AQbD-based UHPLC approach for quantification of everolimus residues in cleaning validation processes

This study aimed to develop and validate a robust reverse-phase ultra-performance liquid chromatography (UHPLC) method for the quantification of everolimus residues on pharmaceutical manufacturing equipment surfaces, ensuring effective cleaning validation.

Cleaning validation is a critical requirement in pharmaceutical manufacturing to prevent cross-contamination and ensure product integrity. The application of the Analytical Quality by Design (AQbD) framework enhances method robustness, reliability, and regulatory compliance while aligning with principles of sustainable and green analytical chemistry.

Risk assessment tools and a central composite design were used to identify and control critical method parameters. Chromatographic separation was achieved using an Acquity UPLC BEH C18 column (50 mm × 2.1 mm; 1.7 μm) with a mobile phase consisting of acetonitrile and water (65:35% v/v) at a flow rate of 0.4 mL/min. The method was validated according to ICH Q2 (R2) guidelines.

The developed method demonstrated a rapid retention time of 1.57 min and excellent linearity over the concentration range of 0.4 to 3.0 μg/mL, with a correlation coefficient (R²) of 0.999. The limits of detection and quantification were found to be 0.100 μg/mL and 0.392 μg/mL, respectively. The method showed high specificity, precision (%RSD within acceptable limits), and recovery accuracy ranging from 91% to 100%.

The developed method provides a sensitive, precise, and eco-friendly solution for the quantification of everolimus residues in cleaning validation processes. It fulfils regulatory expectations and is suitable for routine use in pharmaceutical quality control settings, reinforcing product safety and operational efficiency.