Tag Archives: GTIs

Nitrosamines – An Update


The linkage between nitrosamines and cancer was first postulated by William Lijinsky in 1970. Then, in 2018, N-nitroso-dimethylamine (NDMA)) was detected in an active pharmaceutical ingredient, Valsartan (an Angiotensin-II-receptor antagonist).  Finally, the FDA issued a guidance for the industry, “Control of Nitrosamine Impurities in Human Drugs”, in the fall of 2020. However, the guidelines continue to evolve. There has been an update in March of 2021, with ongoing risk assessments.  Other regulatory agencies have instituted their own, along with updates. For example, since our blog series on nitrosamines, there have been some regulatory updates from the European Medicines Agency (EMA). Their new guidelines are outlined in a document entitled, “Questions and Answers for Marketing Authorization Holders/Applicants on the CHMP Opinion for the Article 5(3) of Regulation (EC) No 726/2004 Referral on Nitrosamine Impurities in Human Medicinal Products.”1 The updated section answers the crucial question, “Which limits apply for nitrosamines in medicinal products?” 

 The answers are provided with a definition of nitrosamines and acceptable exposures: 

 “The ICH M7(R1) guideline defines N-nitrosamines as substances of the “cohort of concern” for which limits in medicinal products refer to the so-called substance-specific acceptable intake (AI) (the Threshold of Toxicological Concern, TTC, value of 1.5 ug/day cannot be routinely applied) which is associated with a negligible risk (theoretical excess cancer risk of <1 in 100,000 over a lifetime of exposure). The calculation of AI assumes a lifelong daily administration of the maximum daily dose of the medicinal product and is based on the approach outlined in the ICH M7(R1) guideline as well as the principles described in relation to the toxicological evaluation in the assessment report of the CHMP’s Article 5(3) opinion on nitrosamine impurities in human medicinal products.”1,2 (A previous blog examined TTC, here.)  

A Useful List of Nitrosamine Limits  

In Appendix 1 found on the EMA site, there is a list of more than eighty nitrosamines listed, along with CAS numbers, known medicinal sources, CPCA (Carcinogenic Potency Categorization Approach) categories, and their guidance publication dates.3,4  

Some Caveats 

There are some exceptions that should be considered. For example, the EMA states, “The ‘less than lifetime’ (LTL) approach should not be applied in calculating the limits as described above but can only be considered after consultation with competent authorities as a temporary measure until further measures can be implemented to reduce the contaminant at or below the limits defined above.”1  

Additionally, those medications intended for advanced cancers also have some exceptions. For example, “If the active substance itself is mutagenic or clastogenic at therapeutic concentrations, N-nitrosamine impurities should be controlled at limits for non-mutagenic impurities according to ICH M7(R1).”  

There is also guidance when one or more than one nitrosamines may be present. For the latter, the guidance advises one of two approaches: 

  1. The total daily intake of all identified N-nitrosamines is not to exceed the AI of the most potent N-nitrosamine identified. 
  1. The total risk level calculated for all identified N-nitrosamines is not to exceed 1 in 100,000. The approach chosen needs to be duly justified by the MAH (Marketing Authorization Holder)/Applicant.1 

Final Thoughts on Nitrosamines 

Nitrosamine guidance worldwide is ever-evolving, yet the impetus to quantify and regulate them is clear. There will doubtlessly be further updates to regulations. AMPAC Analytical Laboratories – an SK pharmteco company (AAL) is an industry leader in the detection of nitrosamines and other genotoxic impurities (GTI), We have the specialized expertise, equipment, and methodologies to detect these impurities by gas chromatography or high-performance liquid chromatography coupled with mass spectrometry to support your API project.  Also, importantly, AAL can assist in navigating those projects within today’s regulatory landscape. Please contact us with any specific questions or to receive a quote for nitrosamines or other GTIs.  


