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 Background, Advantages of, and Considerations for Radiolabeled Peptides The use of radiolabeled peptides is a well-established tool in researching and treating many diseases and conditions. Selective receptor-targeting peptides are utilized as agents due to their rapid circulatory and tissue clearance and the high affinity and specificity to their targets. Peptides also have a relatively small size and low molecular weight compared to proteins and antibodies. There have been innovations and improvements in the design of peptides that incorporate chemical modifications with “impressive diagnostic accuracy and sensitivity.”1 Coupling these peptides with radiolabeling for peptide receptor radionuclide imaging (PRRI) and therapy (PRRT) has yielded remarkable results. In fact, a historical summary of radiolabeled peptides asserts, “The emergence of radiolabeled peptides for use with PET/CT such as 68Ga, 18F, and 64Cu, and the use of new receptor binding ligands…, have revolutionized PRRI and improved its diagnostic power beyond expectation.”2
“Criteria for a successful peptide tracer,” to be utilized for PRRI, “are a high target specificity, a high binding affinity, long metabolic stability, and a high target-to-background ratio.”3
Oncology and Radiolabeled Peptides Oncology has benefited from the “tumor-philic” properties of Arg-Gly-Asp (RGD) peptides “because of their high affinity and selectivity for integrin αvβ3 – one of the most extensively examined targets of angiogenesis. Since the level of integrin αvβ3 expression has been established as a surrogate marker of angiogenic activity, imaging αvβ3 expression can potentially be used as an early indicator of the effectiveness of antiangiogenic therapy at the molecular level.”4 In addition to integrin αvβ3 expression, “tumor angiogenesis…has been well recognized as an essential hallmark for tumor growth, invasion, and metastasis.”4 All this has made RGD-containing peptides “promising molecular agents for imaging angiogenesis.”5
Beyond Oncology Since integrins are “involved in adhesion between cells and the extracellular matrix” and, as such, are associated with normal and pathological states, this family of receptors is useful for targeting a range of diseases.6 Current applications include “cardiovascular imaging, atherosclerosis imaging, remodeling after myocardial infarct or stroke, imaging of myocardial ischemia, identification of abdominal aortic aneurysm, imaging of chronic inflammation, pulmonary inflammation, assistance with bone marrow evaluations, and tissue engineering.”6
A Brief History of RGD Peptides and Radiolabeling Although radiopharmaceutical therapies, which can concurrently detect and treat tumors (i.e., theranostics, a portmanteau of therapeutics and diagnostics), have been around for eighty years, it was not until the combination of these payloads was combined with peptides that the potential for better targeting became a reality. The use of the RGD peptide sequence to attach to cells was first reported by M.D. Pierschbacher and E. Ruoslahti in Nature, nearly forty years ago in 1984, as a feature of fibronectin.5,7 Next, targeting tumors with radioactive peptides began, initiated by OctreoScan’s breakthrough in the early 1990s, wherein somatostatin receptor subtype 2 (SST-2) positive tumors were identified.8,9 After that, the first monomeric integrin-specific PET tracer used in patients was F-Galacto-RGD, a glycosylated RGD-peptide.5,10 Since then, the tripeptide R-G-D sequence has generally been utilized as a tracer, carrying the isotope to integrins that are expressed on both tumor cells and activated endothelial cells of tumor neo-vasculature. In the ensuing years, advancements have continued apace, and going forward, the intersection of PRRI/PRRT from radiolabeled peptides combined with AI, precision, and personalized medicine assures transformative innovations.
Obstacles and Numerous Options for Radiolabeled Peptide Production Because of their established safety, development, and design history, and “the fact that there are many RGD-based tracers with known pharmacokinetics, it can be useful to use them in the imaging of diseases that currently have no accurate method of diagnosis available.”6 However, there are barriers to synthesizing radiolabeled peptides: itcan be a time-consuming, complex, multi-step process. It is also highly variable based on the peptide. Other drawbacks include the intricacies of radiolabeling and the lack of automation for some of these protocols.
