Integrating analytical quality by design into bioanalytical method development: an HPLC-FLD method for quantification of alectinib in biological matrix

This study presents the development and validation of a fluorescence-based high-performance liquid chromatography (HPLC) method for the quantification of alectinib in rat plasma, with a focus on the application of Analytical Quality by Design (AQbD) to bioanalytical method development. Unlike conventional QbD applications, which primarily address synthetic formulations or instrumental settings, this study systematically applied AQbD principles to the complex environment of biological matrices. Critical method parameters, including the organic phase ratio, buffer concentration, and flow rate, were identified through Failure Mode and Effects Analysis, and optimized using a Box-Behnken design. The final method exhibited excellent linearity (R² >0.99) over a concentration range of 5-1,000 ng/mL, with a lower limit of quantification of 5 ng/mL. It also showed high accuracy (95.6-102%), precision (relative standard deviation < 11%), and consistent recovery (98.3-105%), with minimal matrix effects. Alectinib stability was confirmed under various handling conditions. This method was successfully applied in a pharmacokinetic study after intravenous and oral administration of alectinib in rats. These results highlight the value of AQbD in addressing specific challenges of bioanalysis and demonstrate its utility in establishing a sensitive, robust, and regulatory-compliant method suitable for pharmacokinetic applications.

Alectinib; Analytical quality by design; Box–Behnken design; Method development and validation; Taguchi method.