  1. https://www.ema.europa.eu/en/documents/referral/nitrosamines-emea-h-a53-1490-questions-answers-marketing-authorisation-holders/applicants-chmp-opinion-article-53-regulation-ec-no-726/2004-referral-nitrosamine-impurities-human-medicinal-products_en.pdf 
  2. ICH M7 Principles – Impurity Identification and Control (europa.eu) 
  3. https://www.ema.europa.eu/en/human-regulatory/post-authorisation/referral-procedures/nitrosamine-impurities 
  4. Appendix 1 Nitrosamine AIs (europa.eu) 


AMPAC  Analytical

General Information on Nitrosamines 

Nitrosamine and Pharmaceuticals 

Regulatory Experiences with Root Causes and Risk Factors for Nitrosamine Impurities in Pharmaceuticals

TCB* With Your TTC Needs

Triple Quad HPLC

Triple Quad HPLC“The dose makes the poison” – Paracelsus (c. 1493– 1541), born Theophrastus von Hohenheim

The Threshold of Toxicological Concern (TTC) refers to levels of mutagenic impurities expected to pose a negligible carcinogenic risk.1 The US FDA, the EMA (European Medicines Agency), and the European Food Safety Authority (EFSA) all have TTC values and regulations in place for food and active pharmaceutical ingredients (APIs), along with numerous other products.2,3 Originally, these standards were applied to TTC levels from oral ingestion but have expanded to even include cosmetics and fragrances.4,5

One tool to assess risk is the use of Cramer classes for organic impurities. They range from I-III, indicating a low, medium, or high probability of toxicity.5

There are numerous tools and techniques to assess TTC, depending on the product (food, water, and other beverages, APIs, or cosmetics) and the mutagenic impurity. AMPAC Analytical can utilize TTC guidelines and risk assessments to establish method development and validation targets that ensure acceptable levels of mutagenic impurities in your API or food products. Contact us today to learn more about analytical strategies to control mutagenic impurities.

*Taking Care of Business


  1. https://www.fda.gov/media/85885/download
  2. https://www.ema.europa.eu/en/ich-m7-assessment-control-dna-reactive-mutagenic-impurities-pharmaceuticals-limit-potential
  3. https://www.efsa.europa.eu/en/topics/topic/threshold-toxicological-concern
  4. https://www.sciencedirect.com/science/article/abs/pii/S0278691507002207
  5. https://www.sciencedirect.com/science/article/abs/pii/S0273230015300660


  • https://www.fda.gov/media/85885/download
  • https://www.frontiersin.org/articles/10.3389/ftox.2021.655951/full
  • https://academic.oup.com/toxsci/article/86/2/226/1653574
  • https://www.sciencedirect.com/science/article/abs/pii/S027869159600049X

Extractables and Leachables

blank pharmaceutical and drug packaging

Extractables and Leachables (E&L) are essential areas of concern for the pharmaceutical and food industries, specifically regarding their packaging, usage components (e.g., medical devices or syringes), and the manufacturing chain. We will examine testing of analysis of them within pharmaceutical applications. The two terms are related but distinct, each with its own analytical requirements.   

Definitions of Extractables and Leachables 

A handy article published in Pharmaceutical Engineering by the International Society for Pharmaceutical Engineering (ISPE) explains that “Extractables are chemical compounds that migrate from single-use systems (SUS) into model solvent solutions under controlled and exaggerated conditions depending on temperature, pH, polarity, and time.” In other words, this happens when using strong solvents. They note that “SUS are normally not exposed to such conditions in biopharmaceutical processes.”1  

ISPE’s article defines leachables as “chemical compounds that migrate from SUS into process solutions under normal biopharmaceutical process conditions. They further clarify that these compounds “may end up in the final drug product formulation. For the most part, leachables are a subset of extractables, although interaction with product components may produce leachables not seen as extractables.”1 

Guidance on Extractables and Leachables 

The FDA has released a series of guidelines for the pharmaceutical industry, including Container Closure Systems for Packaging Human Drugs and Biologics, that provide guidance for submission in support of an original application for any drug product. It also covers a wide range of forms and delivery systems of drugs:

  • Inhaled 
  • Injected 
  • Liquid-based  
  • Oral  
  • Solid oral dosage forms  
  • Ophthalmic 
  • Topical and topical delivery systems  
  • Powders for reconstitution   
  • And other dosage forms 

Additionally, the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) also has issued the ICH Q3E: Guideline for Extractables and Leachables.2,3 These are both useful in providing direction for E&L concerns and control strategies. 

Plan Against Extractables and Leachables 

To guarantee adherence to all guidelines and regulations while ensuring patient safety, it is crucial to know and utilize materials compatible with your product. To accomplish this, solvent use, packaging, and delivery systems must all be tested and analyzed in cGMP and FDA-compliant laboratories.  This should include the following:

  • A thorough review of all materials used in packaging and production, production, and equipment to predict the compatibility of your packaging system with your product. AAL can provide reports for items from each step. 
  • Extraction studies on the materials used. 
  • Leachable studies to identify any impurity resulting from those materials found in the final product under normal usage conditions. 
  • If impurities are detected, AAL can provide toxicological evaluations, including profiles of the impurities and the risks they pose for the patients, establish safety limits, or adjust for different forms of medication application. 
  • We can assess risks created by various exposure levels due to the impurity in the finished product. 
  • Finally, AAL provides a detailed report of our findings in accordance with the applicable governing bodies (e.g., FDA, EMA, PQRI, PDA). 

