The Continuous Challenge of Nitrosamines

Nitrosamines, a class of potentially cancer-causing chemicals, were found in certain pharmaceuticals, such as heart pressure medications, in 2018. Regulatory agencies have been working with pharmaceutical companies since then to mitigate the risk of these impurities in drug products (1). In January 2025, FDA posted “Determining Recommended Acceptable Intake Limits for N-nitrosamine Impurities in Pharmaceuticals: Development and Application of the Carcinogenic Potency Categorization Approach” in the Spotlight on CDER Science on the FDA.gov website (2).

Pharmaceutical Technology® spoke with Todd Sprouse, associate director, Analytical Services, and Erik Feldmann, PhD, principal technical advisor, Client Solutions & Proposals, both with Cambrex, to find out more about the challenging situation.

What is the carcinogenic potency categorization approach (CPCA)?

PharmTech: What can you tell us about the recommendations FDA published in January 2025 for the development and application of the carcinogenic potency categorization approach (CPCA) to nitrosamines? What is CPCA and how can it be leveraged?

Sprouse (Cambrex): The CPCA approach is a simplified molecular structural assessment for nitrosamines. Many of these compounds, like novel nitrosamine drug substance-related impurities (NDSRIs), are being discovered and characterized as pharmaceutical development progresses. For these new compounds, carcinogenic potency is estimated using data from structurally similar nitrosamines where more data [are] already available, including from pharmaceutical products already on the market and those soon to be on the market. The industry needed a mechanism to quickly assess the potential safety risk of this class of molecules, particularly when limited information is available. This simplified assessment provides an efficient, interim solution in the absence of more advanced health and safety risk data for each specific nitrosamine compound. It establishes a guide to set the initial assessment for any nitrosamine impurity target that drug developers are looking for in their APIs and drug products.

Why is determining acceptable intake for nitrosamines challenging?

PharmTech: Thinking about it in terms of from a client's perspective or from the analytical laboratory's perspective, what can you tell us about what makes determining acceptable intake (AI) for nitrosamines so challenging?

Feldmann (Cambrex): With the increase in regulatory scrutiny around nitrosamines, we’re seeing continued publication of more toxicology data across this class of compounds that extends into studies of novel NDSRIs but also studies that include more well-known nitrosamines, like NDMA (N-nitrosodimethylamine). Recently, the EMA [European Medicines Agency] updated AI limits for over a dozen compounds, so developers must react to these changes (3). For example, we’ve had clients request supplemental screening and further modifications to existing analytical methodologies to screen nitrosamines in their materials at sensitivity levels that previously established methods may not be suitable for.

Clients may also need to be prepared to update the specification for their materials depending on any specific impacts to their control strategy or their program. That next assessment is really looking at how to translate the intake (AI) into an appropriate specification on the drug product itself (or the API, excipient raw materials, etc.) So, for example, how many dosage units is the patient going to be taking in a day? Based on this information, we can develop a testing plan like we do for any other drug product impurity, which is often based on the product label claim. Clients may want us to determine a specification limit based on the weight of the dosage unit as an alternative. The specification is typically defined by the data that’s available and the client’s preference.

We’ve also been asked if the route of administration impacts limits. While the specification limit units are indeed likely to change with the dosing formulation (i.e., ng/mL, parts per million [ppm], etc.), the acceptable intake limit for nitrosamine targets, at least for now, is still the same if you're delivering 1 gram of material orally or 1 gram of material topically, or intravenously for that matter. The dosage form doesn't impact the limit at the moment, with a caveat. Because we do see the dosage form matter in other genotoxic impurity analyses, where one needs to evaluate how much the patient is expected to consume over the lifetime usage of the drug rather than just the maximum daily intake in the case of nitrosamine guidance. This is because for the time being, nitrosamines are not regulated by ICH [International Council for Harmonisation] M7 as is the case for most other potentially genotoxic/mutagenic impurities.

