Hunt for NDSRIs: Unveiling Hidden Threats with the Novel ¹⁵ N-Enriched NAP Test

High Resolution Image Download MS PowerPoint Slide Nitrosamine impurities, particularly nitrosamine drug substance-related impurities (NDSRIs), have emerged as a critical concern in pharmaceutical manufacturing due to their potential carcinogenicity. Regulatory agencies now require rigorous risk assessments and confirmatory testing to ensure product safety, considering all nitrogen atom alert groups. In this study, we present a novel strategy that combines the nitrosation assay procedure (NAP) with 15 N-enriched nitrosating reagents and 15 N NMR spectroscopy to detect and characterize nitrosamine formation. This method enables qualitative analysis of nitrosamines and provides valuable insight into the nitrosation reactivity of pharmaceutical compounds. The diagnostic chemical shift range for N –NO groups was validated, and the method was applied to a series of known nitrosamines and NDSRIs derived from active pharmaceutical ingredients and intermediates. The 15 N-enriched NAP test proved effective in identifying nitrosamines, even in complex matrices, and distinguishing between isomeric and degradation products. This integrated approach provides a robust and rapid tool for nitrosamine risk assessment and supports regulatory compliance by confirming or excluding nitrosamine formation under stress conditions.