Analytical Quality by Design guided RP-HPLC Method Development and Validation for estimation of Palbocilib employing Box-Behnken optimisation

Conventional empirical approaches (trial and error) to HPLC method design tend to yield fragile, non-consistent methods and lack regulatory flexibility, while Analytical Quality by Design offers a structured framework ensuring reliability. In this study a AQbD guided HPLC method was developed and optimised for Palbociclib, a Cyclin-dependent kinase 4/6 inhibitor (CDK4/6i) approved for the treatment of hormone receptor-positive breast cancer. Risk assessment (Ishikawa diagram) tool guided factor selection while Box-Behnken Design enabled optimisation through Design Expert (v.13) software. The optimised method employed a Shimpack C18 column (250 mm x 4.6 mm, 5 µm); mobile phase comprising Buffer (ammonium formate of pH 4.2 adjusted with glacial acetic acid) and Acetonitrile in the ratio 35:65; flow rate of 0.8 mL/min; injection volume of 10 µL; column oven temperature of 35°C and detection wavelength of 357 nm. Validation was performed in accordance with the ICH Q2 (R2) guidelines. The peak was eluted at 4.23 minutes, and the method demonstrated excellent linearity across 3–50 µg/mL with a correlation coefficient (R ² ) of 0.9999, LOD of 0.75 µg/mL and LOQ of 2.27 µg/mL. Accuracy studies demonstrated recoveries between 98.91-100.88% while precision was evaluated through both intra-day and inter-day studies, consistently showing RSD deviations below 2%.