by Andrew James, Marketing Director, Ellutia
N-nitrosamines are a diverse group of organic compounds that may increase the risk of certain types of cancer in humans and have been shown to cause tumors in the liver, lung, nasal cavity, esophagus, pancreas, stomach, bladder, colon, kidneys, and central nervous system.1 People are exposed to nitrosamines in everyday life. They exist in low levels in a wide variety of goods including cosmetics, rubber products, tobacco products, processed meats, brewing and malting, agrochemicals, packaging, and pharmaceutical drugs.
The detection and identification of nitrosamine impurities presents unique challenges and can be complex. Some of the more popular methods for detection are gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-MS (LC-MS) technologies. However, these techniques can produce false positives and don’t have the lower limit levels that are required to see all of the impurities in a sample. A quicker and more straightforward method is now becoming the favored choice for nitrosamine testing – thermal energy analysis.
Nitrosamines and the Pharmaceutical Industry
Nitrosamine is a general term used to designate a vast group of N-nitroso compounds. They are produced by the reaction of a secondary or tertiary amine with a nitrosating agent. Nitrosating agents include nitric oxide, sodium nitrite, dinitrogen tetroxide, and nitrous acid. However, nitrosation can occur independent of these agents; for example, sodium nitrate under certain conditions can act as an indirect nitrosating agent. It has also been found that exposure to reducing agents or oxidative conditions can cause nitrosation. The N-N=O structure is consistent among nitrosamines, but this group is quite varied.
Nitrosamines were first discovered in the pharmaceutical industry in 2018. N-nitrosodimethylamine (NDMA) was found in angiotensin II receptor blockers, or sartans, which then led to the discovery of nitrosamine impurities in other medicines, specifically Zantac – ranitidine and metformin.2 In July 2020, the United States Pharmacopeia (USP) released new reference standards to assist pharmaceutical manufacturers in preventing supply chain contamination. The Food and Drug Administration (FDA) lists several nitrosamines that are concerning for the pharmaceutical industry: NDMA, N-nitrosodiethylamine (NDEA), N-nitrosodiisopropylamine (NDIPA), N-nitrosomethylphenylamine (NMPA), N-nitrosodibutylamine (NDBA), and N-nitrosomethylaminobutyric acid (NMBA). The FDA has a set daily intake limit level for each: 96 nanograms for NDMA and NMBA and 26.5 nanograms for NDEA, NMPA, and NDIPA.
In the EU, all marketing authorization holders of medicines containing chemically synthesized active pharmaceutical ingredients (APIs), and, now, biological active substances, are required to conduct a risk assessment to evaluate the possibility of nitrosamines being present. As part of the risk assessment, any sample showing any amount of nitrosamine must be reported to the national competent authorities for nationally authorized products or EMA for centrally authorized products.
Presenting a unique complication, nitrosamines can be introduced into pharmaceuticals at several stages – before, during and after manufacturing. The drug substance, excipient, water, solvents, manufacturing process and packaging are all potential sources of introduction. The USP states: “Nitrosamines can be introduced into or generated as impurities in pharmaceutical drug products, and examples and sources reported in the literature include active pharmaceutical ingredient (API) processing with certain reagents, solvents and raw materials, the API itself, which may degrade in some conditions, resulting in the formation of nitrosamines, and the degradation of solvents.”2
In the case of valsartan, a medication used to treat high blood pressure, heart failure, and diabetic kidney disease, nitrosamine contamination resulted from a side reaction. The solvent, dimethylformamide, was heated to high temperatures resulting in the formation of dimethylamine that reacted with a nitrite to form NDMA. In other instances, nitrosamine amounts increased over time due to instability. Packaging and printing that contains nitrocellulose, common in cosmetics, can also lead to nitrosamine impurities.1
Along the pharmaceutical chain, there are many opportunities for nitrosamine impurities to form, making their detection challenging.
The Complexity of Nitrosamine Detection
The diversity of individual nitrosamines in terms of size, shape and hydrophilicity, as well as their possible introduction within manufacturing and storage processes, complicates detection and removal efforts. The volatility and small molecular size of nitrosamines is a particular concern. Another challenge is that nitrosamine impurities, if present, are in exceptionally low concentrations requiring sensitive and detailed analysis techniques and instrumentation. All of these factors combine to require the highly sensitive and specific analytical methods necessary for detection.
In a September 2020 article, the USP proposed four analytical methods for drug makers to detect nitrosamine impurities. These were high performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS), GC-MS, HPLC-Tandem MS and GC-Tandem MS. Each of these methods are suited to identify a particular group of nitrosamine impurities e.g., HPLC-HRMS for measuring NDMA, NDEA, NDIPA, NEIPA, NMBA and NDBA.
