Nitrosamine Impurities in Pharmaceuticals: How to Control and Calculate Safe Limits – Learn With FAQs

Nitrosamine Impurities or Nitrosamines are molecules containing a nitroso functional group (N–N=O) that are considered probable human carcinogens. They can be found in certain medications, foods, and water, and may form when a “vulnerable amine” reacts with a nitrosating agent—either during drug manufacturing or storage.

R2N-N=O

Nitrosamine impurities in pharmaceuticals have emerged as a significant challenge due to their toxicity and stringent acceptance limits. Unlike other impurities, nitrosamines cannot be effectively detected using common analytical techniques like chromatography and spectroscopy. These impurities have no industrial use, yet their presence in pharmaceutical products can lead to severe safety concerns. In this article, I will share my expertise on nitrosamine impurities, covering essential topics such as the types of nitrosamines, their toxicity, the formation of nitrosamines during manufacturing, and effective control strategies. Additionally, I will discuss real-world case studies and provide answers to frequently asked questions, offering valuable insights for professionals looking to better understand and manage these critical impurities in drug production.

Related Articles:

1. Relative Response Factor (RRF) in Pharmaceutical Analysis
2. How To Control Impurities In Pharmaceuticals: Get Mastery In 11 Minutes
3. How To Calculate Potency, Purity and Assay In Pharmaceuticals

10. Why are they present?

Nitrosamines appear in pharmaceuticals for several reasons:

• During the synthesis of APIs or intermediates, or during formulation or packaging, amines (secondary, tertiary) may be present, as well as nitrosating agents (nitrites, nitrous acid, nitrosyl halides, nitrogen oxides). Under appropriate conditions (acidic pH, heat), the nitrosation reaction can occur.
• Starting materials, reagents, solvents, catalysts, or even packaging/printing materials may carry residual amines or nitrites, or may be reused/recycled and contaminated. For example, the use of recycled dimethylformamide (DMF), which had dimethylamine (DMA) impurity, plus sodium nitrite quench, contributed to the formation of NDMA in some APIs.
• Packaging materials (such as lidding foil containing nitrocellulose) may contribute nitrosating conditions or amine/nitrite residuals during storage of finished products.
• Drug substances themselves, or excipients, may degrade or react during storage to produce reactive intermediates (e.g., nitrosating species), which then convert amines to nitrosamines.
  Thus, nitrosamines are present because of unintended chemical reactions, cross-contamination, inadequate purge/cleaning, or unrecognised reactive combinations of amines + nitrites + appropriate conditions.

11. How do Nitrosamines form in the process?

Nitrosamine Impurities Formation in the Process

Nitrosamine Impurities may form in the process due to the following reasons: in the Process

• Use of Nitrate, Nitrites and certain amines in the process
• Presence of DMA (dimethyl amine) in DMF (dimethyl formamide (if DMF is the component of the synthetic process)
• General conditions that lead to nitrosamine formation
• Sources of secondary, tertiary, and quaternary amines that can form nitrosamines
• Contamination in vendor-sourced raw materials
• Recovered solvents, catalysts, and reagents as sources of contamination
• Quenching process as a source of Nitrosamine contamination
• Lack of process optimisation and control
• If nitrous acid is used to quench residual azide during tetrazole ring formation
• Introduction of the azide functional group into a molecule
• Presence of Amines (maybe API, degradants, intermediate and purchasing material)
• Presence of Amide solvents: e.g. N,N-dimethylformamide, N-methyl pyrrolidone, N,N-dimethylacetamide, and N,N-dimethylacetamide

How do nitrites turn into nitrosamines?

Nitrosamine Impurities form by following the reaction mechanism:

Expert Tips:

In manufacturing control terms, whenever you have [amine + nitrosating agent + favourable reaction conditions (acidic pH, heat, time)] you have a risk of nitrosamine formation.