How can analytics be used to limit nitrosamine risk?

PharmTech: What are some analytical approaches manufacturers can take to limit the risk of nitrosamines in drug products?

Sprouse (Cambrex): Manufacturers (and developers) need to have a quantitative analytical approach as part of their risk mitigation strategy as they prepare for commercialization. NDA [new drug application], ANDA [annotated new drug application], and BLA [biologic license application] applications must be supplied with confirmatory testing data as part of their application to support the initial risk assessment for nitrosamine formation in their process filed in the clinical phase of development. It’s important to consider [that] current guidance from FDA and EMA requires that methods used must be demonstrated as quantitative in design as opposed to more simplified ‘limit test’-based approaches, despite acceptance of confirmatory testing results from ‘screening’ data. Therefore, the analytical methodology used to report the data must be proven scientifically sound for this intended use. The challenge manufacturers face is finding an analytical laboratory that has suitable instrumentation even capable of quantitating down to sensitivities at the sub-part per million level (i.e., part per billion [ppb]), but also must have the necessary experience to deliver quality results for the applicable sample. Further complicating this challenge, may be the additional requirement of establishing method suitability in a drug product matrix replete with complex components like high-molecular weight polymers, preservatives, and dyes, all in a CGMP [current good manufacturing practice]-regulated environment that meets FDA standards for commercial readiness.

Indeed, our team has been supporting clients with methods demonstrated scientifically sound and providing them with non-GMP/R&D screening data of their registration batches as part of their nitrosamine risk assessment for submissions to regulatory agencies. From there, the decision is based on the results in that assessment as to how much additional analytical work may need to be performed in terms of requiring validation of the analytical method(s) for downstream CGMP testing of their materials to satisfy requirements from regulatory authorities.

For example, if we're reporting results for these batches that show nitrosamine levels at or below 10% of the anticipated specification limit, then CGMP testing may not be required by the agencies. However, if reportable results are observed above that 10% threshold, then CGMP testing is very likely to be required. Regulatory guidance may determine this can be accomplished by ‘skip-lot’ testing or alternatively, more thorough testing of every lot on stability including at every timepoint. We’ve received client feedback that it's quite helpful for them to understand this part of the guidance, where planning ahead to perform historical lot testing as part of analytical method development phase of the risk assessment, can potentially save them a significant amount of time and headache, but also financially, rather than assume CGMP testing is required for all their products going to market before the risk assessment is completed.

Of course, if any products are known to have reportable nitrosamine content, then we can provide assistance where we trace backwards through the drug product and API chemical processes to identify a root cause: what the specific nitrosamine(s) is, when does it form, and at what stage is it becoming enriched? Sources of residual nitrite, the reactive contaminant and predominant source of secondary amine nitrosylation, can be present anywhere throughout the entire chemical process. We take these factors into consideration, and if necessary, we’ll recommend a nitrite analysis of their formulation excipient raw materials if say for example, the impurity is exclusively found in the drug product.

How is ion chromatography used to quantitate nitrite levels?

PharmTech: What are the limitations of using ion chromatography (IC) to quantitate nitrite levels?

Feldmann (Cambrex): In the case of nitrite analytics, we have a lot of first-hand, direct experience with this analyte, not only by IC but other methods as well.

IC is known to be very sensitive for ions, where selectivity becomes particularly important for developing the analytical method because ions are quite ubiquitous. Often, they’re present in the background at levels too prohibitive for trace level IC quantitation with conductivity detection because that background noise adds up into system interference. Excipient raw materials are frequently high in ionic content and this of course often carries over into the finished drug product formulation. Another significant challenge for an IC-based approach is that many of the most common excipients used in solid oral dosage formulations are either poorly soluble or totally insoluble in water. That's really the biggest hindrance there in our experience since IC methods rely heavily on aqueous diluents.

