Chemometrically assisted optimization and validation of the HPLC method for the analysis of alectinib and its related impurity with measurement uncertainty evaluation and whiteness, blueness, and greenness profile assessments.

Abstract

Alectinib, the most common second-generation anaplastic lymphoma kinase inhibitor, is efficacious against various anaplastic lymphoma kinase mutations and is utilized in the treatment of non-small cell lung cancer. In this work, the determination of both alectinib and its impurity 5-trifluoroacetate at the same time was realized by developing a novel high performance liquid chromatography method. The stationary phase was an Ascentis^® Express 90 Å C₈ (10 cm × 4.6 mm, 2.7 µm) HPLC column; the separation was obtained using a gradient system with a mobile phase consisting of acetonitrile and ammonium acetate buffer, and the responses were recorded from the PDA detector at a wavelength of 269 nm. The method's optimization was carried out using the Box-Behnken design, whereas the optimized conditions were selected according to the desirability function. Validation of the method was done with the International Council for Harmonization, ICH Q2(R2) Guidelines. The limit of detection values was 0.1 and 0.3 µg/mL, and the limit of quantification values were 0.3 and 0.5 µg/mL for alectinib and its impurity, respectively. The recovery study was realized in the Alecensa^® capsule (150 mg alectinib/capsule), with high recoveries showing the perfect precision and accuracy of the method. A bottom-up approach was used to estimate the measurement uncertainty. This made it possible to find and measure the sources of uncertainty as well as the factors that affected the chromatographic responses. The resulting regression models were deemed adequate and were then used to define the operable design region using the Monte Carlo method. The eco-friendliness of the proposed methods was validated with the Complex Green Analytical Procedure Index, Analytical Greenness Metric and Blue Applicability Grade Index greenness evaluation tools. Also, the whiteness of the method was calculated with the newly developed White Analytical Chemistry tool. The method can be used to assay alectinib and its impurity agents in its formulation in quality control laboratories.