A green and QbD-optimised UV-spectrophotometric method for the determination of betulin with AGREE-based greenness assessment

Background: Betulin is a naturally occurring pentacyclic triterpenoid predominantly isolated from birch bark and is associated with a wide range of pharmacological activities, including anticancer, anti-inflammatory, antiviral, and antioxidant effects. Despite its growing pharmaceutical importance, systematically optimized and environmentally sustainable UV-spectrophotometric methods incorporating Quality by Design and greenness assessment remain limited. Methods: A green UV-spectrophotometric method was developed using ethanol as an eco-friendly solvent. The maximum absorbance of betulin was observed at 202 nm and selected for quantitative determination. Method optimization was carried out using Central Composite Design to evaluate the influence of critical variables and to establish optimal analytical conditions. The method was validated in accordance with International Council for Harmonisation Q2(R1) guidelines for linearity, accuracy, precision, robustness, limit of detection, and limit of quantification. Environmental performance was assessed using the AGREE metric based on the principles of Green Analytical Chemistry. Results: The developed method exhibited excellent linearity over the concentration range of 2–10 μg/mL with a correlation coefficient of 0.9992. Precision studies demonstrated percentage relative standard deviation values below 2%, confirming reproducibility. The limit of detection and limit of quantification were found to be 0.3700 μg/mL and 1.1212 μg/mL, respectively. Statistical analysis confirmed the significance of the optimization model. The AGREE score of 0.86 indicated strong compliance with green analytical principles. Conclusion: The proposed method provides a simple, reliable, and environmentally responsible approach for the quantitative estimation of betulin. The integration of statistical optimization, regulatory validation, and greenness evaluation supports its suitability for routine pharmaceutical analysis.