Forced Degradation is an important addendum to our previous post on Stability and Storage. Stressors are applied to new APIs and drug products to determine their degradation pathways and products under a variety of environmental conditions, including acid, base, light, heat, and oxidation. Forced degradation studies are also known as stress testing, stress studies, stress decomposition studies, and forced decomposition studies. These conditions “…are more severe than accelerated (stability) conditions and thus generate degradation products that can be studied to determine the stability of the molecule.”1
Regulatory requirements for forced degradation were enacted by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) in 1993.2 However, these guidelines “are very general in (the) conduct of forced degradation and do not provide details about the practical approach towards stress testing. Although forced degradation studies are a regulatory requirement and scientific necessity during drug development, it is not considered as a requirement for (a) formal stability program.”1 However, stability studies have become a requisite for new drug moieties. In the absence of specific guidelines, the amount of stress needs to be representational: “Overstressing a molecule can lead to degradation profiles that are not representative of real storage conditions and perhaps not relevant to method development. Therefore, stress-testing conditions should be realistic and not excessive.”3
AMPAC Analytical (AAL), an SK pharmteco company, can assist with forced degradation studies for products at all phases of development, in tandem with stability, storage, and method development, to ensure the viability of the drug products as they were designed. We introduce forced degradation studies early in method development to ensure your product quality throughout the development lifecycle. Contact AAL today to learn more.
The CGMP guidance for APIs from the FDA states that raw material specifications should be established and documented. The guide’s key line states, “Quality measures should include a system for testing raw materials, packaging materials, intermediates, and APIs. (19.23)”1
All raw materials used in producing APIs for clinical trials must be evaluated by testing or received from the supplier with accompanying analysis and subsequently subjected to identity testing. Raw materials and intermediates need to be designated by names and/or specific codes so that any special quality characteristics can be readily identified. Furthermore, written procedures should provide for the identification, documentation, appropriate review, and approval of any changes to raw materials. Additionally, changes to supply sources of critical raw materials should be treated according to the FDA’s established change control guidelines.
A Range of Tests for Raw Materials Are Available Some of the categories and tests that can be utilized for raw materials testing include:
Determination of Physical Properties (appearance/description, density, refractive index, pH, water content by Karl Fischer titration (coulometric and volumetric), the color and clarity of the solution, conductivity, optical rotation, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), osmolality, particle counting, particle size distribution (wet and dry), total organic carbon (TOC), and various compendial tests)
Identification (appearance/description, infrared spectroscopy – ATR, salt pellets, and salt plates (for liquids), nuclear magnetic resonance (NMR), liquid chromatography – HPLC and UHPLC, gas chromatography (GC), ion chromatography (IC), mass spectrometry (MS), ultraviolet spectroscopy (UV), X-ray powder diffraction (XRPD), residue on ignition/sulfated ash, ICP-MS and ICP-OES for elemental impurities, and various compendial tests)
Assay and Impurity Testing (standard titration methods, liquid chromatography (both HPLC and UHPLC) detection systems including UV, MS, RI, and CAD (charged aerosol detection), residual solvents testing utilizing gas chromatography systems equipped with FID flame-ionization detection), ECD (electron capture detection), TCD (thermal conductivity detection) and MS, ICP-OES, ICP-MS, and a variety of pharmacopeia methods such as residue on ignition/sulfated ash, heavy metals, etc.)
Pharmacopeia Testing (the ability to qualify and implement monographs and testing chapters from the various pharmacopeias and their standards, including USP (United States Pharmacopoeia), EP (European Pharmacopoeia), BP (British Pharmacopoeia), JP (Japanese Pharmacopoeia), FCC (Food Chemical Codex), and ACS (American Chemical Society, Reagent Standards))
Trust – and Verify – Your Raw Materials Testing Solution The range of testing requirements, procedures, and record-keeping can be daunting. It is crucial to have an experienced, reputable, and thorough laboratory available to ensure that each raw material is released in accordance with regulations. It is also important that the partner you choose to perform these tasks does so in a timely manner, communicating every step of the way. AMPAC Analytical has decades of experience along with the resources to provide all the analytical solutions listed above, combined with a responsive customer service attitude, and a demonstrated history of regulatory audit compliance. We urge that you contact AMPAC Analytical today to learn more about you can trust and verify all your raw materials.
