Development and validation of a sustainable UHPLC method for quantification and impurity profiling of carfilzomib.

Carfilzomib, a second-generation proteasome inhibitor, is widely used in the treatment of multiple myeloma. This synthetic tetra-peptide epoxyketone derivative binds irreversibly to the 20 S proteasome, leading to the accumulation of misfolded proteins and apoptosis of malignant cells. Despite its FDA approval, maintaining the purity of carfilzomib remains a challenge due to potential impurities affecting its stability, efficacy, and safety. Various studies have identified key carfilzomib related substances, including Epoxy amine, Epoxy RR isomer, Epoxy SS isomer, Epoxy RS isomer, N-BOC Epoxy impurity, Alkene impurity, D-Phenyl alanine, Diol impurity and N-Oxide impurity, which may arise during synthesis, storage, or degradation. Although mass spectrometry and NMR techniques have been used to identify impurities, a robust analytical method for comprehensive impurity profiling is still lacking. In this study, a gradient UHPLC-UV analytical method was developed to separate and quantify carfilzomib and its impurities efficiently. The method achieved precise separation by resolving impurities and carfilzomib peak within 22 min. Linearity for related substances was observed from LOQ to 150% of the specification level (0.5 µg/mL to 2.25 µg/mL), while the assay method exhibited a correlation coefficient of 0.9983 within a range of 25.54 µg/mL to 76.64 µg/mL. A forced degradation study and mass balance confirmed the accuracy of impurity quantification. Additionally, the method demonstrated superior specificity, minimizing interference from structurally similar contaminants. Environmental sustainability was assessed using GAPI, AGREE, and BAGI tools, validating the method’s eco-friendliness and improved analytical performance.