BioanalysisAhead of Print Technology EvaluationFree AccessNitrosamine impurities: from raw materials to final drug productNaamah MaundrellNaamah Maundrell*Author for correspondence: E-mail Address: n.maundrell@bioanalysis-zone.comFuture Science Group, Unitec House, 2 Albert Place, London, N3 1QB, UKSearch for more papers by this authorPublished Online:8 Nov 2021https://doi.org/10.4155/bio-2021-0238AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail Keywords: active pharmaceutical ingredientsbioanalysisimpuritiesN-nitrosodiethylamineN-nitrosodimethylaminenitrosaminesraw materialstoxicityWhat are nitrosamines?Nitrosamines are a class of mutagenic impurities which contain the nitroso functional group and are formed when a secondary or tertiary amine reacts with a nitrosating agent [1]. Two common nitrosamines include N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA), which have been classified as potential human carcinogens by the International Agency for Research on Cancer [1,2]. A carcinogenic response is induced when the N-nitrosamines are activated by microsomal liver enzymes and react with DNA. Low levels of these compounds can be found in food and drinks, such as roasted meats, cheese and beer, as well as tobacco and pesticides. However, over the past few years, nitrosamines have received a great deal of global attention after being found in medicinal products [1,3].In 2018, the US FDA announced the detection of NDMA contamination in valsartan. Valsartan is an angiotensin II receptor blocker used to treat high blood pressure. Patients taking valsartan with N-nitrosamine impurities may have an increased risk of cancer, which led to lots of the drug being recalled [1–3]. In the case of valsartan, the nitrosamine impurities were only detected when a more sensitive analytical method was used many years after the initial Chemistry, Manufacturing and Controls modification occurred (a change in the synthetic process of the active pharmaceutical ingredient [API]). After impurities were detected in valsartan, global pharmaceutical and regulatory laboratories have discovered nitrosamine impurities in a growing array of pharmaceuticals (e.g., ranitidine and metformin), which have triggered numerous recalls [2,4].The discovery of unacceptable levels of N-nitrosamines in some drugs led to companies receiving warning letters, global drug shortages and new regulatory actions requiring more stringent detection of the impurities [5]. Mi Jang, Lab Manager and Chief Researcher, Pharma & Tech (Korea) remarked on the likelihood of more rigorous regulations:Many pharmaceutical products made in Korea are exported overseas, and the regulations continue to become more stringent. When these new guidelines for nitrosamine testing came out, we knew we needed to move quickly to setup the services necessary.Larger lessons have been learnt from the drug recalls, with pharmaceutical companies and manufacturers changing how they review their own quality processes for risks that could result in a similar contamination issue [6]. Scientific assessment of raw materials, critical products, process changes and the final drug substance or drug product are necessary to ensure patient safety when the supply chain is so complex [2,3].Testing should be carried out in the intermediate stage and semi-finished conditions on both raw materials and finished products, as nitrosamines can be generated at any point in the process, commented Eun-Joo Joe, Senior Researcher, Pharma & Tech (Korea).Regulatory response to nitrosamine impuritiesBoth the FDA and EMA published guidance recommendations for manufacturers of drug products and APIs to detect nitrosamine impurities in pharmaceuticals [7,8]. Key guidance also define the conditions that may introduce nitrosamine impurities, including side reactions from drug syntheses, the breakdown of unstable drug compounds and contamination from recycled solvents used in manufacturing and packaging [3]. The unexpected detection of nitrosamine impurities also highlighted the need for a risk assessment strategy for potential nitrosamine impurities. In a risk assessment all potential sources of contamination should be considered, including the excipient, drug substance, solvents, water, the manufacturing process and packaging components [4,9]. This means that the pharmaceutical industry should examine the entire drug development and manufacturing process, which can include formulation and process development, as well as raw materials and excipients. However, this has proven to be challenging not only due to complicated manufacturing processes and a complex global supply chain, but also because of the number of different impurities and drug products of concern with many possible techniques for detection and quantification.Much of the initial focus during the nitrosamine impurity crisis was on contaminated APIs and drugs, the final product that reaches patients. Therefore, the need to control nitrosamine impurities at lower detection limits necessitated the consideration of MS detection to quantify these trace compounds [4,9].The regulations on nitrosamine impurities in pharmaceutical products are being extended to all products including chemically synthesized drug substances. According to our developed LC–MS/MS analytical method, an LOQ of 0.025 ng/ml was achieved for eight nitrosamines. In addition, data acquisition satisfying the requirements of GMP regarding data integrity is also possible. With the use of this approach, it is possible