Trends and Challenges in Nitrosamine Testing: Part One – Scoping the Problem

When did nitrosamines first enter your radar?
 

Kevin Parker: As a graduate student, I remember reading in science journals and seeing in the news that n-nitrosodimethylamine (NDMA) was found in batches of a major drug called Valsartan. While I wasn’t in the pharmaceutical industry yet, my graduate research utilized mass spectrometry so I was interested in how nitrosamines would impact analytical chemists, as the extremely low levels allowed on a per-day basis require sensitive methodologies. In the years after the discovery of NDMA, conferences at Pittcon, ACS, and the American Society for Mass Spectrometry had a noticeable increase in the attention given to nitrosamines and the analytical challenges they pose.

Naiffer Romero: Nitrosamines entered my radar following the detection of Nitrosodimethylamine (NDMA), a probable human carcinogen in valsartan, a commonly-prescribed medication for hyper-tension, which triggered a global investigation of nitrosamines. This led to the discovery of nitrosamines in other pharmaceutical products like pioglitazone, ranitidine, nizatidine, and metformin, used to treat conditions ranging from heartburn to bacterial infections.

Jessica Hoskins: I first became aware of nitrosamines somewhere around 2018 or 2019. At the time I worked at a surfactant manufacturer, and some customers began to ask about the risk of small molecule nitrosamines or precursors in our products.

Jingyue Yang: Like Jessica, I first became aware of nitrosamines around 2018 when the FDA was notified that N-nitrosodimethylamine (NDMA) had been detected in valsartan drug products. Although nitrosamines had been extensively studied in fields like food and environmental science, this was the first significant instance of nitrosamine impurities being reported in pharmaceuticals, which quickly drew widespread attention.

At that time, our office was asked to develop analytical methods to quantify NDMA and other small-molecule nitrosamines. Although I wasn’t initially involved, I was later brought in to develop an LC-MS method to detect and quantitate those small molecule nitrosamines, which could be challenging for the GC-MS methodology used at the time.

We then developed our first LC-MS method, capable of analyzing six different nitrosamines in valsartan and losartan drug products. As nitrosamine issues in pharmaceutical products continued to expand and evolve, we applied the knowledge gained from these early efforts to develop a variety of methods for nitrosamine analysis.

Alan Thompson: At Almac Sciences, we have been developing mass spectrometric methods to detect and quantify genotoxic impurities at extremely low concentrations in both Active Pharmaceutical Ingredients (APIs) and formulated drug products for a number of years. Nitrosamines have been on our radar for a while, but the scope of our work was largely confined to the monitoring of upstream by-products identified during drug synthesis. As a result our methods were more geared towards ensuring that any nitrosamine by-products were adequately purged. 

They started to come to more prominence a few years back when nitrosamine impurities, including N-nitrosodimethylamine (NDMA), were detected in blood pressure medicines known as “sartans.” This prompted the Committee for Medicinal Products for Human Use (CHMP) to ask marketing authorisation holders to review all chemical and biological human medicines for the presence of nitrosamines and to test products at risk following guidelines issued by the European Medicines Agency (EMA). Since then multiple products have been identified as potential sources of nitrosamines; notably in drug products, where interaction between API and excipient components can increase the probability of their formation. 

Are they dangerous?
 

Jingyue Yang: Nitrosamines are classified as possible or probable carcinogens, but not all nitrosamines carry the same level of risk. Probable carcinogens such as NDEA are assigned an acceptable intake of 26.5 ng/day due to their high potency, while others have lower potencies and may be controlled at the threshold of toxicological concern (1500 ng/day).

Naiffer Romero: I’d add that according to the International Agency for Research on Cancer (IARC), some nitrosamine impurities are identified as probable or possible human carcinogens that may cause harm to patients in cases where acceptable intake (AI) levels are too high, or exposure is sustained over a long period of time. While exposure to nitrosamines can be dangerous, we have come to learn that not all nitrosamines carry the same risk level.

Alan Thompson: There is substantial documented evidence of the carcinogenic and mutagenic properties of these compounds. The wide range of human exposure sources such as food, drugs and cosmetics in addition to occupational sources such as cutting oils and hydraulic fluids, coupled with the presence of endogenous precursors within the digestive system, has prompted the necessity for regulatory action and the search for evermore sensitive methods of analysis. 

Guidance for industry regarding the recommended acceptable intake limits for Nitrosamine Drug Substance Related Impurities (NDSRIs) have been issued by the Food and Drug Administration (FDA), and is graded based on the carcinogenic potency category of the nitrosamine compounds in question. The potency of the nitrosamine is dependent on the structure of the compound and can range from an acceptable intake (AI) of 26.5ng/day for Category 1 to 1500 ng/day for Category 5.

What are the main types of nitrosamines?
 

Kevin Parker: Within n-nitrosamines two classes of molecules arise. One subset is derived from common amine building blocks such as dimethylamine, diethylamine, diisopropylamine, etc. The n-nitrosamines formed from these common secondary amines make up what is dubbed the “cohort of concern”: a group of seven n-nitrosamines which all have stringent daily limits assigned to them as low as 26.5 ng/day. The other group of n-nitrosamines are formed by reactions of APIs or other related compounds along the synthetic route, forming what’s known as “n-nitrosamine drug substance related impurities” (NDSRIs). NDSRIs have varying control limits based on the potential carcinogenicity associated with their structure. 

Jingyue Yang: I’d add that nitrosamines can form in pharmaceuticals through manufacturing processes that involve certain solvents, reagents, or contaminated materials (or from leaching through packaging). Many of these nitrosamines have been extensively studied in other industries. On the other hand, NDSRIs are nitrosamines formed from drug substances or their related impurities that contain vulnerable nitrogen atoms within their molecular structure. 

How does nitrosamine analysis differ between the pharmaceutical industry and in other industries, such as food and environmental?
 

Jessica Hoskins: In the pharmaceutical industry, we must consider the risks for forming both small molecule nitrosamines and NDSRIs. While aliphatic and aromatic amines are important for the drug-like properties that provide efficacy, these are the same functional groups that can be nitrosated to form nitrosamines. While standard methods exist for small molecule nitrosamines, testing for formation of NDSRIs can introduce additional analytical challenges, as NDSRIs are typically unique to each project’s chemistry. In other industries small molecule nitrosamines are typically the main area of concern; and in some cases they may not be regulated to as low of levels as they are in the pharmaceutical industry.

Jingyue Yang: In my experience, there aren’t any significant differences in the core analytical techniques used for the detection, identification, and quantitation of nitrosamines. Mass spectrometry (MS) is the most (and often the only) suitable instrument for nitrosamine analysis due to its high selectivity and sensitivity, which is crucial since nitrosamines are typically present at very low levels. Gas or liquid chromatography is generally used prior to MS analysis to achieve the necessary separation of compounds.

Alan Thompson: I agree, the same analytical techniques including HPLC-UV, GC-MS and LC-MS are used to identify and quantify nitrosamines in the pharma, food and environmental industries, as method sensitivity and specificity are crucial. Where the analysis would differ is in the sample extraction process, with extraction from food and environmental samples seeing more involvement due to a larger number of components in the sample matrix. These samples often require solid phase extraction steps and rigid internal standards to correct for the potential loss of nitrosamines in the extraction process.

Stay tuned for part two, where our experts return to discuss the main analytical challenges associated with nitrosamines 

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