Common Challenges in Nitrosamine Analysis: An LCGC International Peer Exchange

Impurity analysis comes with many complex analytical and regulatory challenges. 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 discuss 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 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 reproducibility 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,” Bhanti said (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 sensitivities. 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.”

Supply Chain Considerations

The point at which nitrosamines are introduced during drug manufacturing has been a longstanding question among experts. During the manufacturing process, for example, nitrite-containing excipients can introduce nitrosamines into the drug (8). The FDA has indicated these compounds have been found in a range of medicines including heartburn products, diabetes medicines, and antibiotics (9). The best approach is for organizations to work closely with stakeholders throughout the process.

“I think it is really important for companies to take a multi-pronged approach to the problem and working collaboratively with both drug product and drug substance suppliers,” Burch said. Proper risk assessment can help mitigate problems, and with the right plans, it can ensure that the materials are free of nitrosamines, Bhanti said.

“I think the industry has to adopt a more proactive approach,” Bhanti said (8). “It’s a responsibility of the product manufacturer, but they have to work collaboratively with active pharmaceutical ingredient (API) manufacturers and to make sure that the product is free from nitrosamines.”

Looming Deadlines

A major concern for drug manufacturers worldwide is the rapidly approaching deadlines set by regulatory agencies—such as the U.S. Food and Drug Administration (FDA)—regarding confirmatory testing for nitrosamine drug substance-related impurities (NDSRIs) in drug products. These regulations require companies to rigorously assess and, if necessary, reformulate their products to ensure safety and compliance. The complexity and scope of the testing, coupled with the high stakes of non-compliance, have placed significant pressure on manufacturers to act swiftly while navigating scientific, logistical, and regulatory challenges.

“It’s a nightmare,” Srinivasan said (8). “You can’t resolve this. Even for simplest of the simple products, and even if you have five products in the market which are nitrosamine prone.

While Bhanti noted that many industries are now working to catch up and complete their confirmatory and risk assessment testing for nitrosamines, he emphasized that these efforts are only part of a broader set of challenges.

“August 1, 2025, is going to be challenging, but I think the industries are now trying to catch up, and they are finalizing the confirmatory testing and risk assessment parts, trying to achieve that date. But as you mentioned, it’s not that easy, and it is going to be a lot more challenging” (8).

The panel’s discussion of these complexities not only shed light on the current landscape but also aimed to spark further dialogue about potential solutions and paths forward for both industry and regulators.

You can watch the full peer-exchange here.

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)

(8) Srinivasan, A.; Bhanti, M.; Burch, A. Addressing Sources, Reformulations, and Upcoming Regulatory Deadlines. LCGC International 2025. https://www.chromatographyonline.com/view/addressing-sources-reformulations-and-upcoming-regulatory-deadlines (accessed 2025-4-10)

(9) Control of Nitrosamine Impurities in Human Drugs: Guidance for Industry. United States Food and Drug Administration 2024. https://www.fda.gov/media/141720/download (accessed 2025-4-10)