A case study on N-nitrosamine investigation in a marketed drug: from analytical procedure validation to risk mitigation decision

Introduction N-nitrosamines, a class of organic compounds recognized for their mutagenic and carcinogenic properties, have been raising major public health concerns following their detection in pharmaceuticals since 2018. These impurities can form during manufacturing processes, particularly through the interaction of amines with nitrosating agents. Regulatory bodies have launched procedures mandating a systematic investigation of medicinal products for N-nitrosamine contamination. This process involves risk evaluation, confirmatory testing, and the implementation of risk mitigation measures. Hyphenated analytical techniques, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), have become the standard for N-nitrosamine detection thanks to their high sensitivity and specificity. Methods In this work, based on a previously developed Quantitative Structure Retention Relationship approach for the determination of N-nitrosamines, sensitive LC-MS/MS methods were proposed and validated. These methods were subsequently applied to confirmatory testing of a marketed drug product. Results The methods were successfully validated in accordance with the ICH Q2 (R2) guidelines as limit tests for trace-level detection of N-nitrosamines. The confirmatory testing demonstrated the absence of most N-nitrosamines throughout the product’s shelf life, except for one potentially problematic impurity that nevertheless remained below the established acceptable limit, thus requiring routine quantitative analysis. Discussion These findings highlight the need for continued monitoring through routine analysis to ensure the safety and quality of pharmaceutical products.