Experts Discuss the Complexities of Nitrosamine Analysis

Nitrosamines are a large group of N-nitroso compounds (NOCs) that bear common functional >N–N=O groups. NOCs can be divided into two classes: N-nitrosamines and N-nitrosamides and related compounds (1). These compounds can be found in various substances, including pharmaceuticals. If humans are exposed to nitrosamines above acceptable levels and over long periods of time, these impurities can increase the risk of cancer (2).

Detecting acceptable intake limits, or the acceptable amount of impurity, such as nitrosamines, in a drug, can be difficult due to the limited availability of safety data for an impurity (3). When safety data is unavailable for nitrosamine impurity, information from nitrosamine comparators, or structurally similar compounds, can be used to identify acceptable intake limits. However, oftentimes appropriate comparators are not available. Further, default acceptable intake limits present challenges to both industry and regulators, significantly impacting drug supply chains.

To address these complex issues, the editors of LCGC International organized a peer exchange of experts to discuss the intricate nature of nitrosamine analysis. The panel was moderated by Aloka Srinivasan, principal and managing partner of Raaha, and also featured Mayank Bhanti, senior director of the Compendial Development Laboratory at the United States Pharmacopeia (USP), and Amber Burch, the senior manager of Technical Business Development at Purisys (4).

The Big Analytical Challenges

The main analytical challenges associate with nitrosamine analysis can be roughly divided into five categories, Bhanti said. One of the main challenges with nitrosamine detection is the increasingly low sensitivities being required by regulatory agencies. This requires the use of techniques such as liquid chromatography–tandem mass spectrometry (LC–MS/MS) technology (5). These instruments can help achieve the target sensitivities, but robustness and reproductibility rates may be a challenge, Bhanti said, especially when handling low levels of the compounds.

“These metrics post a lot of interferences, especially from a mass spectrometry point of view,” said Bhanti (5). Ion sources, by comparison, could potentially lead to an underestimation of nitrosamine impurities.

Sample preparation is also an important part of nitrosamine analysis. Optimizing extraction and clean-up processes may be necessary to help achieve lower sensitivites.

Achieving chromatographic resolution can be especially challenging with nitrosamine drug substance-related impurities (NDSRIs), which are a class of nitrosamine impurities that have been found in both drug products and active pharmaceutical ingredients (APIs) (6). These impurities can form during drug synthesis, storage, or degradation, and hold potential carcinogenic risks (7).

Bhanti calls for higher-resolution technologies, such as high-resolution mass spectrometers (HRMS) to be used for these procedures.

“Pharmaceutical companies have to be flexible in their approach and have to have to change their methods in order to meet the requirements of different regulatory agencies,” he said (5). “Overall, I think a multifaceted approach needs to be adopted to really be able to overcome all these challenges and to get in a nitrosamine-free drug product for the patients.”

Burch emphasized the importance of inter- and intra-laboratory precision of methods.

“Making sure that a method is able to be executed accurately by multiple analysts or by multiple organizations is very key to assuring that the method was going to be suitable for the life of the product,” she said (5). “[Ensuring] that the method can be executed accurately by multiple analysts or by multiple organizations is key to assuring that the method is going to be suitable for the life of the product.”

References

(1) Nitrosamines. ScienceDirect 2005. https://www.sciencedirect.com/topics/chemical-engineering/nitrosamines (accessed 2025-4-2)

(2) Information about Nitrosamine Impurities in Medications. FDA 2025. https://www.fda.gov/drugs/drug-safety-and-availability/information-about-nitrosamine-impurities-medications#updates (accessed 2025-4-2)

(3) Determining Recommended Acceptable Intake Limits for N-nitrosamine Impurities in Pharmaceuticals: Development and Application of the Carcinogenic Potency Categorization Approach. FDA 2025. https://www.fda.gov/drugs/spotlight-cder-science/determining-recommended-acceptable-intake-limits-n-nitrosamine-impurities-pharmaceuticals (accessed 2025-4-8)

(4) Srinivasan, A.; Bhanti, M.; Burch, A. Introducing Our Panel of Experts. LCGC International 2025. https://www.chromatographyonline.com/view/introducing-our-panel-of-experts (accessed 2025-4-2)

(5) Srinivasan, A.; Bhanti, M.; Burch, A. A Look at Major Analytical Challenges. LCGC International 2025. https://www.chromatographyonline.com/view/a-look-at-major-analytical-challenges (accessed 2025-4-2)\

(6) U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER). Recommended Acceptable Intake Limits for Nitrosamine Drug Substance-Related Impurities (NDSRIs): Guidance for Industry. FDA 2023. https://www.fda.gov/media/170794/download (accessed 2025-4-8)

(7) Nitrosamine Impurity Analysis Applications. ThermoFisher Scientific 2025. https://www.thermofisher.com/us/en/home/industrial/pharma-biopharma/biopharma-analytical-solutions/nitrosamine-impurity-analysis/applications.html#nitrosamine-drug-substance-related-impurities (accessed 2025-4-9)