Tag Archives: SK pharmteco

Nitrosamines – An Update


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.  


  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) 


AMPAC  Analytical

General Information on Nitrosamines 

Nitrosamine and Pharmaceuticals 

Regulatory Experiences with Root Causes and Risk Factors for Nitrosamine Impurities in Pharmaceuticals

Dynamic Vapor Sorption

Dynamic Vapor Sorption (DVS) is a gravimetric technique used to measure the change in mass of a material in response to changes to surrounding conditions such as temperature or humidity. DVS is primarily used with water vapor but can be applied to other organic solvents as well for the physicochemical characterization of solids. 

DVS was developed by Daryl Williams, the founder of Surface Measurement Systems Ltd., in 1991. The company then delivered the first working DVS instrument to Pfizer in 1992.1 Since then, many other equipment manufacturers have entered the field. 

Figure 1: A DVS isotherm plot indicating sorption and desorption rates and hysteresis. The isotherm shows a typical hysteresis curve where the adsorption phase is almost identical to the desorption phase (i.e. reversible). Note that at 80 %RH, there is net sorption of 0.9% between adsorption and desorption traces. The material appears to be slightly hygroscopic according to the definition in Ph. Eur.2 

Sorption, Desorption, Absorption, and Adsorption
There are five main physical processes that occur during the DVS experiment. The first, sorption, is when a material takes on moisture due to increased humidity. Conversely, desorption is the process that occurs when the material loses moisture due to decreasing humidity. Sorption can be classified as one of two types. Adsorption is moisture that is observed on a surface of a material, while absorption is moisture that has penetrated the surface of a material. The fifth term and the term that relates sorption and desorption is called hysteresis. The overall chart that includes and tracks the sorption and desorption rates and hysteresis is called the isotherm. These curves are crucial for understanding the physicochemical characteristics of a solid, such as porosity, polymorphic change, or liquefying of a sample.2 


There are numerous reasons to utilize DVS, and some of the most common within the active pharmaceutical ingredient (API) industry are: 

  • To determine the sorption isotherm;  
  • To evaluate the hygroscopicity of an API powder;  
  • To compare the hygroscopicity of different solid-state forms: solvates, polymorphs, salts, amorphicity, and cocrystals; 
  • To determine the deliquescence point of a material
  • To quantify and qualify the amorphous content in drug substance or excipient,3 and 
  • To evaluate packaging materials. 

Of course, there are a variety of other applications in other industries, including for building materials, food science, cosmetics, coatings, and sealants. 

DVS Analysis of APIs
For pharmaceutical development, DVS is used for a variety of applications, including screening early drug and excipient candidates, establishing processing parameters, and identifying packaging and storage requirements (Figure 2).4,5  


Figure 2: A DVS isotherm of an API showing that the material started to gain significant mass after exposure to relative humidity values of more than 60 %RH. The change here was irreversible, as demonstrated by the desorption curve. DVS could be a useful tool to suggest storage conditions in terms of humidity contents in the surrounding environment. 

However, due “to the typically slow establishment of an equilibrium, DVS experiments are rather time-consuming.”4 Nevertheless, “water content of solid active pharmaceutical ingredients and excipients, individually and when formulated in pharmaceutical dosage forms, is a parameter that should be monitored throughout the drug lifecycle.”5  

As an analytical technique for APIs, DVS has become a necessary step within drug development and production, reducing issues that can arise during manufacturing, packaging, transportation, solubility, dissolution rate, stability, or storage. AMPAC Analytical’s sister company, SK biotek Ireland Analytical Services, has the experience, technology (including a Surface Measurement System’s DVS Resolution Dual Vapor Gravimetric Sorption Analyser), and support, to assist with this vital testing. Both companies are part of SK pharmteco and can easily transfer your project from either business unit to ensure the most optimal solution and logistical support are provided to meet your product timeline. We invite you to contact our team members and discuss how we can assist with your sorption testing requirements.  

Figure 3: A SMS DVS instrument like the one located at SK biotek Ireland. 


  1. https://surfacemeasurementsystems.com/our-story/ 
  1. Ph. Eur., 2023, 11.2 Edition, Chapter 2.9.39  
  1. https://www.sciencedirect.com/science/article/abs/pii/S0022354915303348 
  1. https://www.sciencedirect.com/science/article/abs/pii/S0022354918302193 
  1. https://www.sciencedirect.com/science/article/abs/pii/S0022354916325230 

Phase-Appropriate Method Development 

The Cost of Drug Discovery and Development and How to Mitigate It 

The path to successful drug discovery and development is extremely long, expensive, and risky and can take between 10 to 15 years at an average cost of more than $1–2 billion for every new drug that is approved for clinical use.1,2 In fact, preclinical drug discovery alone “typically takes five and a half years and accounts for about one-third of the cost of drug development.”3,4 Therefore, even during the earliest stages of a drug product or active pharmaceutical ingredient project, phase-appropriate method development should be instituted to manage costs. This bolsters the chances for success and ensures reliable results, quality management, and reproducibility while avoiding “unreliable results (that) might not only be contested in court but could also lead to unjustified legal consequences for the defendant or to wrong treatment of the patient.”5 At its most basic, phase-appropriate method development maps the “what is needed” to “when it is needed.”6 Effective phase-appropriate method development can provide long-term product support by introducing mass spectrometry compatibility and forced degradation development to ensure your methods are stability-indicating and amenable to unknown impurity identification. By instituting a phase-appropriate method development process, combined with a quality-by-design approach around each logical sequence of events – and rigorously following it – it is more likely to create a cost-effective, successful outcome as you take the drug product through the regulatory process. 

