Nitrosamine Impurities (NDMA, NDEA): Limits, Testing and Reference Standards

Since 2018, nitrosamine impurities have become one of the most important quality topics in the pharmaceutical industry. Starting with NDMA in valsartan, they have since been found in other medicines, leading to recalls and new testing requirements worldwide. For Indian manufacturers, nitrosamine risk assessment and testing are now a routine part of quality control.

What are nitrosamines?

Nitrosamines (N-nitrosamines) are compounds containing the N-N=O group. Many of them are classified as probable human carcinogens, which is why they are controlled as "cohort of concern" mutagenic impurities under ICH M7, at levels far below ordinary impurities, in nanograms per day.

The two best-known examples are:

• NDMA (N-Nitrosodimethylamine), CAS 62-75-9
• NDEA (N-Nitrosodiethylamine), CAS 55-18-5

Other small-molecule nitrosamines that are commonly tested include NMBA, NDIPA, NEIPA, NDBA, NMPA and N-nitrosopyrrolidine. See our full range of nitrosamine impurities.

How do nitrosamines form in medicines?

Nitrosamines form when a secondary or tertiary amine reacts with a nitrosating agent (such as nitrite) under suitable conditions, often acidic. Common root causes include:

• Use of sodium nitrite in the synthesis (for example to quench azide), in the presence of amines.
• Amine-containing solvents (such as DMF, NMP or triethylamine) and their degradation.
• Recovered solvents, reagents or catalysts contaminated with amines or nitrites.
• Nitrite traces in excipients reacting with an amine-containing API during formulation or storage.
• Packaging materials, for example nitrocellulose-based lidding foil.

NDSRIs: nitrosamines related to the drug itself

If the API itself contains a secondary or tertiary amine, a nitroso version of the drug molecule can form. These are called nitrosamine drug substance-related impurities (NDSRIs). They are specific to each drug, so they usually require custom-synthesised reference standards. Regulators now assign NDSRI limits using a carcinogenic potency categorisation approach, which groups compounds into potency categories with corresponding acceptable intakes.

Acceptable intake limits

Limits are expressed as an acceptable intake (AI) in nanograms per day, corresponding to a very small theoretical lifetime cancer risk. The internationally published values for the two most common nitrosamines are:

To convert an AI into a specification limit in ppm, divide the AI by the maximum daily dose:

When more than one nitrosamine is present, regulators expect the total to be controlled so that the overall risk stays within acceptable levels. Limits and approaches are updated regularly, so always check the current requirements of the authority you file with.

The nitrosamine control process

1. Risk assessment: review the synthesis route, raw materials, solvents, excipients, water and packaging for amine and nitrite sources.
2. Confirmatory testing: if a risk is identified, test representative batches with a validated, sensitive method.
3. Control and mitigation: change the process, suppliers or formulation if needed, and set specifications.

How nitrosamines are tested

Because the limits are so low, nitrosamine testing needs highly sensitive and selective methods:

• LC-MS/MS: the most widely used technique, suitable for a broad range of nitrosamines and NDSRIs.
• GC-MS/MS: well suited to volatile nitrosamines such as NDMA and NDEA.
• LC-HRMS: high-resolution MS that adds selectivity, for example to avoid interference from DMF in NDMA analysis.

The method's limit of quantification (LOQ) must be well below the specification limit, often in the low ppb range.

Reference standards you need for nitrosamine testing

1. Unlabelled nitrosamine standards (for example NDMA, NDEA), for identification, calibration and spiking.
2. Isotope-labelled internal standards: for example NDMA-d6, to correct for matrix effects and recovery in LC-MS/MS and GC-MS/MS.
3. NDSRI standards: custom-synthesised nitroso derivatives of the specific API.

Because nitrosamines are potent and often volatile, handle them with care: use appropriate protection, keep containers tightly closed and store them as instructed on the CoA.

Drugs where nitrosamine testing is common

Nitrosamine risk has been reported in several drug classes, including sartans (valsartan, losartan), ranitidine and metformin, as well as many APIs that contain secondary or tertiary amine groups.

Key takeaways

Need NDMA, NDEA, deuterated internal standards or a custom NDSRI? Request a quote. We deliver across India.