Tag Archives: nitrosamine

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Gas Chromatography – An Introduction

A Brief History of Gas Chromatography
Gas chromatography (GC) is one of the most important and prevalent analytical tools available to chemists. It was invented in 1952 by A.T. James and A.J.P. Martin as an outgrowth of research dating back to the previous decade.1-6 Their early techniques on adsorption and partition enabled some of the later developments that A.J.P. Martin spoke about at the 1957 Lansing Symposium, entitled, “Past, Present and Future of Gas Chromatography.” He concluded his address with the following prediction: “If we tie the gas chromatograph to other pieces of laboratory equipment, we have the possibility of almost the automatic chemist…”2 While the idea of an “automatic chemist” hasn’t quite come to fruition, Martin’s belief “that the uniting instrument of the gas chromatograph in the center” of his lab of the future certainly has.2 With the meteoric rise of GC7, its adoption and adaptation continued unabated in the ensuing decades. Then, “The introduction of robust, efficient, and reproducible fused-silica capillary columns and the provision of relatively inexpensive but reliable equipment for GC-MS provided a crucial new impetus in the 1980s.”1 Interestingly, the GC column saw some major advancements within just a few miles of AMPAC Analytical Laboratories. At that location, Walter Jennings and the company he co-founded, J&W Scientific, were instrumental in the development and manufacturing of capillary GC columns. The company was eventually purchased by Agilent in 2001.8,9 

GC Capabilities
Some of the analyses that GC can do include the separation of compounds in mixtures based on the polarity of the compounds, testing for purity – or for impurities, e.g., and detection of residual solvents. Conversely, with a technique known as preparative chromatography, GC can be used to prepare pure compounds from a mixture.  In pharmaceutical analysis, there are additional applications: 

  • Analysis of various functional groups. 
  • Determining purity of pharmaceutical compounds. 
  • Analysis of drugs that are commonly abused. 
  • Determination in pharmaceutical R & D the identity of natural products that contain  complex mixtures of similar compounds. 
  • Use in metabolomics studies.10 

GC for Testing Residual Solvents
The testing of residual solvents is necessary to ensure potency and, with some solvents, to determine their potential negative effects. GC is an excellent choice to do this. Residual solvents are separated into three classifications by the ICH (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use): 

Class 1 solvents: Solvents to be avoided – Known human carcinogens, strongly suspected human carcinogens and environmental hazards. The Permissible Daily Exposure (PDE) of these solvents used in pharmaceutical products can range from 2 to 1500 parts per million (PPM).  

Class 2 solvents: Solvents to be limited – Non-genotoxic animal carcinogens or possible causative agents of other irreversible toxicity such as neurotoxicity or teratogenicity and solvents suspected of other significant but reversible toxicities. The PDE for these solvents ranges from 50 to 3880 ppm. 

Class 3 solvents: Solvents with low toxic potential – low toxic potential to man requiring no health-based exposure limit. Class 3 solvents have PDEs of 50 mg or more per day.11 

Within these three classifications, some of the most commonly used solvents are listed: 

  • Benzene (class 1)  
  • Acetonitrile, Cyclohexane, Hexane, and Methanol (class 2) 
  • Acetic Acid, Acetone, and Heptane (class 3).11,12 

Headspace GC (HSGC) and Direct Injection GC
In addition to the numerous GC developments such as column type, phase and coating techniques, and multidimensional GC, two types of sampling methods arose: direct injection (“DI”) and headspace (“HSGC”).  In the former, the sample is injected “directly” into a typical sample injector of the GC column. In HSGC, when heated, “the more volatile compounds will tend to move into the gas phase (or headspace) sample. The more volatile the compound, the more concentrated it will be in the headspace. Conversely, the less volatile (and more GC-unfriendly) components that represent the bulk of the sample will tend to remain in the liquid phase.“13 

Therefore, by extracting the “headspace vapor and injecting it into a gas chromatograph, there will be far less of the less-volatile material entering the GC column, making the chromatography much cleaner, easier, and faster.”13 Generally, HSGS is much cleaner and results in less wear on the column. For more on comparative techniques with DI and HSGC, please see the first entry in the “Resources” section below. 

