Unveiling Pretomanid profiling using LC–MS/MS: integrating in-silico toxicity assessment and molecular docking analysis

Abstract

The presence of impurities in pharmaceutical formulations compromises their safety, posing significant concerns. Recent market recalls have highlighted this issue. Pretomanid (PMD), an antitubercular drug, has functional groups such as amine, imidazole, benzene ring substitution, and ether group, making it sensitive to hydrolysis. Stability and forced degradation investigations were conducted following the International Conference for Harmonization (ICH) protocol Q1A (R2). Under various stress conditions such as Hydrolytic (acidic, basic, neutral), Thermal, Photolytic, and Oxidative; total Sixteen new Degradation products (DPs) were identified. These DPs labeled DP 1 to DP 16, were successfully separated using Isocratic elution with a Kromasil C18 (250 × 4.6 mm; 5 μm) column. The LC–MS/MS compatible mobile phase comprise of Acetonitrile (ACN) and 10 mM Ammonium Acetate pH 4.5 (50:50; v/v), at a flow rate of 0.9 mL/min, with a 30-min runtime and a column temperature of 25 °C. The DPs were identified by LC-MSanalysis, and their structures were confirmed with LC–MS/MS fragmentation. Drug Degradation occurred due to majorly Oxidation, Hydrolysis, and Rearrangement mechanisms. Pretomanid was particularly sensitive to Alkali and Oxidative hydrolysis. Therefore, PMD must be protected from aqueous media, temperature, alkaline pH, moisture, and light during formulation and development. In silico toxicity and molecular docking, studies revealed DP 6 as a potential hit molecule with a higher docking score of -151.22 than PMD with comparative less toxicity.

Graphical abstract