Organic Impurities in Active Pharmaceutical Ingredients – Nitrosamines

Excerpt from the GMP Compliance Adviser, Chapter 20.F, Chemical active substances

10 min. reading time | by Stefan Kettelhoit, Norbert Waldöfner
Published in LOGFILE 04/2026 

Since the unexpected discovery of N-nitrosamines in sartans in 2018, what began as isolated recalls has evolved into a permanent, lifecycle-wide compliance challenge for the pharmaceutical industry. With tightening EMA and FDA expectations, substance-specific limits, and intensified GMP scrutiny, companies must now proactively control nitrosamine risks.

N-Nitrosamines attracted worldwide attention in 2018 when they were discovered to be present as un-expected impurities in various active substances. Initially, it were antihypertensive agents from the sartan group that were affected. The discovery of these impurities led to product recalls worldwide. Subsequently, N-nitrosamines were also found in other active ingredients. 

The EMA felt compelled to issue guidelines on preventing of nitrosamines in medicinal products. This has resulted in far-reaching obligations for marketing authorisation holders. Among other things, they are required to check their products for risks associated with the presence of nitrosamines, or the potential for them to be introduced. This involves identifying all active substances that could potentially be contaminated with N-nitrosamines. Analyses must be carried out into the relevant products and the results reported to the EMA. This applies to both chemically synthesised and biological active substances. Deadlines for risk assessments ('step 1'), confirmatory tests ('step 2') and updating marketing authorisations ('step 3') have now expired for medicinal products containing both chemically synthesised and biological active substances. The EMA has made the relevant regulatory processes and templates for these three steps available on its website. 

The EMA's central website "Nitrosamine impurities" contains links to other relevant documents. Extensive documents have been produced on specific medicinal products and active substances, such as sartans, rifampicin, ranitidine, metformin and varenicline (Champix). Of note is document EMA/526934/2019, entitled "Lessons learnt from presence of N-nitrosamine impurities in sartan medicines". This document contains further detailed considerations, including a revision of important international guidelines as well as audits and inspections of active ingredient manufacturers. For example, it was suggested that the ICH Q7 Guideline (Good Manufacturing Practice for Active Pharmaceutical Ingredients) should be more specific regarding the use of reagents or recovery processes (however, the ICH Q7 has not yet been amended). From their own experience, the authors can report that the topic of nitrosamines has now become an established part of GMP audits. Active ingredient manufacturers have generally carried out corresponding risk analyses for all active ingredients manufactured and can refer to specific documents and templates for this purpose. The Active Pharmaceutical Ingredients Committee (APIC) provides the following documents for this purpose:

• Additional guidance on the assessment of the risk assessment for the presence of N-nitrosamines in APIs.
• Report on the risk of potential presence of nitrosamine impurities (template). 

The IPEC Federation provides a questionnaire for the risk assessment of N-nitrosamines in excipients for excipient manufacturers. The Assessment Report EMA/369136/2020 entitled "Nitrosamine impurities in human medicinal products" provides a detailed summary of the current state of knowledge on nitrosamines in human medicinal products. As regulatory developments in the field of nitrosamines are very dynamic, it is essential to research the currently applicable requirements in a timely manner. In the EU, the EMA website and the Q&A document are particularly noteworthy as these two sources of in-formation are updated frequently. Extensive updates were made to the Q&A document. The responsibility of the marketing authorisation holder about controlling nitrosamine impurities throughout the entire life cycle of a medicinal product is referenced. The Q&A document includes approaches for categorising N-nitrosamines in terms of their carcinogenic potential, among other things. Three new appendices have been added for this purpose:

• Appendix 1 lists the N-nitrosamines for which acceptable intakes (AI) have already been established.
• Appendix 2 introduces the Carcinogenic Potency Categorisation Approach (CPCA). This method of classifying the carcinogenic potential of N-nitrosamines is based on an evaluation of the structural features that activate or deactivate the molecule. Five categories, each with a respective AI for N-nitrosamines are specified.
• Appendix 3 describes extended Ames test conditions for N-nitrosamines. 