We Can Assist with your GC Needs  

AMPAC Analytical has years of experience and numerous experts in Gas Chromatography who can assist with method development for analyses for both common and atypical sample matrices that will allow you to stay ahead of evolving regulatory concerns. Please contact us with any specific questions or to receive a quote for your GC needs.   

References  

  1. History of Gas Chromatography – ScienceDirect 
  2. The Development of Gas Chromatography – ScienceDirect 
  3. Gas-Liquid Chromatography: the Separation and Identification of the Methyl Esters of Saturated and Unsaturated Acids from Formic Acid to n-Octadecanoic Acid (PDF) 
  4. James A T & Martin A J P. Gas-liquid partition chromatography: the separation and microestimation of volatile fatty acids from formic acid to dodecanoic acid. Biochem. J. 50:679-90, 1952. (upenn.edu) 
  5. A New Form of Chromatogram Employing Two Liquid Phases – PMC (nih.gov) 
  6. Gas Chromatography | SpringerLink 
  7. Three Early Symposia Showing the Direction for the  Evolution of Gas Chromatography.pdf   
  8. Co-Founder of J&W Scientific, Gas Chromatography Pioneer Walter Jennings Dies | Agilent 
  9. Professor Walter Goodrich Jennings: A Remembrance (chromatographyonline.com) 
  10. Pharmaceutical Applications of Gas Chromatography 
  11. https://database.ich.org/sites/default/files/ICH_Q3C-R8_Guideline_Step4_2021_0422_1.pdf 
  12. ICH_Q3C-R8_Guideline_Step4_2021_0422_1.pdf 
  13. An Introduction to Headspace Sampling in Gas Chromatography Fundamentals and Theory (perkinelmer.com)  

Resources  

 

 

Blog

Nitrosamines – An Update

NDMA

The linkage between nitrosamines and cancer was first postulated by William Lijinsky in 1970. Then, in 2018, N-nitroso-dimethylamine (NDMA)) was detected in an active pharmaceutical ingredient, Valsartan (an Angiotensin-II-receptor antagonist).  Finally, the FDA issued a guidance for the industry, “Control of Nitrosamine Impurities in Human Drugs”, in the fall of 2020. However, the guidelines continue to evolve. There has been an update in March of 2021, with ongoing risk assessments.  Other regulatory agencies have instituted their own, along with updates. For example, since our blog series on nitrosamines, there have been some regulatory updates from the European Medicines Agency (EMA). Their new guidelines are outlined in a document entitled, “Questions and Answers for Marketing Authorization Holders/Applicants on the CHMP Opinion for the Article 5(3) of Regulation (EC) No 726/2004 Referral on Nitrosamine Impurities in Human Medicinal Products.”1 The updated section answers the crucial question, “Which limits apply for nitrosamines in medicinal products?” 

 The answers are provided with a definition of nitrosamines and acceptable exposures: 

 “The ICH M7(R1) guideline defines N-nitrosamines as substances of the “cohort of concern” for which limits in medicinal products refer to the so-called substance-specific acceptable intake (AI) (the Threshold of Toxicological Concern, TTC, value of 1.5 ug/day cannot be routinely applied) which is associated with a negligible risk (theoretical excess cancer risk of <1 in 100,000 over a lifetime of exposure). The calculation of AI assumes a lifelong daily administration of the maximum daily dose of the medicinal product and is based on the approach outlined in the ICH M7(R1) guideline as well as the principles described in relation to the toxicological evaluation in the assessment report of the CHMP’s Article 5(3) opinion on nitrosamine impurities in human medicinal products.”1,2 (A previous blog examined TTC, here.)  

A Useful List of Nitrosamine Limits  

In Appendix 1 found on the EMA site, there is a list of more than eighty nitrosamines listed, along with CAS numbers, known medicinal sources, CPCA (Carcinogenic Potency Categorization Approach) categories, and their guidance publication dates.3,4  

Some Caveats 

There are some exceptions that should be considered. For example, the EMA states, “The ‘less than lifetime’ (LTL) approach should not be applied in calculating the limits as described above but can only be considered after consultation with competent authorities as a temporary measure until further measures can be implemented to reduce the contaminant at or below the limits defined above.”1  

Additionally, those medications intended for advanced cancers also have some exceptions. For example, “If the active substance itself is mutagenic or clastogenic at therapeutic concentrations, N-nitrosamine impurities should be controlled at limits for non-mutagenic impurities according to ICH M7(R1).”  

