Greenness assessment and stability-indicating LC method for determination of alectinib using Box-Behnken design, high-resolution mass spectrometric identification, and molecular docking studies of its novel degradation products

Alectinib is among the second-generation inhibitors prescribed for anaplastic lymphoma kinase (ALK). In the present study, new liquid chromatography (LC) methods have been developed for the quantitative determination of Alectinib and the qualitative determination of its degradation products. The analytical method was optimized using a three-level Box-Behnken response surface design. The optimized analytical method was validated according to ICH (Q2)R1 guidelines, including linearity, sensitivity, accuracy, precision, and robustness. In the study, mixtures of water, acetonitrile and formic acid were used in different ratios in LC-MS/MS and HPLC analyses using Ascentis (R) Express phenyl hexyl (100 x 4.6 mm, 2.7 mu m) as the stationary phase. Briefly, the methods allow for the quantification of alectinib with high accuracy and sensitivity in the ranges of 0.17-6.67 mu g/mL for HPLC and 8.50-340.0 ng/mL for LC-MS/MS. Additionally, the validated method's sensitivity to the degradation products of the active substance was confirmed through stress-induced degradation studies. All degradation products were characterized using LCMS-IT-TOF studies. Five different degradation products, three of which are new, were detected. Comparative molecular docking studies were conducted between these degradation products and the active binding site of the Alectinib compound. Furthermore, the validated method was successfully applied to estimate the content of Alecensa (R) formulation. The greenness of the developed methods was assessed using NEMI, Analytical Eco-scale, AGREE, and GAPI.