This is the third in a series of entries examining nitrosamines in a range of products. Our first of two previous articles presented an overview of nitrosamines, including a historical look at their implication as a probable carcinogen. In the second entry, we reviewed their presence in active pharmaceutical ingredients (APIs), and how to remove them.
Nitrosamines are organic compounds found in the human diet and other environmental sources. These highly potent carcinogens can cause tumors in nearly all organs and have been classified as genotoxic impurities (GTI).
Background on Nitrosamines in Food and Beverages The possible linkage between cancer and the large class of chemical compounds known as nitrosamines was first postulated by William Lijinsky in 1970.1 Since then, they have been detected above recommended intake limits in numerous foods and beverages, both naturally occurring and through additives in processed foods. Nitrosamines have been found in a wide variety of different foods ranging from cheeses, soybean oil, canned fruit, meat products, cured or smoked meats, fish and fish products, spices used for meat curing, beer, and other alcoholic beverages.2,3 Beer, meat products, and fish are considered the main sources of exposure. “Drying, kilning, salting, smoking, or curing promotes the formation of nitrosamines.2,4
Nitrites and nitrates may occur naturally in water or foods such as leafy vegetables due to the use of fertilizer or may be added to foods to prevent (the) growth of Clostridium botulinum, or to add color or flavor.”2,5
The nitrosamines most frequently found in food are N-nitrosodimethylamine (NDMA), N-nitrosopyrrolidine (NPYR), N-nitrosopiperidine (NPIP), and N-nitrosothiazolidine (NTHZ).2,3 NDMA, NPYR, and NPIP are reasonably anticipated to be human carcinogens based on evidence of carcinogenicity in animal experiments.2,6,7Evidence from case-control studies supports an association between nitrosamine intake with gastric cancer, but not esophageal cancer in humans.2,8
Determining Acceptable Levels of Nitrosamine Levels of nitrosamines have been declining during the past three decades, concurrent with a lowering of the nitrite use in food, use of inhibitors such as ascorbic acid, and application of lower operating temperatures and indirect heating during food processing.2,4
A triple quadrupole MS
The FDA provides “action levels” for poisonous or deleterious substances found in human food and animal feed. These action levels and tolerances represent limits at or above which FDA will take legal action to remove products from the market.9 Current FDA regulations do not limit nitrosamine levels in foods, but they have established an action level of 10 ppb for individual nitrosamines in both consumer and hospital rubber baby bottle nipples. They have also limited the approval of nitrites in curing mixes to the FDA-regulated food additive process (21 CFR 170.60), and the approval of sodium nitrite as a food additive (food preservative) (21 CFR 172.175). The USDA monitors finished meat products to ensure that nitrite is not present in amounts exceeding 200 ppm (9 CFR 424.21).2
As investigators summarized in a study published in the World Journal of Gastroenterology, “there is a positive association between nitrite and nitrosamine intake” and gastric cancer, “between meat and processed meat intake and” gastric cancer and esophageal cancer, “and between preserved fish, vegetable, and smoked food intake and” gastric cancer, “but is not conclusive.”8 While there is not an irrefutable link between nitrite and nitrosamine intake to cancer when combined with action-level requirements and guidance from the FDA, the directive for food and beverage producers is certainly clear.
Final Thoughts
Nitrosamines are an inevitable chemical outcome in the manufacturing and processing of many foods, beverages, medicines, and numerous other products. Due to their low concentrations, they are also challenging to detect. Fortunately, rigorous testing services are available to screen and remove them from exposure by the end user. AMPAC Analytical has the specialized expertise, equipment, and methodologies to detect these impurities by gas chromatography or high-performance liquid chromatography coupled with mass spectrometry. Please contact us with any specific questions or to receive a quote for nitrosamines.
References Items marked with an asterisk are open access.
This is the second in a series of entries examining nitrosamines in a range of products. Our first article presented an overview of nitrosamines, including a historical look at their implication as probable carcinogens. This entry will review their presence in active pharmaceutical ingredients (APIs) and process mitigation strategies.
Nitrosamines are organic compounds found in medications, the human diet, and the environment These carcinogens can cause tumors in nearly all organs and have been classified as possible genotoxic impurities (GTI).