There is also guidance when one or more than one nitrosamines may be present. For the latter, the guidance advises one of two approaches: 

  1. The total daily intake of all identified N-nitrosamines is not to exceed the AI of the most potent N-nitrosamine identified. 
  1. The total risk level calculated for all identified N-nitrosamines is not to exceed 1 in 100,000. The approach chosen needs to be duly justified by the MAH (Marketing Authorization Holder)/Applicant.1 

Final Thoughts on Nitrosamines 

Nitrosamine guidance worldwide is ever-evolving, yet the impetus to quantify and regulate them is clear. There will doubtlessly be further updates to regulations. AMPAC Analytical Laboratories – an SK pharmteco company (AAL) is an industry leader in the detection of nitrosamines and other genotoxic impurities (GTI), We have the specialized expertise, equipment, and methodologies to detect these impurities by gas chromatography or high-performance liquid chromatography coupled with mass spectrometry to support your API project.  Also, importantly, AAL can assist in navigating those projects within today’s regulatory landscape. Please contact us with any specific questions or to receive a quote for nitrosamines or other GTIs.  

References  

  1. https://www.ema.europa.eu/en/documents/referral/nitrosamines-emea-h-a53-1490-questions-answers-marketing-authorisation-holders/applicants-chmp-opinion-article-53-regulation-ec-no-726/2004-referral-nitrosamine-impurities-human-medicinal-products_en.pdf 
  2. ICH M7 Principles – Impurity Identification and Control (europa.eu) 
  3. https://www.ema.europa.eu/en/human-regulatory/post-authorisation/referral-procedures/nitrosamine-impurities 
  4. Appendix 1 Nitrosamine AIs (europa.eu) 

Resources 

AMPAC  Analytical

General Information on Nitrosamines  

Nitrosamine and Pharmaceuticals  

Regulatory Experiences with Root Causes and Risk Factors for Nitrosamine Impurities in Pharmaceuticals
https://doi.org/10.1016/j.xphs.2022.12.022 

Blog

Keys To Effective Method Development

Effective method development is crucial for the quality control of Active Pharmaceutical Ingredients (API) and Drug Products (DP). Thorough method development enables successful downstream method validation. 

The regulatory guidance  specifies that: 

  • Method development and validation vary by application (quantitative, qualitative, etc.). 
  • It is phase appropriate. 
  • The client may provide additional guidance/validation criteria. 
  • The validation guidance directs how AMD (analytical method development) is conducted. 

Early Adoption of Forced Degradation Analysis
It is recommended that forced degradation be performed early in the method development lifecycle and that method parameters are suitable for mass spectrometry. This will prevent many issues that could occur in later stages and ensures the primary purity method is stability-indicating (specificity). When performing forced degradation, these considerations should be weighed: 

  • Always utilize a control sample without exposure to stressors.  
  • The stressors generally consist of acid, base, peroxide, heat, and photolytic conditions. Other stressors may be used based on known material incompatibility 
  • After exposing the compound to these stressors, target 5-20% degradation of the main peak.  
  • If degradation is not observed under reasonable conditions, then the material can be considered stable under those conditions. 

Impurity Genesis and Identification Assessment
Acceptance criteria should be scaled to impurity levels. How can the method unequivocally assess the analyte of interest in the presence of likely impurities, degradants, and the sample matrix? Additional considerations include: 

  • Is the method capable of identifying and/or quantifying a specific compound? 
  • Are there solvents present that can interfere with potential impurities? 
  • Known impurities? Are the known impurities stable under the method conditions? 
  • Is the method specific for degradation byproducts for stability-indicating methods? 

Cross-Platform Method Robustness
Robustness refers to a method’s ability to meet its analytical requirements (system suitability requirements) despite small variations of the method’s parameters, such as discrete changes to a column or sample tray temperature, percent organic modifier, flow rate, and detector wavelength. This capability is typically built into the method during method development.  

