In 2018, the detection of N-nitrosodimethylamine (NDMA) in valsartan triggered recalls around the world and led regulators to re-examine how medicines are assessed for a class of highly potent mutagenic impurities. Since then, nitrosamines have become one of the most active areas of pharmaceutical regulation. This article summarises the current regulatory framework, how acceptable intakes are set, and how testing is typically performed. Requirements continue to evolve, so always check the latest guidance from the relevant authority.
What nitrosamines are and why they matter
Nitrosamines contain the N-nitroso group (N-N=O). They form when a secondary or, less readily, tertiary amine reacts with a nitrosating agent, most commonly nitrous acid formed from nitrite under acidic conditions. Many nitrosamines are classified as probable human carcinogens based on animal data. Because they are mutagenic and potent, they are controlled at nanogram-per-day levels, far below the thresholds applied to ordinary impurities under ICH Q3A and Q3B.
Two families of nitrosamine impurities
Small-molecule nitrosamines
These are simple nitrosamines such as NDMA, N-nitrosodiethylamine (NDEA), N-nitroso-N-methyl-4-aminobutyric acid (NMBA), N-nitrosodiisopropylamine (NDIPA), N-nitrosoethylisopropylamine (NEIPA), N-nitrosodibutylamine (NDBA) and 1-methyl-4-nitrosopiperazine (MNP). They can arise from solvents, reagents, recovered materials or contaminated raw materials, and can occur across many different products.
Nitrosamine drug substance-related impurities (NDSRIs)
NDSRIs are nitrosated forms of the drug molecule itself or of a fragment of it. They arise when the API contains a secondary or tertiary amine that can react with trace nitrite present in excipients or water. Because each NDSRI is structurally unique, toxicity data are usually unavailable, and regulators have developed a structure-based approach to set limits.
How acceptable intakes are set
Regulators express limits as an acceptable intake (AI) in nanograms per day, corresponding to a negligible theoretical excess cancer risk over a lifetime of exposure. For well-studied nitrosamines, AIs are derived from carcinogenicity data. Published AIs for some common small-molecule nitrosamines are shown below.
| Nitrosamine | Commonly cited AI (ng/day) |
|---|---|
| NDMA | 96 |
| NDEA | 26.5 |
| NMBA | 96 |
| NDIPA | 26.5 |
| NEIPA | 26.5 |
These values are those published by the FDA and EMA at the time of writing. Regulators update their lists, so confirm current figures before use.
To convert an AI into a concentration limit, divide by the maximum daily dose of the drug. For a medicine with a maximum daily dose of 500 mg and an AI of 96 ng/day, the limit is 96 ng ÷ 500 mg = 0.192 µg/g, or 0.192 ppm, relative to the drug substance.
The Carcinogenic Potency Categorization Approach
For NDSRIs without robust data, both the FDA and EMA use a Carcinogenic Potency Categorization Approach (CPCA). The structure is scored for features that increase potency, such as the number of alpha-hydrogens on the carbons adjacent to the N-nitroso group, and features that decrease it, such as electron-withdrawing groups or steric hindrance. The score places the nitrosamine in one of five potency categories, each with a default AI, ranging from 26.5 ng/day for the most potent category to 1,500 ng/day for the least potent. Where adequate data exist, such as a negative enhanced Ames test or read-across to a well-characterised analogue, a different AI may be justified.
Less-than-lifetime exposure and multiple nitrosamines
For medicines taken for limited periods, regulators may allow higher limits based on less-than-lifetime principles similar to those in ICH M7. Where more than one nitrosamine is present, the total risk should not exceed that of a single nitrosamine at its AI, and authorities describe approaches for applying this. Details differ between regions.
The three-step regulatory expectation
- Risk assessment. Manufacturers evaluate APIs and finished products for potential nitrosamine formation or contamination, considering the synthetic route, reagents, solvents, recovered materials, excipients, water, packaging and storage.
- Confirmatory testing. Where a risk is identified, products are tested using sufficiently sensitive, validated methods.
- Mitigation and reporting. If nitrosamines are found above acceptable levels, manufacturers change the process or formulation and notify the regulator through the appropriate variation or supplement.
The FDA and EMA have set deadlines for completing these steps, and the FDA's guidance on NDSRIs asked manufacturers to meet recommended AIs by August 2025. Timelines and procedural details continue to be updated.
Common root causes
- Use of sodium nitrite, for example to quench azide, in a step where amines are present
- Amide solvents such as DMF, DMAc or NMP, which can degrade to secondary amines
- Recovered solvents, reagents or catalysts contaminated in shared equipment
- Nitrite in excipients reacting with amine-containing APIs during wet granulation or storage
- Nitrocellulose-containing packaging materials in some blister lidding foils
Analytical testing
Because limits are low, nitrosamine testing relies on highly sensitive and selective techniques.
- LC-MS/MS, typically with triple quadrupole instruments using multiple-reaction monitoring, is the workhorse for both small nitrosamines and NDSRIs.
- LC-HRMS offers high selectivity and is useful for distinguishing nitrosamines from isobaric interferences, such as the DMF-related interference in NDMA analysis.
- GC-MS/MS, with headspace or direct injection, suits volatile nitrosamines, although heat can create artefacts for some matrices.
Methods should have a limit of quantitation at or below the acceptable limit, and regulators have indicated that where testing is used for control, the LOQ should typically be well below the limit, often cited as 10% or 30% of it depending on the purpose. Isotopically labelled internal standards, such as NDMA-d6, are used to correct for matrix effects and recovery.
Reference standards for nitrosamine work
Testing requires characterised reference standards of the specific nitrosamines of interest, including NDSRIs that may not be commercially available. These are often prepared by custom synthesis and should be supplied with identity confirmation by NMR and mass spectrometry, HPLC purity and appropriate handling information, since many are potent carcinogens. Labelled analogues are needed as internal standards.
Summary
Nitrosamine control combines chemistry-led risk assessment with ultra-trace analytics. The limits are demanding, the rules continue to evolve, and success depends on understanding where amines and nitrosating agents meet in a process or product, then proving control with sensitive, validated methods and well-characterised standards.
FAQs
What is an NDSRI?
A nitrosamine drug substance-related impurity: a nitrosated form of the drug molecule or a fragment of it, usually formed when an amine-containing API reacts with nitrite.
How is a nitrosamine limit in ppm calculated?
Divide the acceptable intake in nanograms per day by the maximum daily dose in milligrams. The result is in µg/g, which is equivalent to ppm.
Which technique is used to test for nitrosamines?
LC-MS/MS is most common, with LC-HRMS and GC-MS/MS used where appropriate. Methods need LOQs below the acceptable limit and usually labelled internal standards.