Impurities of impurities! Investigation of drug-related nitrosated impurities of omeprazole using optimized forced nitrosation conditions.

Omeprazole (OMP) is-one of the most commonly prescribed proton pump inhibitors for treating acidity, heartburn, and gastric/duodenal ulcers. The present work investigates nitrosation susceptibility of OMP to form drug-related nitroso impurities using optimized protocols employing forced nitrosation workflow in alignment with nitrosation assay procedure (NAP) and modified NAP. This approach allows evaluation of nitrosation propensity of OMP as well as its degradation products. A series of 10 distinct N-nitrosation products (NPs) were successfully identified and separated via gradient elution liquid chromatography on a C18 stationary phase (250 mm × 4.6 mm, 5 µm) using 10 mM ammonium acetate and acetonitrile as mobile phases. Chemical structures of all the NPs were characterized using LC-QTOF-MS/MS analysis. The in-silico toxicity predictions indicated carcinogenic and mutagenic potential of the observed N-nitrosated impurities alongside qualitative risks such as hepatotoxicity, immunotoxicity, and respiratory toxicity in several cases. These findings underscore elevated risks from nitroso-impurities of API degradation products, beyond conventional API-focused assessments, urging expanded monitoring in pharmaceutical manufacturing and stability studies to minimize the patient exposure and enhance consumer safety.