectively indicated excellent
environmental and operational compatibility, with AGREE and BAGI scores of 0.74 and 77.5, respectively.
Conclusion: This verifies the method’s environmental friendliness and practical usefulness, making it a suitable
contender for high-throughput pharmaceutical analysis and sustainable laboratory procedures.