to comply with the regulations for nitrosamines that are becoming stricter by the day, explained Hiroki Ami, Analytical Development Subgroup Leader, Production Technology Department, Shionogi Pharma (Japan).Nitrosamine identification & quantificationIn response to the nitrosamine crisis, effort was put into developing sensitive detection methods that could meet required limits. The correct method and technology to analyze these drugs and impurities depends on the required sensitivity for each individual laboratory. This required sensitivity is based on the drug type and the stage where testing takes place during development and manufacturing. As nitrosamine impurities can occur, and be a concern, at extremely low concentration levels, sensitive and specific analytical techniques are required to enable their detection [10].Traditional LC–UV can be used for quick method analysis of raw materials, solvents or excipients; however, MS has become a significant detection method for nitrosamine analysis as it enables the highly sensitive quantification of known impurities required to meet future low detection limits for final API or drug product [4]. Key MS techniques used include GC–MS/MS, LC–MS/MS and HPLC–high-resolution mass spectrometry. These different instruments cover diverse needs, depending on what is being tested and the analytical challenges that arise [4,11]. But how do you navigate the different methods and technologies?Emmanuel Desmartin, Mass Spectrometry & Bioanalysis Lab Manager, Eurofins Biopharma Product Testing (France) commented on the system his lab implemented: We have implemented in December 2020, a new LC–MS/MS system within our laboratory which is the new Xevo TQ-XS from Waters [12]. Linked to UPLC I-Class system and using MassLynx as a software this system will allow us to carry out analyses from bioanalysis to the screening of nitrosamines but also the assays of genotoxic impurities in your pharmaceutical drug products. Due to its high sensitivity, this equipment will be used for analyses for which we need to have very low limits of quantification.Importantly, regulators will accept any platform that can reach regulatory limits with a method that is fit-for-purpose [13]. However, each laboratory must validate the method on their own instruments to confirm it is suitable for the intended use. LC–MS is the preferred technology for high-sensitive quantification of known compounds (used for volatile and non-volatile nitrosamines); whereas, high-resolution mass spectrometry is most appropriate when screening for new and unexpected impurities [1,14]. GC–MS/MS is not an appropriate technique for detecting NDMA levels in ranitidine, due to the possibility of high temperature degradation. Given the complexity and challenges of nitrosamine analysis in both API and drug products, it is important to find the best solution and method based on individual circumstances [1,10].Gabriela Grijalva, Chemical Engineer, Donovan Werke (Guatemala) stated: We looked for an instrument that worked well and was appropriately suited to our needs, to detect these types of impurities at established working concentrations. We also wanted a vendor with experience in the detection of these compounds, as well as support from the brand to implement an accurate, reliable and sensitive analytical method for the determination of nitrosamines in our raw materials, excipients and finished products.For these reasons, the Waters ACQUITY UPLC H-Class with QDa Mass Detector was chosen for our needs, becoming a useful and reliable tool. The UPLC-QDa is sensitive enough for our low working concentrations, so it is reliable for the identification and quantification of impurities in general. The shorter retention time, and therefore shorter run time, had a direct impact on cost and time savings by reducing the amount of solvents and reagents consumed, reducing the time of preparation and analysis and saving work time that can be used to perform other dedicated tasks. With this instrument, the analysis of raw materials, excipients and finished product is carried out.SummaryKey lessons have been learnt from the presence of N-nitrosamines in medicines, most importantly how crucial it is to maintain a safe supply of medications [9,15]. Scientists and regulators must work together to ensure that analytical platforms used for nitrosamine analysis can reach regulatory limits with a method that is fit-for-purpose. Although traditional LC–UV can be used for quick method analysis of raw materials, solvents or excipients, MS has become the analytical platform of choice for these genotoxic impurities as it enables the highly sensitive quantification of known impurities required to meet future low detection limits for final API or drug product. Since nitrosamine contamination can have multiple possible sources, either with the API, final drug product, excipients or solvents, it is important that the chosen method is fit for its intended purpose to accurately detect and quantify nitrosamine impurities [4].DisclaimerThis article has been drawn from the discussions from the Technology Digest article published in Bioanalysis Zone, which was sponsored by Waters Corporation. The opinions expressed in this feature are those of the author and do not necessarily reflect the views of Future Science Group.Financial & competing interests disclosureN Maundrell is an employee of Future Science Group and Editor-in-Chief of Bioanalysis Zone. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.