Precision or Accuracy – You Need Both in Strong AMD
AMPAC Analytical development strategy involves the early adoption of forced degradation studies with the goal of every primary purity method being stability-indicating (specificity) and mass spectrometry compatible. The validation strategy is phase-appropriate and application-specific and guides the development strategy. The validation acceptance criteria guidelines are specific to the test methodology, intended use, and level. Finally, method lifecycle performance is assessed, and reevaluation or revalidation can occur.  

 

 

 

 

Contact us today to learn how we can create accurate, precise method development for your API and DP pipeline.  

Blog

TCB* With Your TTC Needs

Triple Quad HPLC

Triple Quad HPLC“The dose makes the poison” – Paracelsus (c. 1493– 1541), born Theophrastus von Hohenheim

The Threshold of Toxicological Concern (TTC) refers to levels of mutagenic impurities expected to pose a negligible carcinogenic risk.1 The US FDA, the EMA (European Medicines Agency), and the European Food Safety Authority (EFSA) all have TTC values and regulations in place for food and active pharmaceutical ingredients (APIs), along with numerous other products.2,3 Originally, these standards were applied to TTC levels from oral ingestion but have expanded to even include cosmetics and fragrances.4,5

One tool to assess risk is the use of Cramer classes for organic impurities. They range from I-III, indicating a low, medium, or high probability of toxicity.5

There are numerous tools and techniques to assess TTC, depending on the product (food, water, and other beverages, APIs, or cosmetics) and the mutagenic impurity. AMPAC Analytical can utilize TTC guidelines and risk assessments to establish method development and validation targets that ensure acceptable levels of mutagenic impurities in your API or food products. Contact us today to learn more about analytical strategies to control mutagenic impurities.

*Taking Care of Business

References

  1. https://www.fda.gov/media/85885/download
  2. https://www.ema.europa.eu/en/ich-m7-assessment-control-dna-reactive-mutagenic-impurities-pharmaceuticals-limit-potential
  3. https://www.efsa.europa.eu/en/topics/topic/threshold-toxicological-concern
  4. https://www.sciencedirect.com/science/article/abs/pii/S0278691507002207
  5. https://www.sciencedirect.com/science/article/abs/pii/S0273230015300660

Resources

  • https://www.fda.gov/media/85885/download
  • https://www.frontiersin.org/articles/10.3389/ftox.2021.655951/full
  • https://academic.oup.com/toxsci/article/86/2/226/1653574
  • https://www.sciencedirect.com/science/article/abs/pii/S027869159600049X
Blog

Forced Degradation Studies Can Reduce Stress(ors)

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.  

References 

  1. https://www.sciencedirect.com/science/article/pii/S2095177913001007 
  2. http://www.columbiapharma.com/reg_updates/international/ich/q1a.pdf 
  3. https://www.researchgate.net/profile/Dan-Reynolds-3/publication/279607256_Available_Guidance_and_Best_Practices_for_Conducting_Forced_Degradation_Studies/links/5afd6a2ca6fdcc3a5a44c50f/Available-Guidance-and-Best-Practices-for-Conducting-Forced-Degradation-Studies.pdf 

Learn more: https://ampacanalytical.com/laboratory-services/stability-program/ 

Blog

Raw Materials Testing: Trust – and Verify – Your Sources

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 

Medical products, pharmacology, dietary supplements

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. 

 References 

  1. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/guidance-industry-q7a-good-manufacturing-practice-guidance-active-pharmaceutical-ingredients#P309_13037 

 

Blog

Nitrosamines in Food and Beverages

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.2,6,7 Evidence 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.  

  1. https://doi.org/10.1038/225021a0 
  2. * https://doi.org/10.3390/toxins2092289 
  3. https://ntp.niehs.nih.gov/whatwestudy/assessments/cancer/roc/index.html 
  4. https://onlinelibrary.wiley.com/doi/book/10.1002/9780470430101#page=369 
  5. https://onlinelibrary.wiley.com/doi/book/10.1002/9780470430101#page=566 
  6. * http://ntp.niehs.nih.gov/ntp/roc/eleventh/profiles/s137nsop.pdf
  7.  http://ntp.niehs.nih.gov/ntp/roc/eleventh/profiles/s136nsop.pdf
  8. * https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4087738/ 
  9. * https://www.fda.gov/regulatory-information/search-fda-guidance-documents/guidance-industry-action-levels-poisonous-or-deleterious-substances-human-food-and-animal-feed 

Resources & Further Reading 

AMPAC 

General Information on Nitrosamines 

Nitrosamine and the Diet 

Blog

Nitrosamines in Active Pharmaceutical Ingredients 

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 June 2018, 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.3,4 The cost of recalls could be high.5 APIs 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.6,7  As 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.  

