Since 2018, most pharmaceutical companies, drug manufacturers and marketing authorization holders (MAHs) have been assessing the risk of N-Nitrosamine contamination in their active pharmaceutical ingredients (APIs) and drug products. If you belong to any of these groups you probably think you know everything you need about nitrosamine impurities, but Conscio Group have noticed our clients are often facing a new and serious challenge that requires everyone’s attention: Nitrosamine analogues.
This white paper captures expert insights on API-derived nitrosamines (nitrosamine analogues)—an emerging analytical and quality challenge across both APIs and drug products. It outlines typical formation scenarios and practical testing pathways, setting the stage for the following expert perspective from Libor Hoplíček, Head of R&D.
Expert Perspective: Libor Hoplíček, Head of R&D
Mr. Libor Hoplíček, Head of R&D department at our Prague site, has been working on analytical method development, reverse engineering, determination and isolation of unknown impurities since long before nitrosamines became a major concern. His team develops and pre-validates methods for nitrosamine determination, whether they are quantification methods or limit test methods, while other Conscio departments perform GMP validations and analysis. As a result, he has first-hand knowledge of the unusual requests Conscio has been receiving lately:
“More and more often we are being contacted about new types of nitrosamines, the so-called active substance-derived nitrosamines (NDSRIs) or nitrosamine analogues. In some cases, nitrosamine impurities may also originate from the formulation matrix or excipient components, if they contain amine functionalities capable of undergoing nitrosation reactions, for example meglumine, as observed in recent recalls of contrast agents. As of today, we have been asked to analyze around 30 different analogues by different clients worldwide” Mr. Hoplíček begins.
“Some clients have requested rapid limit tests, others GMP-validated quantifications, some come with APIs or substances while others with finished drug products. The problem is that even if a tested API is free of nitrosamine analogues, its related drug product may contain significant, often above-specification, amounts of these impurities. Especially, when drug products are stored in stability chambers, concentration of nitrosamine analogues could quite easily exceed product specification. We have seen a few cases where the sponsor thought the confirmatory screening wouldn’t be an issue, to then receive the unpleasant news that limits had been exceeded” Libor Hoplíček states.
Regulatory Context and When to Act
Regulatory authorities are aware of the occurrence of nitrosamine analogues and advise all MAHs for EU medicines to prioritize this risk factor in their evaluations and if confirmed, proceed with confirmatory testing. Screening for such analogues, however, is more difficult than for simple N-Nitrosamines.
What Are Nitrosamine Analogues (API-Derived Nitrosamines)?
To properly understand the challenge, it helps to start with their origin and molecular nature.
Mr. Hoplíček says:
“These nitrosamine analogues are nothing but the original API or its impurity with some of its amine functional groups replaced by an N-Nitrosamine residue: This is a reaction called nitrosation. Nowadays, even API which have tertiary amine group and is “protected” from nitrosation reaction could be considered by authorities as possible nitrosamine source. As possible, nitrosamine impurity could originate from N-dealkylated impurity of API, which does not have its amino group protected. We have developed several methods for APIs that should not be susceptible to nitrosation such as Zopiclone, Levofloxacine, Citalopram and other. Nitroso-desmethyl analogues of these APIs were considered by authorities in risk assessment as possible contaminants to be monitored”
Libor Hoplíček explains:
“It is unclear which step of the manufacturing process is to be blamed but the general idea is that vulnerable, nitrosatable amine groups in the API react with trace nitrite impurities from excipients or other inactive ingredients. In any case, the outcome will be an analytical sample with a mixture of the original API and its nitrosamine analogue, the latter sometimes in high amounts. Given the similarities between both, more complex extraction procedures and improvement of the chromatographic steps are needed to properly discriminate the analogue from its API” he defends.”
Capability: A Dedicated Nitrosamine Analogue Team
Conscio operates a cross-functional nitrosamine analytics team with laboratories in Prague (CZ), Brno (CZ) and Eisenstadt (AT). The team is supported by multiple LC-MS/MS Triple Quads, HS-GC-MS and four UHPLC-Orbitrap systems, with customizable in-house methods built around isotopically labeled internal standards, an industry benchmark for robust quantification. Where such standards are not readily available, alternative quantification strategies are carefully developed and scientifically justified to ensure reliable and accurate results. Where appropriate, we also offer streamlined approaches for rapid exploratory screening, backed by first-hand experience with over 50 different nitrosamines, including analogues.
Method Development in Practice
Libor Hoplíček explains how team expertise is essential for handling nitrosamine analogues:
“At the start of the Nitrosamine situation we had to adapt to limits of quantification (LOQs) from 0.03ppm to 0.1ppb, but now analogues demand more than just boosting sensitivity. Method setup and development usually take for 1-3 weeks and are more straightforward with conventional nitrosamines since only slight modifications are needed to customize our methods to each matrix. But for nitrosamine analogues we must carefully perfect the chromatography step (choice of column, mobile phases and their gradient) based on the structure of the API and tested nitrosamines. Even API impurities that are far below specification and not detected by UV detection can cause issues during method development, negatively influencing accuracy of method.
Choice of instrument is also conditioned by the API, as well as required limits of quantification and potential matrix effects. For many applications, particularly when analysing larger analogue molecules that show a strong mass spectrometric response, triple quadrupole systems are well suited to achieve the required sensitivity and selectivity. Their targeted approach enables robust and reliable quantification, making them an efficient solution for routine analysis of known compounds.
However, in cases where background noise, smaller peaks or more pronounced matrix effects are present, high-resolution mass spectrometry provides an additional level of confidence. These systems allow the measurement of exact mass with high resolving power and have proven particularly valuable in complex samples, where co-eluting compounds or impurities generate signals with very similar mass-to-charge ratios that cannot be sufficiently distinguished using unit-resolution techniques.
We have encountered such situations even for small nitrosamines such as nitrosodimethylamine and nitrosodiethylamine, where interferences from the product matrix or related impurities required the selectivity of high-resolution MS for unambiguous identification and accurate quantification.
Overall, both approaches are highly complementary, and the availability of both technologies allows selection of the most suitable method depending on the analytical challenge, ensuring robust, sensitive and reliable results across a wide range of projects.”
Conclusion
API-derived nitrosamines are so similar to the original API that they pose an immediate challenge to precision, selectivity, sensitivity as well as accuracy but in the end, the key to their successful screening and quantification is proper method setup, having the right know-how, experience with multiple nitrosamines and instrumental alternatives to choose from.
Mr. Hoplíček finally sentences:
“Currently, our team is exposed to so many different molecules, projects and clients that this is our greatest strength: Our versatility”.
When asked about some closing words for companies with drug substances or products at a higher risk of formation of nitrosamine analogues, Libor Hoplíček has the following advice:
“We are experiencing an increasing number of nitrosamine analogue requests. If your risk assessment indicates a potential source of conventional nitrosamines the process is more clear-cut: You may opt for rapid preliminary screening to simultaneously test different nitrosamine limits; or else go straight for a GMP quantification for more accurate data. But if you suspect the presence of nitrosamine analogues, then take care to doublecheck all your APIs, finished products and excipients, always aiming for early detection.”
Remember: a clean API does not necessarily mean your drug product will be free of analogues. If you identify vulnerable amine groups likely to undergo nitrosation, please contact us.
We’ll be happy to support your assessment and testing strategy.
