Oxidative stress-induced degradation of Brinzolamide: Isolation and in-depth characterization of unique hydroxylamine and oxime degradation products

Abstract

Since the safety and efficacy of therapeutic products are strongly related to their stability and purity, impurities including the unavoidable degradation products may affect the pharmacological effect. The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines Q3A requires the identification of process impurities and as well as degradation products in any drug substance to assess the inherent stability of the drug. The present work involves an ICH-guided degradation study for the Brinzolamide (BRZ), a topical ophthalmic drug which is generally used to lower the intraocular pressure (IOP) during glaucoma. Under oxidative stress at room temperature for 20 h, four degradation products (namely BRZ-Pk1, BRZ-PK2, BRZ-Pk3, and BRZ-Pk4) are isolated using advanced chromatographic techniques. Upon confirming the masses of the compounds using High-resolution mass spectrometry (HRMS), functional groups are identified with the help of Fourier-transform infrared spectroscopy (FT-IR). Extensive 1-dimensional (1D) and 2-dimensional (2D) Nuclear Magnetic Resonance spectroscopic (NMR) experiments especially 1D nOe, 1H-13C-HSQC and 1H-13C-HMBC unequivocally confirm the structures. Among the four compounds analyzed, three (BRZ-Pk1, BRZ-Pk2, and BRZ-Pk4) are novel, while BRZ-Pk3 was previously reported solely with mass spectrometric data. Nitrogen-based 2D NMR experiments are crucial for determining the oxidation state of hydroxylamine and oxime products within the molecules, and 1D nOe measurements help confirming E/Z isomerism (geometrical isomerism) for BRZ-Pk2 and BRZ-Pk4. All the proposed structures are justified with appropriate analytical data. The proposed mechanisms are expected to help in identifying the possible degradation pathways for similar pharmaceutical candidates.