12. What is Nitrosamines’ impact?

• Many nitrosamines are genotoxic (damage DNA) and carcinogenic in animal studies. Therefore their presence in pharmaceuticals is of high regulatory concern.
• Their impact in the context of medicine: if patients are exposed repeatedly (especially long‐term) to nitrosamines above safe levels, there is a theoretical increased risk of cancer.
• For exposures below the acceptable intake limits defined by regulators, the additional cancer risk is considered negligible (often described as a lifetime exposure scenario). For example, the US Food and Drug Administration states that a person taking a drug containing nitrosamines at or below the acceptable intake every day for 70 years is not expected to have an increased cancer risk.
• Practical impact: Many drug batches/products have been recalled because nitrosamine levels were above acceptable limits; manufacturing processes needed to be revised; supply disruptions; regulatory scrutiny increased.

13. What is being done for Nitrosamines?

Regulatory, manufacturing and analytical actions include:

• Regulatory agencies (FDA, European Medicines Agency [EMA], Therapeutic Goods Administration [TGA], World Health Organisation [WHO]) have issued guidance documents requiring risk assessments of all drug products (APIs, excipients, finished products) for nitrosamine formation risk.
• They have defined “acceptable intake” (AI) limits for known nitrosamine impurities (in ng/day) and specified conversion to ppm limits with respect to max daily dose.
• They have required manufacturers to test batches (both API and finished product) for nitrosamine impurities using validated sensitive analytical methods.
• Manufacturers are required to identify root causes (e.g., process steps, raw materials, recycled solvents, packaging), implement mitigation (change process, purge steps, alternative reagents, remove nitrite/amine sources), and establish a control strategy.
• Many recalls have been implemented for affected products (e.g., certain ARBs, ranitidine, metformin). Regulators continue to monitor and update guidance.
• Industry organisations (e.g., United States Pharmacopoeia [USP]) have developed reference standards for nitrosamines, published general chapters, and tools for detecting and controlling nitrosamines.
• Ongoing research: improved analytical methods, better predictive/supply-chain understanding, addressing emerging nitrosamine drug-substance–related impurities (NDSRIs).

14. What are the 8 nitrosamine impurities?

Different sources list slightly different sets, but common “small-molecule nitrosamines” flagged in pharmaceuticals include (from FDA/EMA/WHO sources):

• N-nitrosodimethylamine (NDMA)
• N-nitrosodiethylamine (NDEA)
• N-nitroso-N-methyl-4-aminobutyric acid (NMBA)
• N-nitrosodiisopropylamine (NDIPA)
• N-nitrosoisopropylethylamine (NEIPA)
• N-nitrosomethylphenylamine (NMPA)
• N-nitrosodibutylamine (NDBA)
• N-nitroso-varenicline (NNV) (or other nitrosamine drug‐substance–related impurities [NDSRIs])

Expert Tips:

Some documents say “seven” nitrosamines, but more are emerging. For pharmaceuticals, you might see “eight” flagged for a specific drug e.g., metformin: NDMA, NDEA, NEIPA, NDIPA, NMPA, NDBA, NMBA, etc.

15. What are the symptoms of nitrosamines?

From a pharmaceutical regulatory perspective, nitrosamines do not cause immediate acute symptoms when present at trace levels in medicines. Their hazard is primarily long‐term (carcinogenic risk). So there are no specific “symptoms of nitrosamine exposure” in the context of drug impurities documented in patients.
However, in general toxicology of nitrosamines (in larger exposures), they can cause liver damage, kidney damage, respiratory issues, gastrointestinal effects and ultimately tumour formation in multiple organs (in animals).
In pharmaceuticals, If a product is found to contain nitrosamine above acceptable limits, the concern is cancer risk over time rather than an acute “symptom”. Patients are usually advised not to discontinue essential medications without consulting their physician, because risk of underlying disease may outweigh the nitrosamine risk.