 AMPAC Analytical can review your analysis and testing needs for extractables and leachables for any forms and delivery systems listed in the table above, complying with the strictest standards necessary. 


  1. https://ispe.org/pharmaceutical-engineering/may-june-2017/extractables-leachables-not-same 
  2. https://www.fda.gov/media/70788/download 
  3. https://database.ich.org/sites/default/files/ICH_Q3E_ConceptPaper_2020_0710.pdf 



Nitrosamines in Active Pharmaceutical Ingredients 

This is the second in a series of entries examining nitrosamines in a range of products. Our first article presented an overview of nitrosamines, including a historical look at their implication as probable carcinogens. This entry will review their presence in active pharmaceutical ingredients (APIs) and process mitigation strategies. 

Nitrosamines are organic compounds found in medications, the human diet, and the environment These carcinogens can cause tumors in nearly all organs and have been classified as possible genotoxic impurities (GTI).  

Background on Nitrosamines in Active Pharmaceutical Ingredients
The linkage between cancer and a large class of chemical compounds known as nitrosamines was postulated by William Lijinsky in 1970.1 Then, in June 2018, their presence (specifically, N-nitroso-dimethylamine (NDMA)) was detected in the API Valsartan, an Angiotensin-II-receptor antagonist.  


It later became “obvious that the issue may not only occur with sartans but, in principle, with any API containing a vulnerable amine and a nitrosation source. Hence not only NDMA but a plethora of potential nitrosamines could be created.”2 They have been subsequently detected in other medicines resulting in 250 product recalls, affecting more than 1400 lots.3,4 The cost of recalls could be high.5 APIs or their impurities can become nitrosated “during the later stages of the synthetic process of the drug product manufacturing or even while in the completed, packaged product.”6 As discussed in our previous entry, primary amines are not a concern, as they have limited stability.6 However, secondary and tertiary amines, along with quaternary ammonium compounds, are considered potential nitrosamine precursors, according to the current guidelines of the FDA and EMA.6,7  As a useful reference for amine components, there is a central system for the ingredients in medicinal products known as the Global Substance Registration System (GSRS https://gsrs.ncats.nih.gov/.)8   Some of the possible causes for the presence of nitrosamines are:  

  • The use of sodium nitrite (NaNO2), or other nitrosating agents. 
  • The use of raw materials and intermediates contaminated by nitrosamines 
  • Degradation processes of starting materials, intermediates, and drug substances during formulation or storage 
  • The use of certain contaminated packaging materials 

Detection Tools 

Fortunately, there are many tools to detect nitrosamines. NDMA, NDEA, and other nitrosamine impurities can be detected at ppb level using gas chromatography, such as with a QTOF (Quadrupole Time of Flight Mass Spectrometer) or triple quadrupole.  

 Ways to Mitigate Nitrosamine Formation 

There are numerous ways that nitrosamines can be mitigated through API process design. For example, the FDA’s Control of Nitrosamine Impurities in Human Drugs Guidance for Industry, issued by the Center for Drug Evaluation and Research, states that:  “The following factors should be considered during process development:  

  • Avoiding reaction conditions that may produce nitrosamines whenever possible; when not possible, demonstrating that the process is adequately controlled and is capable of consistently reducing nitrosamine impurities through appropriate and robust fate and purge studies.  
  •  Using bases other than secondary, tertiary, or quaternary amines (when possible) if ROS (Route of Synthesis) conditions may form nitrosamines 
  • Using caution when the ROS involves the use of amide solvents (e.g., N,N-dimethylformamide, N,N-dimethylacetamide, and N-methylpyrrolidone) 
  • Replacing nitrites with other quenching agents for azide decomposition processes 
  • Optimizing and consistently controlling the sequences of reactions, processes, and reaction conditions (such as pH, temperature, and reaction time) 
  • Designing a manufacturing process that facilitates the purge of nitrosamine impurities in the subsequent processing steps. 
  • Auditing API supply chains accompanied by continuous monitoring for any at-risk raw materials, starting materials and intermediates, and avoiding cross-contamination when using recovered materials such as solvents, reagents, and catalysts in the manufacturing process.  
  • Recovered material should be used only in the same step or in an earlier step. API manufacturers should be aware that potable water used in API manufacture may contain low levels of nitrite and even nitrosamines from environmental contamination”.9,10 