Fortuitously, RGDs radiopeptides are not the only ones exhibiting exciting potential for diagnostic imaging and targeted radionuclide therapies.An extensive review article from Paweł Kręcis et al. that appeared in Bioconjugate Chemistry is recommended, as it presents some developments and perspectives in both aforementioned areas regarding the research on somatostatin, bombesin, vasoactive intestinal peptide, gastrin, neurotensin, and exendin peptide analogs, among others.11 It includes the application of radiolabeled peptides and antibodies and an analysis of clinical trials reported in 2008-2018.
Finally, just a few of the current companies doing interesting things in radiopharmaceuticals include Aktis Oncology, Mariana Oncology, Point Biopharma, and Rayze Bio. AMPAC Fine Chemical and AMPAC Analytical, both SK pharmteco companies, have been at the vanguard of small molecule and analytical developments utilizing innovative technologies and techniques directed by teams with decades of experience. If you have a radiolabeled peptide targeted for development, our teams can design, validate, optimize, and analyze your project, ranging from research to commercial quantities. Contact us today for more information.
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.
Extractables and Leachables (E&L) are essential areas of concern for the pharmaceutical and food industries, specifically regarding their packaging, usage components (e.g., medical devices or syringes), and the manufacturing chain. We will examine testing of analysis of them within pharmaceutical applications. The two terms are related but distinct, each with its own analytical requirements.
Definitions of Extractables and Leachables
A handy article published in Pharmaceutical Engineering by the International Society for Pharmaceutical Engineering (ISPE) explains that “Extractables are chemical compounds that migrate from single-use systems (SUS) into model solvent solutions under controlled and exaggerated conditions depending on temperature, pH, polarity, and time.” In other words, this happens when using strong solvents. They note that “SUS are normally not exposed to such conditions in biopharmaceutical processes.”1
ISPE’s article defines leachables as “chemical compounds that migrate from SUS into process solutions under normal biopharmaceutical process conditions. They further clarify that these compounds “may end up in the final drug product formulation. For the most part, leachables are a subset of extractables, although interaction with product components may produce leachables not seen as extractables.”1
Guidance on Extractables and Leachables
The FDA has released a series of guidelines for the pharmaceutical industry, including Container Closure Systems for Packaging Human Drugs and Biologics, that provide guidance for submission in support of an original application for any drug product. It also covers a wide range of forms and delivery systems of drugs:
Solid oral dosage forms
Topical and topical delivery systems
Powders for reconstitution
And other dosage forms
Additionally, the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) also has issued the ICH Q3E: Guideline for Extractables and Leachables.2,3 These are both useful in providing direction for E&L concerns and control strategies.
Plan Against Extractables and Leachables
To guarantee adherence to all guidelines and regulations while ensuring patient safety, it is crucial to know and utilize materials compatible with your product. To accomplish this, solvent use, packaging, and delivery systems must all be tested and analyzed in cGMP and FDA-compliant laboratories. This should include the following:
A thorough review of all materials used in packaging and production, production,and equipment to predict the compatibility of your packaging system with your product. AAL can provide reports for items from each step.
Extraction studies on the materials used.
Leachable studies to identify any impurity resulting from those materials found in the final product under normal usage conditions.
If impurities are detected, AAL can provide toxicological evaluations, including profiles of the impurities and the risks they pose for the patients, establish safety limits, or adjust for different forms of medication application.
We can assess risks created by various exposure levels due to the impurity in the finished product.
Finally, AAL provides a detailed report of our findings in accordance with the applicable governing bodies (e.g., FDA, EMA, PQRI, PDA).
AMPAC Analytical can review your analysis and testing needs for extractables and leachables for any forms and delivery systems listed in the table above, complying with the strictest standards necessary.
A special thanks to Matt Webberley, Associate Director Analytical Research and Development at our sister company, SK biotek Ireland Limited, for his assistance with the profiles of AAS, ICP-OES, ICP-MS, and XRD.
A Definition of These Newsmakers Elemental impurities in food have been in the news recently, with reports of everything from lead being found in baby food to arsenic, cadmium, and assorted heavy metals in dark chocolate and other foods.1-8 Of course, the FDA and Congress are taking notice.3The US Pharmacopeia definition of elemental impurities states they “include catalysts and environmental contaminants that may be present in drug substances, excipients, or drug products. These impurities may occur naturally, be added intentionally, or be introduced inadvertently (e.g., by interactions with processing equipment and the container closure system).”9 As recent news reports show, the concern can be expanded beyond drugs and APIs (active pharmaceutical ingredients) to include food and beverages.
