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). 

 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 

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. 

References  

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 

AMPAC 

• * Watch Our On-Demand Webinar on Genotoxic Impurities
  https://ampacanalytical.com/events/on-demand-webinar/ 

• * AMPAC Analytical’s Nitrosamine Impurities Testing Webpage
  https://ampacanalytical.com/laboratory-services/ampac-analytical-offers-ndma-analyses-of-api-and-dp/#1644962917933-ed2cb310-b026 

General Information on Nitrosamines 

• * An Organic Chemist’s Guide to N‑Nitrosamines: Their Structure, Reactivity, and Role as Contaminants (PDF)
  https://pubs.acs.org/doi/10.1021/acs.joc.0c02774?ref=pdf 

Nitrosamine Exposure and Environmental Concerns 

• Nitrosamines as Environmental Carcinogens
  https://doi.org/10.1038/225021a0 

• Nitrosamines as Potential Environmental Carcinogens in Man
  https://doi.org/10.1016/0009-9120(90)90489-H 
• * Critical Review of Major Sources of Human Exposure to N-nitrosamines
  https://doi.org/10.1016/j.chemosphere.2018.07.098 
• * Environmental Guidelines and Regulations for Nitramines: A Policy Summary (PDF)
  https://tcmda.com/app/uploads/sites/5/2020/09/MIT-nitramine_report_final_July2011.pdf 

Nitrosamine and Pharmaceuticals 

• * FDA Guidance Document: Control of Nitrosamine Impurities in Human Drugs
  https://www.fda.gov/regulatory-information/search-fda-guidance-documents/control-nitrosamine-impurities-human-drugs 

• * Information about Nitrosamine Impurities in Medications
  https://www.fda.gov/drugs/drug-safety-and-availability/information-about-nitrosamine-impurities-medications 
• * Prevalence of Nitrosamine Contaminants in Drug Samples: Has the Crisis Been Overcome?
  https://doi.org/10.1002/ardp.202200484 
• * Nitrosamines Impurities in Pharmaceuticals, the Abrupt Challenges They Bring, and Approaches to Tackle the Risk
  https://doi.org/10.1016/j.xphs.2022.07.016 
• Critical Analysis of Drug Product Recalls Due to Nitrosamine Impurities
  https://doi.org/10.1021/acs.jmedchem.0c02120 
• * Estimated Cancer Risks Associated with Nitrosamine Contamination in Commonly Used Medications
  https://doi.org/10.3390/ijerph18189465  

Nitrosamine and the Diet 

• Carcinogenic N-nitrosamines in the Diet: Occurrence, Formation, Mechanisms and Carcinogenic Potential
  https://doi.org/10.1016/0165-1218(91)90123-4 
• History of Nitrite and Nitrate in Food (book chapter)
  https://doi.org/10.1007/978-1-60761-616-0_5