Nitrosamine impurity analysis of pharmaceuticals by EMA and FDA guidelines

Many nitrosamines are highly potent carcinogens that can be present in pharmaceutical products as impurities. To manage the associated risks, both the European Medicines Agency (EMA) and the U.S. Food and Drug Administration (FDA) require pharmaceutical companies to implement rigorous nitrosamine mitigation strategies.¹  

This article summarizes EMA and FDA recommendations for nitrosamine impurity analysis, including the steps to determine whether testing is necessary, the determination of acceptable intake levels, and appropriate analytical methods to ensure these levels are not exceeded. If you require testing for your company’s products, do not hesitate to contact our experts for a quote.

When is nitrosamine impurity analysis required?

Both the EMA and the FDA require pharmaceutical manufacturers to adopt similar nitrosamine control strategies, consisting of the following steps:

1. Evaluating the risk of nitrosamine presence.

2. Performing confirmatory testing if a risk is identified. 

3. Implementing mitigation measures if nitrosamine presence is confirmed. 

During the initial risk assessment, companies should review their products’ composition and production processes in detail. Since trace-level concentrations of nitrosamine impurities can pose health risks, even a small likelihood of nitrosamine formation warrants proceeding to the testing step.²

Examples of nitrosamine impurity formation routes

Nitrosamines are usually formed by a nitrosating reaction between certain amines and nitrosating agents in acidic conditions. These nitrosamine precursors can be present in active pharmaceutical ingredients (APIs) or other substances used in the drug manufacturing process, both intentionally and as unintentional contaminants or degradation products. Examples of practices that increase the risk of nitrosamine impurity formation include using sodium nitrite in the presence of secondary or tertiary amines, using recovered solvents, catalysts, or reagents, and not fully implementing GMP principles throughout the production chain.³ 

Most pharmaceutical products containing chemically synthesized APIs are manufactured in a way where at least some nitrosamine precursors are likely to be present at certain stages of the multi-step production process. Biological medicines are also at risk when they contain chemically synthesized fragments, are produced using nitrosating reagents, or are packaged in certain high-risk packaging materials, such as nitrocellulose-containing blister packs.⁴

Which compounds should be targeted during confirmatory testing?

The composition of the API and the finished drug product will determine which types of nitrosamine impurities are at risk of being formed during the manufacturing process:⁵

• Nitrosamine drug substance-related impurities (NDSRIs) are structurally similar to the APIs in specific drugs and are usually unique to those products. Examples include N-nitroso-ramipril in ramipril, N-nitroso-fluoxetine in fluoxetine, and N-nitroso-duloxetine in duloxetine drugs.  

• Small-molecule nitrosamines are not structurally similar to any specific active pharmaceutical ingredient (API) and can form in various drug products. Several of the most common small-molecule nitrosamines, such as NDMA, NDEA, NDIPA, and NEIPA, can often be analyzed simultaneously.

Determination of acceptable intake levels for compliance testing

The acceptable intake (AI) limits for nitrosamine compounds are determined based on their mutagenic and carcinogenic potency. To minimize the likelihood of detrimental health effects, the limits are set very conservatively at a level where the excess cancer risk is not expected to exceed 1 per 100,000 people over daily exposure lasting 70 years.⁶

Some small-molecule nitrosamines, such as NDMA and NDEA, have been studied extensively, and their mutagenic and carcinogenic potential is well established. For these compounds, the acceptable intake limit is based on data from substance-specific toxicology studies. When compound-specific data are not available, the EMA and the FDA recommend using a predicted carcinogenic potency categorization approach to determine the AI limit. This is the case with most NDSRIs and less-studied small-molecule nitrosamines.

The AI limits set by the FDA and the EMA for selected nitrosamine impurities are listed in Table 1. These limits apply when the drug contains a single nitrosamine.⁷

To convert the AI limit to a parts-per-million (ppm) concentration, it is divided by the daily maximum dose of the drug. 

Requirements for nitrosamine impurity testing methods

Nitrosamine impurities are not typically identified during routine quality control tests intended to confirm API identity or screen it for known impurities, which means that specifically targeted methods are required. Due to the low limits for acceptable exposure, these methods must be highly sensitive. 

The FDA lists recommended GC-MS- and LC-HRMS-based analysis methods for specific drugs known to be particularly susceptible to nitrosamine impurities. These include methods to screen angiotensin II receptor blockers (ARB drugs or “sartans”) for common small-molecule nitrosamines, as well as methods to quantify NDSRIs in selected drugs where they have been known to form. In Europe, the European Directorate for the Quality of Medicines & HealthCare (EDQM) maintains a similar list, specifying methods developed at Official Medicines Control Laboratories in several countries.⁸

Similar high-performance chromatography techniques can be applied to other pharmaceutical products and nitrosamines, but the method has to be specifically validated for each target compound and sample matrix. This makes nitrosamine impurity analysis a highly specialized field, where a significant degree of expertise is required from the analyzing laboratory.

Measurlabs provides accredited and GMP-certified nitrosamine impurity analyses for a wide range of pharmaceutical products. You can use the form below to request more details about our service offering.