Facing Up to the New Nemesis of Pharma: Nitrosamines

What are nitrosamines and how are they formed?

Nitrosamines are a class of organic compounds that can be found in water and some foods, such as dairy products, meats and vegetables. The presence of nitrosamines is not uncommon, but they are not intentionally added to medications. However, nitrosamine impurities can form during manufacturing through a complex interplay of three main factors:

1. Presence of a vulnerable amine. A “vulnerable” amine is capable of reacting with a nitrosating agent to form a stable N-nitrosamine. Many pharmaceutical products and some excipients have these amines. Secondary, tertiary, and quaternary amines are most susceptible to nitrosation.
2. Presence of a nitrosating agent. These are chemicals that can transfer a nitro (NO2) group to the amine, forming a nitrosamine. A common culprit is nitrite (NO₂^-), which can be present as an impurity in some starting materials or excipients. Other nitrosating agents might also be introduced during the manufacturing process.
3. Favorable reaction conditions. Nitrosamine formation is more likely under acidic conditions (low pH). Additionally, higher temperatures and specific solvents or catalysts can accelerate the reaction.

When were nitrosamines found in medicines? Are they dangerous?
 

The presence of nitrosamines in some medications was first detected in 2018 – and came as a surprise. The impurities had inadvertently made their way into pharmaceutical products primarily because of manufacturing processes and raw material contamination.

Concern lies in the potential for nitrosamines to cause harm if exposure is high and sustained over a long period of time because some nitrosamines are classified as a possible or probable human carcinogen. Key risk factors to consider include the specific nitrosamine, the dose, and duration of exposure. The level of nitrosamines present in a medication is crucial. Regulatory bodies continue to establish acceptable daily intake (ADI) limits for different nitrosamines in medications, considering potential risks and exposure levels that could cause harm. Frequent exposure to nitrosamines leads to greater potential risk, so even low levels of nitrosamines can be concerning if a medication is taken for many years.

In response to the situation, regulatory agencies worldwide have created guidelines for manufacturers to monitor, quantify, and mitigate their presence in pharmaceutical products. These measures include implementing robust risk assessment strategies, conducting thorough investigations into the root causes of the impurities, enhancing analytical methods for detection, and implementing corrective actions to prevent future occurrences.

As more information becomes available about nitrosamines and their ADI limits, manufacturers will be able to implement more effective controls and improved testing methods.

What types of medicines have been found to contain unsafe levels of nitrosamine impurities?
 

Given the right conditions, a considerable proportion of active ingredients and finished drug products could potentially be susceptible to the formation of nitrosamine impurities. They have been detected at levels considered potentially unsafe and triggered recalls in the following drug products, according to the FDA:

• Valsartan, losartan and irbesartan, angiotensin II receptor blockers used to treat high blood pressure and heart failure.
• Ranitidine and nizatidine, histamine-2 (H2) blockers used to treat heartburn and other gastrointestinal conditions.
• Certain metformin formulations, used to control high blood sugar in patients with type 2 diabetes.
• Rifampin and rifapentine, antibacterial drugs used to treat tuberculosis and other infections.

How are regulatory frameworks changing?
 

Regulatory frameworks will continue to change over time. The discovery of a new class of nitrosamines called nitroso drug substance-related impurities (NDSRIs), for example, introduced a new set of challenges. NDSRIs are nitrosamines that can potentially form directly on the active ingredient molecule structure. 

Suggested ADI limits are provided based on the predicted carcinogenic potency of the nitrosamine and may not be consistent across all regulatory bodies. Health agencies from around the world are periodically updating their frameworks, so it can be challenging to keep up with these changes. To facilitate continuous conversation, USP has created the Nitrosamines Exchange, a virtual knowledge-based community for industry members to share information and best practices to carry out effective nitrosamines risk assessments. This includes a dashboard where – as a community – members can contribute and help keep information on ADI limits from a variety of agencies up to date. 

What are the biggest challenges in detecting nitrosamines? 
 

Some of the most significant challenges include:

• Low concentrations. Nitrosamine impurities are typically present at very low concentrations, often in the parts per billion (ppb) range. Detecting trace levels requires analytical methods with exceptional sensitivity and precision.
• Complexity of matrix. Pharmaceutical products are complex matrices containing various excipients, degradation products, and other impurities. Detection can be complicated by interference from matrix components, which may hinder accurate quantification.
• Diversity of nitrosamines. Nitrosamines encompass a wide range of chemical structures, making it challenging to develop universal analytical methods capable of detecting all nitrosamine impurities; some may require unique approaches for detection and quantification, especially for NDSRIs.
• Matrix effects. Matrix effects, including ion suppression or enhancement, can impact the performance of analytical instruments, particularly in techniques such as mass spectrometry. Careful method validation is essential for mitigating these effects.
• Analytical sensitivity and selectivity. Achieving the necessary sensitivity and selectivity for nitrosamine detection requires advanced instrumentation and analytical techniques. High-performance liquid chromatography coupled with tandem mass spectrometry, or high-resolution mass spectrometry, is commonly employed for nitrosamine analysis because of the superior sensitivity and selectivity of these techniques.
• Reference materials availability. Access to high-quality reference materials is essential for accurately quantifying nitrosamine impurities. However, obtaining reference materials of reliable quality for all relevant nitrosamines can be challenging. USP continues to invest resources to prioritize the availability of trustworthy reference materials that can be reliably used for analytical research and testing of NDSRIs and other nitrosamines.

The most critical evolution in nitrosamine testing over the past few years has been the transition from reactive to proactive approaches. Initially, testing for nitrosamine impurities was often triggered by specific incidents or regulatory alerts. However, as awareness of the potential risks associated with nitrosamines has increased, along with identification of these impurities in various pharmaceutical products, there has been a shift towards proactive testing strategies. 

What else can drug companies do to mitigate the risks?
 

Manufacturers have implemented a range of critical changes to mitigate the risks, including: 

• Enhanced risk assessments. Pharmaceutical manufacturers continue to develop comprehensive risk assessments to evaluate potential for nitrosamine formations across product portfolios, considering factors such as chemical structure, manufacturing processes, raw materials, and historical data to identify products at higher risk of nitrosamine formation. 
• Process optimization. The industry is optimizing manufacturing processes to minimize the risk associated with the formation of nitrosamines and mitigate potential sources of contamination to improve product quality and safety. This includes modifying reaction conditions, controlling process parameters, and selecting alternative synthetic routes or reagents that reduce the likelihood of nitrosamine impurities.
• Advanced analytical testing. There has been significant investment in advanced analytical testing capabilities for nitrosamine detection. State-of-the-art instrumentation can improve sensitivity, selectivity, and accuracy when analyzing nitrosamines, enabling detection of nitrosamine impurities at lower levels.
• Increased testing frequency. Manufacturers are implementing more frequent testing protocols for nitrosamine impurities throughout the drug development and manufacturing processes to ensure compliance with regulatory requirements and quality standards.
• Stronger supplier qualification and oversight. Strengthening supplier qualification and oversight processes helps ensure the integrity of raw materials and components used in drug manufacturing. 
• Regulatory compliance and transparency. Members of industry are proactively engaging with agencies to ensure compliance with evolving regulatory requirements. This includes adhering to regulatory guidelines, submitting comprehensive data and documentation and participating in regulatory discussions and consultations.

How is USP helping with the fight against nitrosamines?
 

Through international and domestic work with regulators, policymakers and drug manufacturers, USP collects information to understand the potential implications of nitrosamines in medicines. 

USP has developed tools and solutions to help manufacturers analyze, monitor, and control nitrosamine impurities in medicines to strengthen the global medicines supply chain. Our existing resources include an informational General Chapter on nitrosamine impurities, associated Reference Standards and materials, and on-demand educational webcasts. We are also helping regulatory agencies from around the world by offering our expertise and tools through customized training. These efforts are intended to increase the capabilities of global regulatory agencies that may be at earlier stages in their journey to understand and mitigate the risks for their medicines supply chains.

What other challenges does the industry face with nitrosamines and how can we find a path forward?
 

As the industry and regulatory agencies continue to address nitrosamine impurities, we anticipate several upcoming challenges that reflect the evolving nature of the situation. 

Pharmaceutical companies and regulators must remain vigilant when monitoring for emerging nitrosamine precursors that may pose risks to drug products. With new synthetic routes, starting materials and manufacturing processes continuing to develop, novel precursors can be introduced. Identifying and assessing precursors requires ongoing research, analytical method development, and risk assessment to ensure they are addressed in testing protocols and regulatory guidelines.

Determining nitrosamine potency or safety risks has proven to be complex. Industry and regulators must continue collaborating to generate robust data and a mechanistic understanding of the mutagenicity and carcinogenicity of these impurities. The continued contribution to this safety knowledge will allow regulatory evolution to ensure accurate safety requirements.

Managing nitrosamine risks throughout the global supply chain presents challenges for both industry and regulators. Pharmaceutical companies rely on a network of suppliers and contract manufacturers to source raw materials and components for drug manufacturing, increasing the complexity of supply chain oversight. Ensuring the integrity of raw materials, implementing quality agreements, and conducting supplier audits are essential to mitigate nitrosamine risks. Regulators must also harmonize and standardize requirements for supply chain management to promote consistency and transparency across regions.