References  

  1. https://doi.org/10.1038/225021a0   
  2. https://jpharmsci.org/article/S0022-3549(23)00018-7/fulltext  
  3. https://doi.org/10.1021/acs.jmedchem.0c02120  
  4. https://doi.org/10.1016/j.xphs.2022.11.013 
  5. https://www.bloomberg.com/news/articles/2022-09-01/drug-recalls-for-nitrosamines-could-cost-big-pharma-millions  
  6. https://www.fda.gov/media/141720/download 
  7. https://www.ema.europa.eu/en/documents/referral/nitrosamines-emea-h-a53-1490-assessment-report_en.pdf https://doi.org/10.1093/nar/gkaa962  
  8. https://doi.org/10.1093/nar/gkaa962   
  9. https://ampacanalytical.com/wp-content/uploads/2023/01/Control-of-Nitrosamine-Impurities-in-Human-Drugs-Guidance-for-Industry.pdf  
  10. https://www.who.int/water_sanitation_health/water-quality/guidelines/en/ 

Resources & Further Reading  

AMPAC  

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Nitrosamines: An Overview

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 as genotoxic 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. Halden wrote 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).”1 Concern 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.”2 Primary 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”.”1 Almost 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 APIs from 66 manufacturers.   

Figure 1
(N/A = not applicable/interim limits not published yet). 

Name  Abbreviation  CAS-No.  IL Interim Limits (ng/day) 
N-Nitrosodimethylamine  NDMA  62-75-9  96 
N-Nitrosomethylethylamine  NMEA  10595-95-6  NA 
N-Nitrosodiethylamine  NDEA  55-18-5  26.5 
N-Nitrosodiethanolamine  NDELA  1116-54-7  NA 
N-Nitrosoethylisopropylamine  NEiPA  16339-04-1  26.5 
N-Nitrosodiisopropylamine  NDiPA  601-77-4  26.5 
N-Nitrosodi-n-propylamine  NDPA  621-64-7  26.5 
N-Nitrosodi-n-butylamine  NDBA  924-16-3  26.5 
N-Methyl-N-nitrosoaniline (N-nitrosomethylphenylamine)  NMPhA  614-00-6  34.3 
N-Nitrosomethyl(2-phenylethyl)amine  NMEPhA  13256-11-6  8 
N-Nitrosodiphenylamine  NDPhA  86-30-6  NA 
N-Nitrosopyrrolidine  NPyr  930-55-2  NA 
N-Nitrosopiperidine  NPip  100-75-4  1300 
N-Nitrosomorpholine  NMor  59-89-2  127 
1-Methyl-4-nitrosopiperazine  MNPaz  16339-07-4  26.5 
N-Nitroso-N-methyl-4-aminobutyric acid  NMBA  61445-55-4  96 

 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 medicines resulting 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 

carmustine structure

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. 

References  

  1. * https://doi.org/10.1016/j.chemosphere.2018.07.098 
  2. https://pubmed.ncbi.nlm.nih.gov/2184959/ 
  3. * https://doi.org/10.1016/j.xphs.2022.11.013 
  4. * https://doi.org/10.1002/ardp.202200484 
  5. https://doi.org/10.1021/acs.jmedchem.0c02120 
  6. https://www.bloomberg.com/news/articles/2022-09-01/drug-recalls-for-nitrosamines-could-cost-big-pharma-millions 
  7. * https://pubchem.ncbi.nlm.nih.gov/compound/Carmustine 
  8. * https://medlineplus.gov/druginfo/meds/a682060.html 

Resources & Further Reading 

AMPAC 

General Information on Nitrosamines 

Nitrosamine Exposure and Environmental Concerns 

Nitrosamine and Pharmaceuticals 

Nitrosamine and the Diet