Encequidar Mesylate Novel Degradation Products: Isolation, Characterization, and Structural Evaluation Using Mass‐Triggered Preparative HPLC, HRMS, and NMR Techniques

Abstract

Encequidar mesylate salt is a novel, little‐absorbed P‐glycoprotein (P‐gp) that facilitates oral absorption. It is a substance that stops the P‐gp adenosine triphosphate‐binding cassette transporter from working. To ascertain the drug substance's inherent stability, it underwent stress testing under a variety of degradation conditions, including acidic hydrolysis, alkaline hydrolysis, oxidative, thermal, and photolytic, in accordance with regulatory requirements. It was discovered that the drug substance is highly unstable in alkaline conditions, and the degradation leads to the formation of two unique, unknown degradation products. Utilizing UHPLC–ESI/MS for identification and mass‐triggered preparative HPLC for isolation, the degradants’ structures were definitively determined by using high‐resolution mass spectrometry and 2D NMR methodologies. These unique degradation products were determined to be novel impurities of the encequidar drug substance and were firmly proven to be its hydrolysis components on the basis of spectral and chromatographic data. Possible potential mechanisms have been proposed to explain the formation of the degradants.