The EDQM has defined measures for CEP holders, which it has summarised in its newsroom. 

The guidance published by the FDA (Control of Nitrosamine Impurities in Human Drugs) provides valuable advice on how to avoid nitrosamine impurities. This guideline is available in Revision 1. Another recent FDA guideline worth mentioning is "Recommended Acceptable Intake Limits for Nitrosamine Drug Substance-Related Impurities (NDSRIs)". Like the EMA, the FDA provides a summary of current information on its website. 

The European Pharmacopoeia addresses the topic of N-nitrosamines in several monographs/texts: 

• Monograph 2034 (substances for pharmaceutical use)
• Monograph 2619 (pharmaceutical preparations)
• Chapter 2.5.42 (N-nitrosamines in active substances) 

The monographs set out the requirements relevant to manufacturers of active substances and medicinal products. Affected manufacturers should minimise risk as far as possible, for example by adapting the manufacturing process. In addition, they should implement a control strategy to enable the detection and control of N-nitrosamine impurities. Chapter 2.5.42 lists various analytical methods. 

How should limits for N-nitrosamines be set? 

N-nitrosamines are considered substances of "cohort of concern" according to ICH M7(R2). For these substances, the "Threshold of Toxicological Concern" (TTC) of 1.5 µg/day cannot be routinely applied Therefore, acceptable intake levels (AIs) must be established on a substance-specific basis, for which a negligible risk (theoretical increased cancer risk of less than 1 in 100000 with lifetime exposure) can be expected. The calculation of AIs is based on the maximum daily dose of the medicinal product being administered for a lifetime. According to the EMA Q&A document, the 'less than lifetime' (LTL) approach should only be considered as a temporary measure, after consultation with the competent authorities, until further measures can be taken to reduce the level of impurities. Annex 1 of the EMA Q&A document contains nitrosamines for which acceptable intake levels have already been established. Based on the AIs, limit values for medicinal products can be determined. The exact calculation is described in detail in Q&A No. 10 of the EMA Q&A document.

How are N-nitrosamines formed? 

Q&A 4 of the EMA Q&A document sets out the risk factors relating to the manufacture of active substances in detail. Seven points are mentioned. The FDA guideline (Control of Nitrosamine Impurities in Human Drugs) also lists numerous mechanisms and examples. In general, N-nitrosamines can form when certain amines and a nitrosating agent are combined under specific conditions. The EMA Q&A document (Q&A No. 4) also mentions other possible formation pathways, such as oxidation and reduction processes from hydrazine-like compounds and N-nitro derivatives. The main reason for the formation of nitrosamines during chemical synthesis of active substances is the presence of nitrite salts and esters or other nitrosating agents in the presence of secondary and tertiary amines. In addition to the direct use of these compounds within a manufacturing step, other pathways within a manufacturing process are also conceivable that can lead to such a constellation. 

Examples of nitrosating agents are: sodium nitrite, alkyl nitrites, nitrosohalides, nitrosonium salts, nitrogen oxides, nitroalkanes, halogenated nitroalkanes, potassium nitrosodisulphonate (Frémy's salt) and nitroso sulfonamides. 

Sources of secondary, tertiary or quaternary amines in active ingredient synthesis can include: The active ingredient itself, intermediates, starting materials, raw materials & reagents, catalysts and solvents. 

The amine functionalities may be a component of these substances or may be present as impurities. They may also arise through the degradation of certain substances (e.g. amide solvents). 

In general, certain technical or GMP aspects of active ingredient production can also lead to increased risks:

• Use of multipurpose equipment,
• Inefficient manufacturing instructions or cleaning instructions that lead to the carryover of impurities between process steps,
• Use of contaminated recycled or reused materials (e.g. solvents). If solvent recovery is carried out externally and/or multipurpose equipment is used, there may be a significant risk of contamination, even if the actual synthesis of the active ingredient presents a low risk. 

