The term nitrosamine describes a class of compounds having the chemical structure of a nitroso group bonded to an amine. The compounds can form by a nitrosating reaction between secondary, tertiary, or quaternary amines and nitrous acid in acidic conditions. Nitrosamine compounds are potent genotoxic agents in several animal species, and some are classified as probable or possible human carcinogens by the International Agency for Research on Cancer (IARC). Almost all regulatory bodies have established deadlines for confirmatory testing and mitigation plan submission for nitrosamine control. Regulatory audits have revealed real GMP gaps in nitrosamine control for API and drug products, primarily due to manufacturers failing to implement a dynamic, lifelong process to control nitrosamine levels in both the API and finished products.

How Nitrosamines Form

Formation of nitrosamine is possible in the presence of secondary, tertiary, or quaternary amines and nitrite salts under acidic conditions. Under these conditions, nitrite salts may form nitrous acid, which can react with an amine to form a nitrosamine. There is a greater risk of nitrosamine formation if nitrous acid is used to quench residual azide, or if the azide functional group is introduced into the molecule in the presence of precursor amines.

Amines may be present in the manufacturing process for a variety of reasons. The API intermediate or API raw materials may contain secondary or tertiary amine functional groups. Tertiary or quaternary amines may also be added intentionally as a reagent or catalyst. All of these types of amines can react with nitrous acid or other nitrosating agents and may form nitrosamines in the API.

In finished products, nitrites are common nitrosating impurities that have been reported in many excipients at PPM levels. Nitrite impurities are found in commonly used excipients, which may lead to nitrosamine impurities forming in the drug product during manufacturing and may increase over the shelf life.

Real GMP Gaps

Under GMP, inspectors expect companies to manage high-risk impurity concerns through clear risk assessments, justified controls, and traceable decisions. They focus less on document volume and more on whether control strategies clearly link to identified risks.

Recurring Audit Findings

Regulatory audits have identified several recurring mistakes in nitrosamine risk assessments for APIs and drug products.

• Failure to identify the general conditions that lead to nitrosamine formation, indicating gaps in process understanding.
• Insufficient verification of sources of secondary, tertiary, and quaternary amines that can form nitrosamines during synthesis and formulation.
• Over-reliance on excipient vendor Certificates of Analysis (CoAs) without independent assessment, which can lead to variable nitrosamine impurity levels batch by batch.
• Risk of nitrosamine formation when a quenching step is conducted directly in the main reaction mixture, indicating a lack of process optimisation and control.
• Recovered solvents not being analysed for nitrosamine impurities.
• Risk documented and conclusions drawn without adequate rationale.

Three-Stage Risk Assessment Policy

To address these gaps, API and drug product manufacturers should adopt a three-stage risk assessment policy:

1. Assess the risk of nitrosamine impurities in APIs, marketed products, and products under approved and pending applications. Risk assessments should be conducted in a timely manner and with priority.
2. Perform confirmatory testing when there is any risk for the presence of nitrosamine impurities.
3. Report changes implemented to prevent or reduce nitrosamine impurities in APIs and drug products to the FDA.

Common Mistakes in Risk Assessments

Improper Supply Chain Management

Most organisations fail to identify and analyse the secondary and tertiary amines present in reagents, catalysts, and solvents as impurities. Although these are not part of the API directly, they can play a part in the formation of nitrosamines in the API. It is therefore risky to overlook secondary amines or nitrites.

Proper Identification of Nitrite Levels in Excipients

There is a risk of varying levels of nitrite in the excipients used for formulation. Companies tend to rely on the CoA provided by the excipient manufacturer for nitrite levels and often fail to identify gaps. These varying levels can act as nitrosating agents and have a significant impact on formulation, which may be observed during shelf life. Over-reliance on vendor CoAs therefore represents a potential threat of nitrosamine formation.

Method-Related and Technical Errors

Analytical methodologies often ignore the risk of nitrosamine formation during sample preparation. Over-sonication or highly acidic diluents can trigger false-positive formation of nitrosamine impurities. Extreme care should be taken during the selection of analytical methods.

Overestimating the synthetic or manufacturing process and its ability to remove nitrosamines without analytical confirmation is a common mistake that is often made, which may also contribute to nitrosamine impurity failures. It is critical to develop and validate analytical methods with high sensitivity and a low limit of detection (LOD). If the LOD is too high, nitrosamine results may be reported as ‘Not Detected’ even when present above the LOD or above acceptable intake (AI) limits.

Gaps in Assessment: Cross-Contamination and Packaging Material

Most organisations perform a one-time risk assessment as a formality for documentation purposes and never revisit it, even when there are changes in the synthetic process, changes in supplier for different raw materials, changes in manufacturing equipment, or the introduction of specialised packaging materials. Each such change should be evaluated, and a revised risk assessment and mitigation plan should be prepared accordingly. This operational gap can become a serious compliance concern if not addressed on time and properly.

Impact on Regulatory Compliance

The above failures in risk assessment may lead to failures in regulatory compliance, as many organisations still fail to properly report risk assessments in the Chemistry, Manufacturing and Controls (CMC) sections of their regulatory submissions.

Proactive Strategies for QC Labs

In the challenging pharmaceutical environment, nitrosamine control must be managed by QC laboratories in a proactive manner. The focus should shift towards in-depth testing using modern analytical techniques, rather than confirmatory tests alone.

Analytical Capabilities

• QC labs are increasingly shifting to highly sensitive LC-MS/MS platforms, which are required to detect nitrosamine impurities at very low levels, given that nitrosamines have very low AI limits.
• Labs should proactively attempt to identify unknown nitrosamines during reaction monitoring, not only the previously identified compounds.
• Sample preparation techniques should be developed in a way that minimises the probability of in situ nitrosamine formation.

Stability Testing and Routine Monitoring

QC labs should not rely solely on a supplier’s Certificate of Analysis (CoA). Independent testing should be performed on raw materials, reagents, and recovered solvents.

Nitrosamine testing during stability studies is an integral part of the stability protocol and should not be limited to batch release only, as there is always a risk of nitrosamine formation and increase during shelf life.

Routine nitrosamine monitoring may be waived if levels are below 10% of the AI limit. However, if results are above 30% of the AI limit, batch-by-batch testing becomes mandatory.

FAQ:

Reference:

• Center. “Control of Nitrosamine Impurities in Human Drugs.” U.S. Food and Drug Administration, 2024, https://www.fda.gov/regulatory-information/search-fda-guidance-documents/control-nitrosamine-impurities-human-drugs.
• “Nitrosamine Impurities | European Medicines Agency (EMA).” European Medicines Agency (EMA), 28 Oct. 2019, https://www.ema.europa.eu/en/human-regulatory-overview/post-authorisation/referral-procedures-human-medicines/nitrosamine-impurities.
• “ICH M7 Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk – Scientific Guideline | European Medicines Agency (EMA).” European Medicines Agency (EMA), 24 Sept. 2014, https://www.ema.europa.eu/en/ich-m7-assessment-control-dna-reactive-mutagenic-impurities-pharmaceuticals-limit-potential-carcinogenic-risk-scientific-guideline.