Nitrosamines are an inevitable chemical outcome in the manufacturing and processing of many items, including APIs. Due to their low concentrations, they are also challenging to detect. AMPAC Analytical has rigorous testing services available to screen to trace levels in challenging sample matrices, including process intermediates, drug substances, and drug products. We have the specialized expertise, equipment, and methodologies to detect these impurities by gas chromatography or high-performance liquid chromatography coupled with mass spectrometry. Please contact us with any specific questions or to receive a quote for nitrosamines screening.  


  1. https://doi.org/10.1038/225021a0   
  2. https://jpharmsci.org/article/S0022-3549(23)00018-7/fulltext  
  3. https://doi.org/10.1021/acs.jmedchem.0c02120  
  4. https://doi.org/10.1016/j.xphs.2022.11.013 
  5. https://www.bloomberg.com/news/articles/2022-09-01/drug-recalls-for-nitrosamines-could-cost-big-pharma-millions  
  6. https://www.fda.gov/media/141720/download 
  7. https://www.ema.europa.eu/en/documents/referral/nitrosamines-emea-h-a53-1490-assessment-report_en.pdf https://doi.org/10.1093/nar/gkaa962  
  8. https://doi.org/10.1093/nar/gkaa962   
  9. https://ampacanalytical.com/wp-content/uploads/2023/01/Control-of-Nitrosamine-Impurities-in-Human-Drugs-Guidance-for-Industry.pdf  
  10. https://www.who.int/water_sanitation_health/water-quality/guidelines/en/ 

Resources & Further Reading 


General Information on Nitrosamines 

Nitrosamine and Pharmaceuticals 

Nitrosamines: An Overview

This is the first in a series of entries examining nitrosamines in a range of products.  

 Nitrosamines are organic compounds found in the human diet and other environmental outlets. Being potent carcinogens that can cause tumors in nearly all organs, they have been classified as genotoxic impurities (GTIs). There are guidelines and rulings by various regulatory organizations, including the FDA, EPA, EMA, and the IARC (International Agency for Research on Cancer). Their presence and attendant concerns have been noted for many years. A.J. Gushgari and R.U. Halden wrote in Chemosphere,  Nitrosamines were first proposed as environmental carcinogens by William Lijinsky in 1970, which fostered research on N-nitrosamine occurrences in environmental media.”1 These included “ambient water, aquatic sediments, and municipal sewage sludge (Schreiber and Mitch, 2006; Venkatesan et al., 2014; Zeng and Mitch, 2015; Gushgari et al., 2017).”1 Concern about their presence has significantly expanded to include food and active pharmaceutical ingredients (APIs). Our next two blog entries will explore the effects and mitigation of nitrosamines in these two areas. 

Background on Nitrosamines
Basically, “Nitrosamines are formed from the reaction of nitrite with primary, secondary, or tertiary amines in an acidic medium.”2 Primary and tertiary amines are typically not concerns for nitrosamines, but should be part of the chemical evaluation as there are cases where they can be impacted to form these impurities. 

 Since nitrates and the conditions are common in a wide range of products, vigilance is warranted. The reaction between nitrous acid and primary aromatic amines was first observed and reported in 1864 by Peter Griess. The work of Baeyer and Caro, and Otto Witt in the 1870s further researched the reaction. As Gushgari and Halden state, it was Witt in his 1878 publication that the term “nitrosamine” was first introduced to describe ““any substituted ammonia which contains, instead of at least one atom of hydrogen, the univalent nitrosyl group, NO, in immediate connection with the ammoniacal nitrogen”.”1 Almost one hundred years later, the aforementioned William Lijinsky, studying the environmental causes of cancer and specifically chemical carcinogens, began his decades-long examination of nitrosamines, eventually leading him to appear before multiple congressional committees and to work with the FDA. As a result, the FDA issued numerous guidelines in the following decades, with many released in the last few years. The FDA’s guideline of a current acceptable intake limit is 26.5 ng/day for APIs. For drinking water, it is 7 ng/L. Along with many other resources, they published Control of Nitrosamine Impurities in Human Drugs (PDF) for “immediate implementation” on September 1, 2020.  The European Medicines Agency (EMA) has also been active in this area, with many resources found here 

 Many Types and an Increasing Concern 
Of course, there is more than one type of nitrosamine to contend with since there are countless combinations of the structural elements available. Sebastian Schmidtsdorff et al. listed a table (Figure 1) of sixteen investigated nitrosamines with their attendant CAS numbers, abbreviations, and interim limits (IL).4 These were discovered during their research using 249 different, randomly selected samples of APIs from 66 manufacturers.   