16. What products are recalled due to nitrosamine impurities?

Examples include:

• Certain batches of angiotensin II receptor blockers (ARBs) such as Valsartan, Losartan, and Irbesartan because of NDMA / NDEA.
• Products containing Ranitidine (commonly marketed as Zantac) – due to NDMA formation on storage/degradation.
• Some formulations of Metformin (an antidiabetic drug) are due to nitrosamine impurities.
• Other drugs have also been flagged for nitrosamine drug substance-related impurities (NDSRIs). For instance, some antidepressants, etc. (though less widely publicised).
  Manufacturers, in collaboration with regulators, issued voluntary recalls or warnings when nitrosamine levels exceeded acceptable intake limits.

17. What are the sources of Nitrosamines?

Sources can be grouped under manufacturing, formulation/storage, supply chain or packaging. Some of the common sources:

A. Manufacturing / API synthesis sources

• Use of sodium nitrite (NaNO₂) or nitrous acid in synthesis (e.g., tetrazole formation in ARBs) combined with amine‐containing solvents or reagents → nitrosamine formation.
• Residual amine impurities in solvents (e.g., dimethylamine in DMF) or catalysts, which then become nitrosated.
• Recycled or recovered solvents/catalysts or shared equipment that have cross‐contamination of nitrosamines or nitrite/amine species.
• Poor control of reaction steps, pH, temperature, purification/purge, which allows side reactions to form nitrosating species and amines.

B. Finished product / Formulation / Storage sources

• Excipients that contain or form nitrite or amine functionality; residual nitrites from excipient manufacture.
• Packaging materials: e.g., lidding foil, printing ink or nitrocellulose foil that may release reactive nitrogen oxides/nitrites.
• Drug product degradation during storage: a drug or excipient may degrade to form amine or nitrosating agent, leading to nitrosamine formation over time.

C. Cross‐contamination / supply-chain sources

• Multi‐product manufacturing facilities where equipment or lines are shared and residual nitrites/amines carryover occurs.
• Outsourced or third‐party solvent recovery operations without strict purge/clean controls → recycled solvent carries nitrosamine risk.

D. Food, drinking water and environmental sources (though less relevant for immediate pharma control)

• Nitrosamines are found in certain foods (cured meats, smoked fish), in water supplies (nitrites in water) and in tobacco smoke. Regulatory agencies note everyone is exposed to some baseline nitrosamines from diet/environment.

Expert Tips:

the presence of amines + nitrosating agents + suitable reaction conditions (acid, heat, time) + possible cross‐contamination/purging issues = sources of nitrosamines.

18. How are the limits of Nitrosamines calculated?

The regulatory calculation approach typically involves the following steps:

1. Determine the Acceptable Intake (AI) for the specific nitrosamine impurity (in ng/day) based on toxicology (carcinogenic potency) if available, or default “cohort of concern” approach (e.g., ICH M7(R1)) or Carcinogenic Potency Categorisation Approach (CPCA).
   For example, NDMA has AI ~96 ng/day; NDEA ~26.5 ng/day.
2. Identify the Maximum Daily Dose (MDD) of the drug product (in mg/day) – e.g., a patient takes X mg per day.
3. Convert AI (ng/day) to a parts per million (ppm) level allowable in the drug product using the formula (since 1 mg = 1 000 µg = 1 000,000 ng, the conversion factor is 1000 in some contexts)

1. Set Specification for the drug batch: Ensure that the measured level of nitrosamine in the product (or API) is ≤ the calculated ppm (or ng/unit), giving ≤ AI/ng‐day exposure at the MDD.
2. For multiple nitrosamines present, you use a “sum of ratios” or additive risk approach (see next section).
3. If you have compound‐specific toxicology data (e.g., TD₅₀, rodent carcinogenicity) you may derive a more refined AI rather than the default.

Thus, the limits are calculated based on toxicology (AI), daily dose of drug, and conversion to a limit in the product (ppm or ng per unit).

