Nitrosamines are potent cancer-causing chemicals generated by a variety of natural and industrial processes, and found in a wide range of environments around the world. In this article, we examine some of the main sources of nitrosamines, discuss key challenges in environmental n-nitrosamine analysis, and outline how using reliable reference materials can help laboratories to achieve accurate, compliant, and efficient nitrosamine testing.

What are nitrosamines, and why are they dangerous?

Nitrosamines – also known as n-nitrosamines – are a diverse group of around 300 compounds that share the same basic N-N=O chemical structure. They are formed from a wide range of precursors under numerous different conditions, but typically when amines react with nitrosating agents in acidic conditions or at high temperatures. As a class, they have gained notoriety “both for the potent carcinogenicity of many …members and for their widespread occurrence throughout the human environment” - from air and water to soils, sediments, food, drink, and drug impurities.

The International Agency for Research on Cancer (IARC), the United States Environmental Protection Agency (USEPA), and the US National Toxicology Program have all listed a number of nitrosamines as possible or known human carcinogens. IARC has classified 24 n-nitrosamines with regard to their cancer-causing potential, with N-nitrosonornicotine and NNK ranked as Group 1 known carcinogens, and N-Nitrosodimethylamine (NDMA) identified as “probably” a human carcinogen, based primarily on robust evidence in animals.

Alkyl nitrosamines are generally more likely to be carcinogenic, via a mechanism in which they undergo enzymatic α-hydroxylation with cytochrome P450, and subsequently form an unstable, dealkylated primary nitrosamine. This compound further decomposes to diazonium 3 - a DNA alkylating agent that causes damage potentially leading to cancer. Studies have also linked nitrosamines to developmental or reproductive toxicity, immunotoxicity, neurotoxicity and systemic toxicity, while there is also evidence that n-nitrosamines cause endocrine disruption and hepatoxicity.

Sources of environmental nitrosamines

Nitrosamines are created in a multitude of ways, “from daily human life to industrial activity,” and their sources include foods, cosmetics, cigarettes, urine, disinfectants, rubber additives, polymers, dyes, and steels. For example, urine and faeces contain compounds such as dimethylamine and trimethylamine-N-oxide – both of which both possess amine groups that can leach out in matrices like water, and then form nitrosamines by undergoing nitrosylation in the presence of nitrites and nitrates. Among the industrial processes that help to create environmental nitrosamines are printing, dyeing, electroplating, and tanning, as well as pharmaceutical, rubber and pesticide manufacturing. In addition, nitrosamines can potentially form in “nearly every kind of personal care product” – usually when amine compounds like diethanolamine (DEA) or triethanolamine (TEA) are used to adjust pH levels or as wetting agents. Some lesser-known nitrosamine precursors include alkanolamine compounds used as carbon capture solvents which can ultimately degrade to form dozens of individual nitrosamines, including NDELA (N-Nitroso-diethanolamine). Finally, “third-hand” cigarette smoke trapped in clothing, furniture and wallpaper has also been found to produce hazardous tobacco-specific nitrosamines (TSNAs) after reacting with airborne nitrous acid from gas appliances and car exhausts.

Nitrosamines in water

Despite the many ways that environmental nitrosamines can be created, and the various places in which they can be found, experts agree that “it is their presence in water that has drawn the most concern.” While many waterborne nitrosamines are effectively removed by biological treatment processes, and some decompose naturally through photolysis, NDMA and other nitrosamine compounds can also be formed as disinfection by-products (DBPs) during treatment processes such as chlorination and chloramination. Chlorination is still widely used in many developing countries and is perhaps safer in drinking water, which typically has low nitrite concentrations. On the other hand, it “may be significant in other water matrices with elevated amounts of nitrite and amine precursors.” Even more concerningly, chloramination – or the process of adding chloramines to untreated water to destroy pathogenic microbes – “produces high levels of nitrogenous DBPs, in particular nitrosamines such as… NDMA.” For this reason, the USEPA includes NDMA and four other nitrosamines on its CCL4 list of water contaminants that may require regulation under the Safe Drinking Act (SDWA), and describes them all as potential disinfection byproducts.

Challenges in environmental nitrosamine testing

The dangers posed by nitrosamines have led to the establishment of several global, regional, and national guidelines on water quality – many of which set recommended or mandatory limits on the maximum concentration of NDMA and other n-nitrosamine compounds. There are also a number of approved nitrosamine testing methods – notably EPA Method 521 in the US, and HJ 809-2016 in China - and yet it is widely recognised that nitrosamine analysis is a particularly demanding task for analytical laboratories. As one report puts it, “Our ability to understand how N-nitrosamines form and spread in our environment… is inherently limited by our methods for detecting these contaminants.

Among the factors that make nitrosamine analysis particularly challenging are:

Extremely low concentrations - typically ng/l or part-per-trillion levels – at which nitrosamines are present in water and soil, meaning that expensive instrumentation and time-consuming sample preparation are required (notably in EPA Method 521).

Complex environmental matrices containing organic matter, metals, and other pollutants that can complicate analysis by masking or otherwise interfering with nitrosamine detection.

Analytical method challenges, such as carefully using extraction and concentration steps like Solid Phase Extraction (SPE) to isolate nitrosamines without losing them or causing contamination. This must then be followed by painstaking instrument calibration and method validation when using techniques like GC-MS or LC-MS/MS.

Lack of globally standardised methods, which can lead to variability in results across labs, and makes validating methods for each nitrosamine and sample type resource intensive.

Interferences and false positives, such as when nitrosamine precursors like amines and nitrites react during analysis to artificially form nitrosamines, or when laboratory materials or reagents containing nitrosamines or their precursors lead to false positives.