Figure 1
(N/A = not applicable/interim limits not published yet). 

Name  Abbreviation  CAS-No.  IL Interim Limits (ng/day) 
N-Nitrosodimethylamine  NDMA  62-75-9  96 
N-Nitrosomethylethylamine  NMEA  10595-95-6  NA 
N-Nitrosodiethylamine  NDEA  55-18-5  26.5 
N-Nitrosodiethanolamine  NDELA  1116-54-7  NA 
N-Nitrosoethylisopropylamine  NEiPA  16339-04-1  26.5 
N-Nitrosodiisopropylamine  NDiPA  601-77-4  26.5 
N-Nitrosodi-n-propylamine  NDPA  621-64-7  26.5 
N-Nitrosodi-n-butylamine  NDBA  924-16-3  26.5 
N-Methyl-N-nitrosoaniline (N-nitrosomethylphenylamine)  NMPhA  614-00-6  34.3 
N-Nitrosomethyl(2-phenylethyl)amine  NMEPhA  13256-11-6  8 
N-Nitrosodiphenylamine  NDPhA  86-30-6  NA 
N-Nitrosopyrrolidine  NPyr  930-55-2  NA 
N-Nitrosopiperidine  NPip  100-75-4  1300 
N-Nitrosomorpholine  NMor  59-89-2  127 
1-Methyl-4-nitrosopiperazine  MNPaz  16339-07-4  26.5 
N-Nitroso-N-methyl-4-aminobutyric acid  NMBA  61445-55-4  96 

 The most commonly occurring nitrosamines in APIs are NDMA, NDEA, NMBA, NDPA, NEIPA, NDBA, and NMPA. In addition to the number of nitrosamines, the products where they have been detected have increased dramatically. For example, since the discovery of their presence in an API, Valsartan (an Angiotensin-II-receptor antagonist) in 2018, they have been detected in other medicines resulting in 250 product recalls, affecting more than 1400 lots.5,6 In addition to the financial impact of these recalls costly litigation has risen too. 

 A Positive Note
Interestingly, although nitrosamine impurities in products are an ever-present concern, at least one medication, Carmustine [154-93-8] (Figure 2), is an antineoplastic nitrosourea [13010-20-3] and is used in treating several forms of cancer.7,8 

Figure 2 

carmustine structure

Final Thoughts
Nitrosamines can form during the manufacturing and processing of foods, beverages, medicines, and numerous other products.  In addition, they can form upon storage.5 Despite detection challenges, rigorous testing and mitigation services are available to screen and avoid their formation, thereby protecting consumers. In fact, AMPAC Analytical (AAL) has the specialized expertise, equipment, and implemented stringent methodologies to detect these impurities, utilizing gas chromatography or high-performance liquid chromatography coupled with tandem or high-resolution mass spectrometry. AAL currently maintains three validated procedures for general nitrosamines screening. Please feel free to contact us with any specific questions or to receive a quote for nitrosamine screening in your product. 

 Items marked with an asterisk are open access or available without registering. 


  1. * https://doi.org/10.1016/j.chemosphere.2018.07.098 
  2. https://pubmed.ncbi.nlm.nih.gov/2184959/ 
  3. * https://doi.org/10.1016/j.xphs.2022.11.013 
  4. * https://doi.org/10.1002/ardp.202200484 
  5. https://doi.org/10.1021/acs.jmedchem.0c02120 
  6. https://www.bloomberg.com/news/articles/2022-09-01/drug-recalls-for-nitrosamines-could-cost-big-pharma-millions 
  7. * https://pubchem.ncbi.nlm.nih.gov/compound/Carmustine 
  8. * https://medlineplus.gov/druginfo/meds/a682060.html 

Resources & Further Reading 


General Information on Nitrosamines 

Nitrosamine Exposure and Environmental Concerns 

Nitrosamine and Pharmaceuticals 

Nitrosamine and the Diet