Novel Genotoxic Impurities in the First-Line Oral Anticoagulant Drug, Apixaban: Computational Toxicological Evaluation and Development of a Highly Sensitive LC-MS/MS Quantification Method

Abstract

Apixaban is an oral anticoagulant classified as a direct factor Xa inhibitor. It is widely utilized for prophylaxis and management of thromboembolic conditions, including stroke, deep vein thrombosis, and pulmonary embolism, as a primary therapeutic option. Apixaban is subjected to stringent testing for potential genotoxic impurities during its manufacturing, due to its structure indicating the possibility of such impurity's formation. Setting limits in accordance with ICH M7 guidelines is essential to reduce patient exposure and also recommended an advanced analytical tool for testing. The present study aims to establish limits based on computational toxicological evaluation, and develop and validate a highly sensitive LS-MS/MS method for quantifying three novel genotoxic impurities of apixaban, namely impurities F, G, and H. The method was established utilizing a mobile phase comprising a pH 5.5 acetate buffer and acetonitrile in a gradient mode. Used a C18 column, of 150 mm (length) × 3.0 mm (width), 2.7 μm of particle size as stationary phase. Quantification was performed with multi-response monitoring in mass spectrometry, employing precursor ions of m/z 437.2, 525.2, and 569.1 for impurities F, G, and H, respectively. The established method is validated for its intended use in accordance with regulatory guidelines and found suitable.