19. What are the different analytical techniques available for testing the presence of nitrosamines?

The following techniques are widely used for testing the presence of nitrosamines :

• GC-MS (Gas Chromatography–Mass Spectrometry): Frequently used for volatile/smaller nitrosamines (e.g., NDMA, NDEA) via headspace or direct injection.
• LC–MS/MS (Liquid Chromatography–Tandem Mass Spectrometry): Especially for less volatile, more polar nitrosamines or NDSRIs; allows high sensitivity and selectivity.
• LC-HRMS (Liquid Chromatography–High Resolution Mass Spectrometry): Provides high resolution, accurate mass detection, useful for confirmatory testing and for novel nitrosamine impurities.
• HPLC with UV or Fluorescence Detection (less common for nitrosamines because sensitivity may not always suffice, but some studies use derivatisation plus fluorescence detection).
• TLC
• HPTLC

Therefore, manufacturers and control labs typically implement advanced GC–MS or LC–MS methods, establish DL (detection limit) sufficiently below the specification, ensure sample preparation avoids artifact generation of nitrosamines, and monitor for the relevant nitrosamine impurities.

20. What are the available guidelines on the control of Nitrosamine impurities in pharmaceutical products?

The following are the major guidelines:

• US Food and Drug Administration (FDA) – Guidance for Industry: Control of Nitrosamine Impurities in Human Drugs (final version September 2024, Revision 2), which describes root causes, risk assessment, testing/mitigation strategies.
• FDA – Recommended Acceptable Intake Limits for Nitrosamine Drug Substance-Related Impurities (NDSRIs) (August 2023) – framework for AI limits.
• Therapeutic Goods Administration (TGA, Australia) – Guidance on nitrosamine impurities in medicines: acceptable intake, risk assessment etc.
• World Health Organization (WHO) – Good Practice Considerations for the Prevention and Control of Nitrosamines in Pharmaceutical Products (Annex 2 of WHO TRS No. 1060)
• European Medicines Agency (EMA) – Various assessment/review documents and Q&A for marketing authorisation holders regarding nitrosamine impurities.
• Pharmacopeia standards (e.g., USP <1469> “Nitrosamine Impurities”) – reference standards, method development.

These guidelines generally cover: risk assessment of all APIs/excipients/product for nitrosamine risk, identification of potential nitrosamine impurities (small-molecule and NDSRIs), acceptable intake limits, requirement for testing and validation of methods, specification setting, process/packaging mitigation, documentation and regulatory submission/changes.

21 What are Common sources of nitrosamine impurities?

Common sources

• Amines/amine-containing reagents, solvents or catalysts (e.g., dimethylamine from DMF)
• Nitrites/nitrosating agents used or generated in process (e.g., sodium nitrite quenching, nitrous acid from NOx)
• Recycled solvents or catalysts that carry over nitrite/amine or nitrosamine residues
• Shared equipment or multi-purpose manufacturing lines where cross‐contamination occurs
• Excipients or packaging materials that contain nitrite/amine or which generate reactive nitrosating species (e.g., nitrocellulose foil)
• Storage and stability issues: drug/excipient degradation generating amine/nitrosating species during shelf life
• Incomplete purge steps or inadequate cleaning between batches/processes

These sources are often identified in root-cause analysis of nitrosamine findings.

22. What are the examples of Nitrosamine impurities?

Examples of specific nitrosamines found or flagged in pharmaceuticals include:

• N-nitrosodimethylamine (NDMA)
• N-nitrosodiethylamine (NDEA)
• N-nitroso-N-methyl-4-aminobutyric acid (NMBA)
• N-nitrosodiisopropylamine (NDIPA)
• N-nitrosoisopropylethylamine (NEIPA)
• N-nitrosomethylphenylamine (NMPA)
• N-nitrosodibutylamine (NDBA)
• N-nitroso-varenicline (NNV) (an NDSRI)

These provide concrete instances of nitrosamine impurities which have been detected or are of regulatory interest.

23. What is the Nitrosamine impurities limit calculation procedure?

The following is the calculation procedure for the Nitrosamine impurities limit:

• For each nitrosamine impurity, identify the acceptable intake (AI, ng/day) as per guidance or toxicity data.
• Identify the maximum daily dose (MDD) of the medicinal product (mg/day).
• Use the formula:

• Express result as ppm (parts per million) in the drug substance or product:
  ppm = {AI (ng/day)/MDD (mg/day)} x1000
• Set specification for the product (or API) such that measured nitrosamine ≤ calculated limit (or stricter if appropriate) throughout shelf-life.
• If multiple nitrosamines are present, use a combined risk (sum of ratios) approach:

• Ensure the sum of ratios remains ≤ 1.
• Document and justify any deviation, apply mitigation/controls, monitor shelf-life trends, and submit to regulatory authorities as required under guidance.