In the authors' view, using contaminated raw materials ordered from suppliers is a risk that is often overlooked. Such materials may contain residual nitrosamines or nitrosating agents, and it is not entirely possible for the active substance manufacturer to control these risks. This makes careful supplier qualification, taking these aspects into account, even more important. 

Other specific risks in active substance manufacturing mentioned in the EMA Q&A document are:

• Nitrite formation through the oxidation of hydroxylamine, or nitrite release from nitroaromatic precursors in the presence of secondary or tertiary amines within the same or different steps of the manufacturing process. 
• Use of disinfected water (chlorination, chloramination, ozonation) in the presence of secondary or tertiary amines within the same or different steps of the manufacturing process.
• Oxidation of hydrazines, hydrazides and hydrazones by hypochlorite, air, oxygen, ozone and peroxides in the manufacturing process or during storage. Use of contaminated raw materials in the API manufacturing process (e.g. solvents, reagents and catalysts).
• Carryover of nitrosamines intentionally generated during the manufacturing process (e.g. as starting materials or intermediates). 

In addition, the EMA Q&A document identifies specific risks associated with drug manufacturing (e.g. certain packaging materials). However, these aspects will not be discussed further here. 

How are nitrosamines determined? 

Normally, the exact determination of the concentration of N-nitrosamines in active substances is naturally of particular importance. It is the responsibility of the marketing authorisation holder to develop a suitable analytical method with the required sensitivity. This sensitivity should be based on the acceptable intake levels (AIs) of the respective N-nitrosamines. Q&As Nos. 8 and 9 of the EMA Q&A document lay down several principles in this regard, particularly with regard to the required limit of quantification (LoQ). 

The pharmacopoeia monographs mentioned above refer to Chapter 2.5.42 of the European Pharmacopoeia for the determination of N-nitrosamines, which lists three analytical methods for the detection/determination of various N-nitrosamines:

• Method A: LC-MS/MS
• Method B: GC-MS
• Method C: GC-MS/MS 

The methods are considered validated for certain combinations of N-nitrosamines and active substances, in accordance with Table 2.5.42-1 of the Pharmacopoeia. For other combinations, the methods must be validated. In general, the recently amended Chapter 5.26 on the implementation of Pharmacopoeia methods must also be considered. 

The official medicinal product control laboratories (OMCLs) of the General European OMCL Network (GEON) have developed methods for determining N-nitrosamines. These methods were subsequently published on the EDQM website for informational purposes. 

How can the formation of nitrosamines be prevented? 

Cross-contamination or errors caused by production personnel during the production of active substances can cause nitrosamine formation (see the section 'How are N-nitrosamines formed?' in this chapter for more information). To prevent N-nitrosamine formation during the manufacture of active substances, the aforementioned reaction conditions should be avoided, for example by using alternative solvents or reagents where possible. Additionally, if there is a risk of contamination by nitrosamines, the raw materials used should be subjected to intensive evaluation and testing. Suppliers of raw materials for active ingredient production should also be qualified. The use of recovered solvents should be carefully evaluated, or it should be clearly demonstrated that any nitrosamines contained in the solvents can be completely removed by purification processes. The commissioning of external companies for solvent recovery should also be reviewed. Regarding solvent recovery, the following scenarios are recommended:

• Use of solvents recovered from the same process step in the synthesis of the active substance.
• Carrying out solvent recovery internally (not outsourcing).
• Use dedicated equipment for solvent recovery.
• Evaluate and, if necessary, validate cleaning procedures for the equipment used for recovery.
• Carefully document cleaning steps (cleaning batch record) and check them in accordance with the dual control principle. 

Further necessary measures depend on the specific results of the respective root cause analysis and must be initiated considering the current state of knowledge and the principles of quality risk management.

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