While chromatography works well to identify nitrosamines, there is a risk that the API will overlap chromatographically with the target analyte, therefore complicating quantification. False positives caused by interfering compounds are also a concern, as is the cost of this equipment. Additionally, chromatography analysis is time consuming and requires a highly skilled technician to monitor the column at all times. An alternative technique is highly specific chemiluminescent detection using thermal energy analysis (TEA).
Screening for Nitrosamines with Thermal Energy Analysis
TEA is able to rapidly identify and analyze N-nitroso compounds without the matrix interference that is common with MS detection. Due to the sensitivity and selectivity for nitroso-containing compounds, TEA has been the preferred method of testing in many industries since the 1960s.
Figure 1: Process showing the breakdown and detection of nitrosamines using gas chromatography-thermal energy analysis.
A typical analysis workflow separates compounds using GC before the effluent is directed through a 500°C pyrolyser to initiate a series of reactions (Figure 1) to speciate the nitrosamines. For non-speciated, total nitrosamine analysis, TEA is used after a chemical reaction where the nitrosamines are broken down chemically. The nitric oxide (NO) radical is released due to heat breaking the N-N bond. The gas is passed through a TEA detector. The NO reacts with ozone, which is produced in the unit, to form an electronically excited NO2 molecule. Once this is relaxed, it releases a photon above 600 nm. The NO2 rapidly decays and emits near infrared light that is then detected by a sensitive photomultiplier.
TEA uses selective thermal cleavage of the N-NO bond and the detection of the liberated NO radical reacting with ozone to generate a chemiluminescence signal. TEA systems have a nitrogen chemiluminescence sensitivity of less that 2pgN/second. In addition, TEA detectors can alternate between nitrogen and nitroso/nitro modes, with the latter eliminating nitrogen compound interference.1
An added advantage to TEA is the selection of temperature settings within the pyrolyser to break specific bonds. Bond breakage is largely limited to N-N bonds at 500°C enabling TEA to excel at identifying nitrosamines. Carbon-nitrogen bonds can also be broken by increasing the temperature to 650°C, enabling nitro group identification.3
TEA can also be configured to offer rapid, routine pre-screening of total nitrosamine content in pharmaceutical samples as well as detailed, speciated analysis. Receiving the apparent total nitrosamine content (ATNC), showing both volatile and non-volatile compounds, will enable those compounds below the limit of detection level to be quickly deemed safe. Those compounds with a positive level of ATNC can be further tested to identify the specific nitrosamine compound present.2 The ATNC test allows pharmaceutical manufacturers to meet the requirements of the EMA risk assessment without needing to outsource testing of raw materials or of products at different stages of manufacturing.
Samples for ATNC analysis, rather than undergoing pyrolysis, are injected into refluxing ethyl acetate containing concentrated hydrobromic acid (HBr). This chemical stripping method causes a reaction with the HBr and produces NO, a secondary amine and bromine. The NO is carried toward the TEA by a flow of nitrogen above the headspace of the reaction vessel. A condenser coil prevents the loss of the solvent, and the vapor is cleaned by a secondary cold trap before entering the TEA for analysis.3
Safeguarding Public Health
With nitrosamines linked to increased rates of cancer, safeguarding public health begins with identifying, minimizing, and eliminating nitrosamine impurities from pharmaceuticals and other consumer products. However, nitrosamines impurity detection is far from straightforward with new root causes of contamination continuing to be identified.
The detection of nitrosamines also presents a problem due to their diversity and the methods employed to identify them, with most being time consuming procedures that can delay production. However, using TEA provides a relatively low cost, low maintenance technique that has the sensitivity and selectivity adjustments to identify and speciate nitrosamines at extremely low levels of contamination. TEA allows pharmaceutical manufacturers to quickly and correctly test drug products to indicate the ANTC and mitigate any risk of nitrosamine impurities entering into the process, thus maintaining public health.
References
- Food and Drug Administration, Office of New Drugs. (March 29-30, 2021). Nitrosamines as Impurities in Drugs – Health Risk Assessment and Mitigation Public Workshop https://www.fda.gov/media/150932/download
- Eglovitch, Joanne, (Sept 2020). USP Proposes Analytical Methods for Drug Makers To Detect Nitrosamine Impurities. Pharma Intelligence. Retrieved from https://pink.pharmaintelligence.informa.com/PS142872/USP-Proposes-Analytical-Methods-For-Drug-Makers-To-Detect-Nitrosamine-Impurities
- James, Andrew (July 2020). Identifying Nitrosamines in Pharmaceutical Products. Manufacturing Chemist. Retrieved at https://www.manufacturingchemist.com/news/article_page/Identifying_nitrosamines_in_pharmaceutical_products/167249
- James, Andrew (June 2020). Testing for Nitrosamines in Pharmaceutical Drugs. Pharmaceutical Technology’s In the Lab E Newsletter. Retrieved from https://www.pharmtech.com/view/testing-nitrosamines-pharmaceutical-drugs