24. What is the Formula for a single nitrosamine impurity calculation? Also, give one example

Formula:

Example: Suppose regulatory AI for NDMA = 96 ng/day . Let’s say the MDD of a drug is 320 mg/day.
Then:
Limit (ppm) = 96/320 = 0.3ppm

Thus the API or finished product must contain ≤ 0.3 ppm NDMA (when dosed at 320 mg/day) to stay within AI

Alternatively, if each tablet is 160 mg and taken 2 tablets/day = 320 mg/day, then permitted nitrosamine per tablet = 96 ng/day ÷ 2 tablets = 48 ng/tablet.

25. What are the approaches for multiple nitrosamine impurity calculations? Explain with an example.

When more than one nitrosamine impurity is present in a product (for example NDMA and NDEA and NMBA), you cannot just consider each individually; you must consider combined exposure because each contributes to potential genotoxic/carcinogenic risk. The typical approach is the “sum of ratios” approach:

Example:

Suppose a finished product’s daily dose results in:

• NDMA = 30 ng/day (AI_NDMA = 96 ng/day)
• NDEA = 10 ng/day (AI_NDEA = 26.5 ng/day)
• NMBA = 50 ng/day (AI_NMBA = 96 ng/day)

Compute the sum of ratios:

(30/96) + (10/26.5) + (50/96) = 0.3125 + 0.3774 + 0.5208 = 1.2107

Since 1.2107 > 1, the combined exposure exceeds the acceptable combined risk threshold — in regulatory terms this would not be acceptable. Mitigation would be required (reduce levels of impurities) or justify benefit‐risk.

If instead exposures were NDMA=20 ng, NDEA=5 ng, NMBA=40 ng →

(20/96) + (5/26.5) + (40/96) = 0.2083 + 0.1887 + 0.4167 = 0.8137 < 1

Then the combined exposure is within acceptable risk envelope.

Note: Some regulators may allow different control strategies (e.g., specification per impurity plus total, time trend monitoring), but the sum of ratios is a widely accepted conservative approach.

Expert Tips:

• It is important to emphasise that nitrosamines are controlled with very low allowable levels (ng/day) because of their genotoxic/carcinogenic nature.
• Manufacturers have to take a risk-based approach: identify any plausible formation or contamination routes, test appropriately, set specifications, monitor over shelf‐life, put in place mitigation.
• For finished pharmaceuticals, controlling nitrosamines is now an integral part of quality assurance / good manufacturing practice (GMP) and regulatory compliance.
• From a patient perspective: the presence of nitrosamine impurity at or below acceptable limits does not necessarily mean immediate harm; however, exceeding limits triggers action. Regulatory agencies stress that patients should not stop essential medicines without consulting healthcare providers.

26. What is the chemistry behind the toxicity of Nitrosamine Impurities?

Nitrosamines contain the nitroso group, and molecules having this functional group fall into the cohort of concern due to their high potential carcinogenic nature. Their intake, even below the TTC would theoretically be associated with the potential for a significant carcinogenic risk. That is why they are toxic and are excluded from the TTC approach.

27. What are the common sources of Nitrosamine impurities?

There are various sources of Nitrosamine impurities, such as:

1. Processed meat
2. Can form in the stomach: If a food contains precarser of the corresponding nitrosamine
3. Alcoholic beverages, cosmetics and cigarette
4. During the disinfection of drinking water: Dichloramines are used during disinfection of the drinking water and it may result in the formation of a trace amount of nitrosamine
5. Nitrosamine may form in the synthesis of pharmaceuticals : Nitrates, Nitrites and certain amines may lead to the formation of Nitrosamine impurities

28. Can the formation of nitrosamine impurities be prevented?

No but some extent. Formation of Nitrosamines can be inhibited by Ascorbic acid. Ascorbic acid (vitamin C) is known to prevent the formation of nitrosamines.

