Box-bhenken Design Combined with 3D Surface Methodology for Optimization of an Eco-friendly HPLC Method to Determine Venetoclax in Human Plasma, and its Bioanalytical Method Validation According to ICH M10 Guideline

Aims: Venetoclax is a selective inhibitor of the prosurvival protein BCL-2 approved by the Food Drug Administration in 2016, restoring the apoptic ability of malignant cells. In this study, a fast, highly accurate and precise HPLC method was developed for the analysis of Venetoclax in human plasma. Methods: The optimization of the method was investigated according to Box-Bhenken Design combined with 3D surface methodology. The chromatographic separation was performed in gradient mode with an Ascentis Express C8 column (2.7 μm, 4.6 mm × 10 cm). Agomelatine was used as an internal standard to increase accuracy. The method was completely validated according to ICH guideline M10 bioanalytical method validation. Additionally, the greenness of the method was scaled with NEMI, Analytical Ecoscale, AGREE, and GAPI greenness metrics. Results: The method was linear in the range of 1.67-12.50 μg/mL with a calculated R2 of 0.99; LOD and LOQ were 0.34 μg/mL and 1.02 μg/mL, respectively. The recovery was in the range of 102.6% to 99.08%, and with an RSD% of less than 1.00%. The analytical eco scale and AGREE score of the current method were 85 and 0.55, respectively. Conclusion: The approach that was developed herein exhibits green, rapidity, high levels of accuracy and precision, cost-effectiveness, and ease of use in the context of clinical and pharmacokinetic investigations.