Nitrosamine Control: From Risk Assessment to Analytical Testing with Emphasis on Sample Preparation and Phase-Appropriate Method Validation

In recent years, the detection of nitrosamine impurities in pharmaceuticals has emerged as a significant safety concern, primarily due to their potential carcinogenic and mutagenic effects on patients. These impurities may be introduced as byproducts from synthetic steps or formed in the presence of specific precursors. Although pharmaceutical manufacturers have methodologies for source identification, risk assessment, and mitigation strategies at their disposal, implementing rigorous analytical testing is paramount for quantifying nitrosamine levels and ensuring compliance with regulatory standards. This Review provides a comprehensive overview of integrating robust analytical testing within risk assessment frameworks, focusing on methods to detect nitrosamines in both drug products and substances. Sample preparation is highlighted as a pivotal step in ensuring robust nitrosamine quantitation, as it directly impacts analytical sensitivity and specificity by isolating trace-level impurities from complex pharmaceutical matrices while minimizing matrix effects, analyte instability, and artifactual formation. Despite its critical importance, no prior reviews have comprehensively addressed the challenges and strategies associated with sample preparation for nitrosamine analysis. This review fills this gap by emphasizing the role of optimized sample preparation techniques, discussing key challenges such as matrix interference and contamination risks, and presents a compilation of solutions published in the literature. Additionally, a phase-appropriate method validation strategy is proposed, tailored to drug development stages, ensuring that methods are fit for purpose while meeting regulatory expectations. By presenting a comprehensive framework for optimizing sample preparation and validation approaches, this Review aims to support accurate nitrosamine quantitation and ensure pharmaceutical product safety.