29. How to control nitrosamine impurities in pharmaceuticals?

The following Nitrosamine Impurities control strategies should be adopted:

1. Pharmaceuticals manufacturers should optimize the design of the manufacturing process for pharmaceuticals during the route of synthesis (ROS) selection to minimize or prevent the formation of nitrosamine impurities.
2. Avoid the reaction conditions that may produce nitrosamines whenever possible; When not possible, demonstrate that the process is adequately controlled and capable of consistently reducing nitrosamine impurities through appropriate and robust fate and purification studies.
3. Use bases other than secondary, tertiary, or quaternary amines (when possible) if ROS (route of synthesis) conditions can form nitrosamines.
4. Be careful when ROS involve the use of amide solvents (for example, N,N-dimethylformamide, N,N-dimethylacetamide, and N-methylpyrrolidone)
5. Replace nitrites with other quenching agents for azide decomposition processes
6. Design a manufacturing process that facilitates purification of nitrosamine impurities in subsequent processing steps.
7. Remove the quenching steps (when there is a risk of nitrosamine formation)
8. Audit supply chains from the raw materials, starting materials, and intermediates are used in the process
9. Avoid cross-contamination when recovered materials such as solvents, reagents and catalysts are used in the manufacturing process. Recovered material should be used only at the same stage or in an earlier stage of the same process from which it was collected.
10. Be aware that potable water used in API manufacturing should be free from nitrites.
11. Pharmaceutical batches may be reprocessed or reworked to control the levels of nitrosamine impurities as provided in ICH Q7 for modifications and controls in such operations.
12. If a nitrosamine impurity is detected above the detection limit (DL), the Pharmaceutical manufacturer should develop a vision-based strategy to control the same
13. Make sure Nitrosamine level reliably remains well below the AI limit in the Pharmaceuticals
14. Any Pharmaceuticals batch found to contain levels of nitrosamine impurities above the recommended AI should not be released.
15. Apply an effective lesson-learned approach

30 What are the Analytical Challenges in Controlling Nitrosamine Impurities?

Nitroso amine Impurities can not be controlled by common analytical techniques like chromatographic techniques and spectroscopic techniques due to their low limit. LC-MS and GC-MS are widely used in the industries to control these impurities.

Case studies related to Nitrosamine Impurities

APIs prone to Nitrosamine Impurities

• For which azide or nitrite is used in synthesis such as Metronidazole, Cilostazol, Cefazolin etc.
• Sartans with a tetrazole ring such as Valsartan, irbesartan, Losartan, Olmesartan, candesartan etc

Warning letters

Several warning letters have been given in past by different authorities due to inadequate control of Nitrosamine impurities

Deficiency letters

Several Deficiency letters (DLs) have been given in past by different authorities due to failure to control of Nitrosamine impurities

Conclusion

In conclusion, controlling nitrosamine impurities in pharmaceuticals is a critical challenge that requires careful attention to manufacturing processes, rigorous testing, and adherence to regulatory standards. Given their toxicity and the difficulties in detecting them using traditional analytical methods, it’s essential for pharmaceutical companies to implement robust control strategies to minimise the risk of contamination. By understanding the types of nitrosamines, their formation pathways, and effective mitigation techniques, companies can safeguard the safety and quality of their products. The case studies and strategies discussed in this article provide valuable guidance for tackling these impurities, ensuring compliance, and ultimately protecting public health

You may also want to check out other articles on my blog, such as:

• How to identify Genotoxic Impurities in pharmaceuticals?

References

• FDA: Control of Nitrosamine Impurities in Human Drugs
• General Chapter <1469> Nitrosamine Impurities
• ICH Q7

Abbreviations

• DMA: Dimethyl amine
• DMF: Dimethyl
• TTC: Threshold for toxicological concern
• API: Active pharmaceutical ingredient