Classifying Elemental Impurities In addition to the recalls and resources, there are a variety of impurity classification levels, too. In the following table, the FDA and EMA sort them by elements.12,13They are classified into three categories based on their toxicity and also based on occurrence in the drug product (note: this is usually assumed to be the same as for the drug substance but not always).
Class 1: The elements, As, Cd, Hg, and Pb are human toxicants that have limited or no use in the manufacture of pharmaceuticals. Their presence in drug products typically comes from commonly used materials (e.g., mined excipients). Because of their unique nature, these four elements require evaluation during the risk assessment across all potential sources of elemental impurities and routes of administration. The outcome of the risk assessment will determine those components that may require additional controls, which may, in some cases, include testing for Class 1 elements. It is not expected that all components will require testing for Class 1 elemental impurities; testing should only be applied when the risk assessment identifies it as the appropriate control to ensure that the PDE will be met.
Class 2: Elements in this class are generally considered as route-dependent human toxicants. Class 2 elements are further divided into sub-classes 2A and 2B based on their relative likelihood of occurrence in the drug product.
• Class 2A elements have [a] relatively high probability of occurrence in the drug product and thus require risk assessment across all potential sources of elemental impurities and routes of administration (as indicated). The class 2A elements are: Co, Ni, and V.
• Class 2B elements have a reduced probability of occurrence in the drug product related to their low abundance and low potential to be co-isolated with other materials. As a result, they may be excluded from the risk assessment unless they are intentionally added during the manufacture of drug substances, excipients, or other components of the drug product. The elemental impurities in class 2B include: Ag, Au, Ir, Os, Pd, Pt, Rh, Ru, Se, and Tl.
Class 3: The elements in this class have relatively low toxicities by the oral route of administration (high PDEs, generally > 500 μg/day) but may require consideration in the risk assessment for inhalation and parenteral routes. For oral routes of administration, unless these elements are intentionally added, they do not need to be considered during the risk assessment. For parenteral and inhalation products, the potential for inclusion of these elemental impurities should be evaluated during the risk assessment unless the route-specific PDE is above 500 μg/day. The elements in this class include: Ba, Cr, Cu, Li, Mo, Sb, and Sn.
Other elements: Some elemental impurities for which PDEs have not been established due to their low inherent toxicity and/or differences in regional regulations are not addressed in this guidance. If these elemental impurities are present or included in the drug product, they are addressed by other guidance and/or regional regulations and practices that may be applicable for particular elements (e.g., Al for compromised renal function; Mn and Zn for patients with compromised hepatic function), or quality considerations (e.g., presence of W impurities in therapeutic proteins) for the final drug product. Some of the elements considered include: Al, B, Ca, Fe, K, Mg, Mn, Na, W, and Zn.
Beyond the FDA, EMA, and the USP, other assets are available to industries, including testing for and mitigating these elemental impurities. Detection by testing plays a crucial role in ensuring the quality and safety of food, beverages, and medical products. In a very relevant article in Pharmaceutical Technology, published two years ago, Felicity Thomas states, “The most commonly used techniques to analyze elemental impurities are inductively coupled plasma–mass spectrometry (ICP–MS) or inductively coupled plasma–optical emission spectroscopy (ICP–OES).” Both systems utilize high-energy plasma charges that ionize any elements present in the sample preparation and detect them using elemental masses or emission bands. The authors quote Paul Kippax, Pharmaceutical Sector director at Malvern Panalytical, who says, “The advantage of using ICP is that it can detect a wide range of elemental impurities at very low concentrations. This [capability] enables the product safety requirements for the main product types (oral solid dose, inhaled, and injectable products) to be assessed.”14However, elemental impurity testing is not limited to ICP-MS or ICP-OES. Other techniques available include material characterization (including particle size and thermal analysis), chromatography, x‐ray diffraction and foreign matter identification, and NMR (nuclear magnetic resonance) spectroscopy, each with specific advantages or limitations depending on factors such as time, budget, material or impurities and levels being tested. Here is each profile:
Atomic Absorption Spectroscopy (AAS) Atomic absorption spectrometry (AAS) detects elements in either liquid or solid samples through the application of characteristic wavelengths of electromagnetic radiation from a light source. Individual elements will absorb wavelengths differently, and these absorbances are measured against standards. In effect, AAS takes advantage of the different radiation wavelengths that are absorbed by different atoms. In AAS, analytes are atomized by an Air/Acetylene or Nitrous oxide/Acetylene flame so that their characteristic wavelengths are emitted and recorded. When a hollow cathode lamp is passed into the cloud of atoms, the selected metals to monitor absorb the light from the lamp, and the concentration is measured by a detector. Most of the elements reach excitation temperature using this source, which has a maximum temperature of 2,600 °C. For a few elements, such as V, Zr, Mo, and B, the source temperature is not sufficient to atomize the molecules, and as a result, sensitivity is reduced. Moderate detection limits and not all elements can be determined by AAS are some of the limitations of atomic absorption spectroscopy.
Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES or ICP-AES) The source in ICP-OES is plasma at temperatures as high as 10,000 °C, where all elements, including refractory elements, atomize with higher efficiencies than in AAS. As a result, elements can be determined more precisely, and lower limits of detection levels are possible. There are two variants in ICP-OES, radial and axial. Axial viewing increases the path length and reduces the plasma background signal [over radial viewing], resulting in lower detection limits. ICP-OES is a multi-element technique. Under the source of plasma, the sample dissociates into its atoms and ions. At their excitation level, they emit light at characteristic wavelengths. The concentration of a particular element in the sample can be measured from the intensity of the emitted light with a detector. However, the detection limits in ICP-OES are moderate to low.
Inductively Coupled Plasma-Mass Spectrometry ICP-MS (ICP-MS) The same source as in ICP-OES is used to dissociate a sample into atoms and ions. It is also a multi-element technique. The basic difference between ICP-OES and ICP-MS is that ions are directly detected by an MS detector rather than by emission of light, as in the case of ICP-OES. The ions are separated by a Quadrupole based on the mass-to-charge ratio. The best detection limits are available for most of the elements in ICP-MS as the number of ions produced is high, and although some spectral interference is seen, these are defined and limited.
X-ray fluorescence (XRF) X-ray fluorescence (XRF) is a non-destructive analytical technique used to determine the elemental composition of materials. Analyzers determine the chemistry of a sample by measuring the fluorescent (or secondary) X-rays emitted from a sample when it is excited by a primary X-ray source. Each element in a sample produces a set of characteristic fluorescent X-rays (“a fingerprint”) unique for that specific element, which is why XRF spectroscopy is an excellent technology for qualitative and quantitative analysis of material composition. The ICP-OES technique has better sensitivity and lower detection limits compared to XRF. Therefore, using XRF for determining lower levels has higher errors, and the correlation with ICP-OES is weaker.
Recalls and Resources The FDA’s actions on contamination from elemental impurities range from issuing guidance to product recalls. There are three class recall levels (I-III) and two related activities: a market withdrawal and a medical device safety alert.10 The former of these are voluntary, while the latter can be considered, in some cases, a recall.
Additionally, the FDA has a range of resources available for both consumers and manufacturers. For consumers, these include:
The European Medicines Agency also provides numerous guides and requirements on their site, including the International Council for Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use’s ICH Q3D Elemental impurities – Scientific Guideline.11
Final Thoughts on Elemental Impurities
Whether it is in food and beverages or drug products, there is increased scrutiny by regulatory agencies and consumers on the presence of elemental impurities. Industries hoping to avoid costly product withdrawals, recalls, and possible litigation is advised to safeguard the quality and safety of food, beverages, and medical products by testing for elemental impurities. AMPAC Analytical has decades of experience and the full array of equipment and methods to ensure our products avoid these worst-case scenarios. Furthermore, these analytical services are accompanied by a devoted customer focus, with communication and guidance at each step to assist with all regulatory and filling requirements.
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.
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.
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
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
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
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.
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