So, if the goal is getting a suitable method at the levels that are needed, you’re potentially facing insurmountable limitations from a combination insufficient signal of the target analyte due to poorly soluble sample coupled with background noise that is too high that trace level analysis just isn’t practical or may even be impossible to adequately pass method accuracy in a validation. Furthermore, if you need to test for nitrite across multiple sample matrices, whether it’s each individual formulation excipient, the API, or the finished drug product, the method onboarding and testing costs can certainly add up and strain the contracted outsourcing budget.

To mitigate some of the challenges with IC, we adopted a headspace GC-MS [gas chromatography-mass spectrometry] approach that is designed as a platform method that minimizes the amount of sample prep changes required to analyze a wide variety of different sample matrices. With this headspace technique, it eliminates many of the negative impacts we just discussed on formulation excipients. Namely the sample solubility issue but also it reduces interference from the matrix components from the sample injection onto the instrument, a notorious problem for chromatographic analyses of drug product and complex polymers. By reacting nitrite with cyclamate to make cyclohexene in a quick and complete reaction, we avoid the matrix interference present in an IC sample preparation.

Our method uses derivatization, because nitrite isn’t volatile. We derivatize any residual nitrite in the sample into cyclohexene and quantitate nitrite indirectly from the converted product. Since we use MS detection, we can achieve trace level sensitivity with great selectivity from the single ion response of the derivatized analyte and with minimal background interference. Our clients prefer this approach because, as mentioned, it’s a platform approach that can be used across a battery of sample types like common industry excipients used in many drug products, translating into a highly economical and novel solution for this growing market demand. We can perform method evaluations instead of full developments for each sample matrix. This shortens the timeline and lowers the expense of performing nitrite testing to assess vulnerable areas for nitrosamine formation.

The method is already validated and is highly amenable to multiple client programs and different formulations. We’re providing the marketplace a competitive and powerful approach to solve a difficult analytical challenge with minimal method onboarding time to help our clients mitigate risk and achieve their regulatory goals.

References

1. FDA. FDA Statement on the FDA’s Ongoing Investigation into Valsartan and ARB Class Impurities and the Agency’s Steps to Address the Root Causes of the Safety Issues. Press Release. Jan. 25, 2019.
2. FDA. Determining Recommended Acceptable Intake Limits for N-nitrosamine Impurities in Pharmaceuticals: Development and Application of the Carcinogenic Potency Categorization Approach. Spotlight on CDER Science. FDA.gov. Jan. 10, 2025.
3. EMA.Nitrosamine Impurities: Guidance for Marketing Authorisation Holders. Appendix 1: Acceptable Intakes Established for N-nitrosamines. Reference Number: EMA/245074/2025 Rev. 10. Last Updated 02Sep2025. https://www.ema.europa.eu/en/human-regulatory-overview/post-authorisation/pharmacovigilance-post-authorisation/referral-procedures-human-medicines/nitrosamine-impurities/nitrosamine-impurities-guidance-marketing-authorisation-holders#related-documents-69

About the interviewees

Todd Sprouse is associate director of the Analytical Department for Cambrex at the Durham, North Carolina site. He has a master's degree in organic chemistry and has been supporting analytical development in the pharmaceutical CDMO space for drug substance and drug products for 20-plus years. He has supported developing and validating complete analytical packages for clients now for more than 12 FDA submissions. In recent years, he has been responsible for leading a specialized mass spec team primarily focused on genotoxic impurities, that has included extensive support around control of nitrosamines for Cambrex clients. Cambrex has developed dozens of methods to cover their nitrosamine risk assessments to satisfy FDA (and EMA) responses.

Erik Feldmann is principal technical advisor for Cambrex at its Durham, NC site. He has a PhD in biochemistry and previously served as a lead analytical development scientist at the site before moving over to the commercial side where he supported the proposal management team and the business development team as technical director from 2023-2025. He’s been in CDMO pharma and biotech for 12-plus years and has held senior scientist and management roles at multiple start-up companies in the local NC Research Triangle area.