Background on Nitrosamines in Active Pharmaceutical Ingredients The linkage between cancer and a large class of chemical compounds known as nitrosamines was postulated by William Lijinsky in 1970.1 Then, in June2018, their presence (specifically, N-nitroso-dimethylamine (NDMA)) was detected in the API Valsartan, an Angiotensin-II-receptor antagonist.
NDMA
It later became “obvious that the issue may not only occur with sartans but, in principle, with any API containing a vulnerable amine and a nitrosation source. Hence not only NDMA but a plethora of potential nitrosamines could be created.”2 They have been subsequently detected in other medicines resulting in 250 product recalls, affecting more than 1400 lots.3,4The cost of recalls could be high.5APIs or their impurities can become nitrosated “during the later stages of the synthetic process of the drug product manufacturing or even while in the completed, packaged product.”6 As discussed in our previous entry, primary amines are not a concern, as they have limited stability.6 However, secondary and tertiary amines, along with quaternary ammonium compounds, are considered potential nitrosamine precursors, according to the current guidelines of the FDA and EMA.6,7As a useful reference for amine components, there is a central system for the ingredients in medicinal products known as the Global Substance Registration System (GSRS https://gsrs.ncats.nih.gov/.)8 Some of the possible causes for the presence of nitrosamines are:
The use of sodium nitrite (NaNO2), or other nitrosating agents.
The use of raw materials and intermediates contaminated by nitrosamines
Degradation processes of starting materials, intermediates, and drug substances during formulation or storage
The use of certain contaminated packaging materials
Detection Tools
Fortunately, there are many tools to detect nitrosamines. NDMA, NDEA, and other nitrosamine impurities can be detected at ppb level using gas chromatography, such as with a QTOF (Quadrupole Time of Flight Mass Spectrometer) or triple quadrupole.
Ways to Mitigate Nitrosamine Formation
There are numerous ways that nitrosamines can be mitigated through API process design. For example, the FDA’s Control of Nitrosamine Impurities in Human Drugs Guidance for Industry, issued by the Center for Drug Evaluation and Research, states that: “The following factors should be considered during process development:
Avoiding reaction conditions that may produce nitrosamines whenever possible; when not possible, demonstrating that the process is adequately controlled and is capable of consistently reducing nitrosamine impurities through appropriate and robust fate and purge studies.
Using bases other than secondary, tertiary, or quaternary amines (when possible) if ROS (Route of Synthesis) conditions may form nitrosamines
Using caution when the ROS involves the use of amide solvents (e.g., N,N-dimethylformamide, N,N-dimethylacetamide, and N-methylpyrrolidone)
Replacing nitrites with other quenching agents for azide decomposition processes.
Optimizing and consistently controlling the sequences of reactions, processes, and reaction conditions (such as pH, temperature, and reaction time)
Designing a manufacturing process that facilitates the purge of nitrosamine impurities in the subsequent processing steps.
Auditing API supply chains accompanied by continuous monitoring for any at-risk raw materials, starting materials and intermediates, and avoiding cross-contamination when using recovered materials such as solvents, reagents, and catalysts in the manufacturing process.
Recovered material should be used only in the same step or in an earlier step. API manufacturers should be aware that potable water used in API manufacture may contain low levels of nitrite and even nitrosamines from environmental contamination”.9,10
Solutions
Nitrosamines are an inevitable chemical outcome in the manufacturing and processing of many items, including APIs. Due to their low concentrations, they are also challenging to detect. AMPAC Analytical has rigorous testing services available to screen to trace levels in challenging sample matrices, including process intermediates, drug substances, and drug products. We have the specialized expertise, equipment, and methodologies to detect these impurities by gas chromatography or high-performance liquid chromatography coupled with mass spectrometry. Please contact us with any specific questions or to receive a quote for nitrosamines screening.
This is the first in a series of entries examining nitrosamines in a range of products.
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 asgenotoxic 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. Haldenwrote 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).”1Concern 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.”2Primary 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”.”1Almost 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 APIsfrom 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 medicinesresulting 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.
Forced Degradation is an important addendum to our previous post on Stability and Storage. Stressors are applied to new APIs and drug products to determine their degradation pathways and products under a variety of environmental conditions, including acid, base, light, heat, and oxidation. Forced degradation studies are also known as stress testing, stress studies, stress decomposition studies, and forced decomposition studies. These conditions “…are more severe than accelerated (stability) conditions and thus generate degradation products that can be studied to determine the stability of the molecule.”1 
NDMA