It Can Pay to Outsource 

As the incentives for strong phase-appropriate method development increase, so too has the recognition of its value. Unfortunately, “it is not uncommon…to find pharmaceutical companies and contract research organizations (CROs) that are not taking advantage of the phase-appropriate approach and simply reference the typical ICH guidance for analytical items, such as method validation.”7 However, while FDA guidance encourages the use of a phase-appropriate approach, it is lacking in details and requirements. This leaves many companies to seek out ICH guidance as an alternative, conservative approach. Also, within their CGMP quality system, they may find it difficult to accommodate differing levels of CGMP compliance throughout the various clinical phases of development. This is when it might be an opportune moment to consider an outside expert that specializes in phase-appropriate method development processes for drug discovery and validation.  A successful yet robust phase-appropriate method development program can balance competing interests and requirements and still provide a regimen that meets the overall development goals without sacrificing any of the requirements of the program.  

AMPAC Analytical Laboratories (AAL), an SK pharmteco company, has decades of experience in providing a wide array of release testing services for raw materials, intermediates, APIs, and drug products. Our labs are equipped to handle hazardous, cytotoxic/high potency compounds as well as controlled substances for schedule II through V. Additionally, we have not only the expertise to conduct forced degradation experiments but also appropriate instrumentation like mass spectrometers to support later phases of development for your products. Please contact us to discuss how we can ensure the success of your drug discovery and development project and simultaneously reduce risks. 


  1. https://www.sciencedirect.com/science/article/pii/S2211383522000521 
  2. https://www.frontiersin.org/articles/10.3389/fphar.2020.00770/full 
  3. https://www.frontiersin.org/articles/10.3389/fphar.2020.00770/full 
  4. https://www.nature.com/articles/nrd3078 
  5. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3658022/ 
  6. https://www.pharm-int.com/2020/08/27/phase-appropriate-drug-development-validation-process/ 
  7. https://www.pharmtech.com/view/designing-phase-appropriate-cmc-analytical-programs 



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


  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


  • 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

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.  


  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/ 

The Background, Advantages of, and Considerations for Radiolabeled Peptides

Integrin αvβ3 and RGD Binding

Integrin αvβ3 and RGD Binding

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: it can 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.     


  1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5235994/ 
  2. https://link.springer.com/article/10.1007/s00259-012-2064-5 
  3. https://link.springer.com/article/10.1007/s00259-011-2038-z 
  4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4679356/ 
  5. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8924613/ 
  6. https://link.springer.com/article/10.1007/s00259-020-04975-9 
  7. https://pubmed.ncbi.nlm.nih.gov/6325925/ 
  8. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789871/ 
  9. https://www.cancer.gov/news-events/cancer-currents-blog/2018/lutathera-fda-gastrointestinal-nets 
  10. https://pubs.acs.org/doi/10.1021/ja9603721 
  11. https://pubs.acs.org/doi/full/10.1021/acs.bioconjchem.0c00617 

Related Articles and Resources  

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. 


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


Extractables and Leachables

blank pharmaceutical and drug packaging

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:

  • Inhaled 
  • Injected 
  • Liquid-based  
  • Oral  
  • Solid oral dosage forms  
  • Ophthalmic 
  • 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. 


  1. https://ispe.org/pharmaceutical-engineering/may-june-2017/extractables-leachables-not-same 
  2. https://www.fda.gov/media/70788/download 
  3. https://database.ich.org/sites/default/files/ICH_Q3E_ConceptPaper_2020_0710.pdf 



Elemental Impurities

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.3 The 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.12,13 They 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. 
Ag, Silver; Al, Aluminum; As, Arsenic; Au, Gold;  B, Boron; Ba, Barium; Ca, Calcium;
Cd, Cadmium; Co, Cobalt; Cr, Chromium;
Cu, Copper; Hg, Mercury; Fe, Iron;  
Ir, Iridium; K, Potassium; Li, Lithium;
Mg, Magnesium; Mn, Manganese;
Mo, Molybdenum; Na, Sodium; Ni, Nickel;
Os, Osmium;  
Pb, Lead; Pd, Palladium; Pt, Platinum;
Rh, Rhodium; Ru, Ruthenium; Sb, Antimony; Se, Selenium; Sn Tin; Tl, Thallium; V, Vanadium;
W, Tungsten; Zn, Zinc 

Tools for Testing Elemental Impurities 

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.”14 However, 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:  

For industry resources, the offerings consist of the following: 

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. 


  1. https://cen.acs.org/safety/consumer-safety/FDA-seeks-limit-lead-baby 
  2. https://www.nytimes.com/2023/01/26/health/baby-food-metals-lead.html 
  3. https://oversightdemocrats.house.gov/sites/democrats.oversight.house.gov/files/ECP%20Second%20Baby%20Food%20Report%209.29.21%20FINAL.pdf 
  4. https://www.nytimes.com/2023/02/09/well/eat/dark-chocolate-metal-lead.html 
  5. https://www.reuters.com/business/retail-consumer/consumer-reports-urges-dark-chocolate-makers-reduce-lead-cadmium-levels-2023-01-23/ 
  6. https://www.npr.org/2022/12/30/1146254933/hersheys-lawsuit-dark-chocolate-heavy-metals-lead 
  7. https://www.usnews.com/news/health-news/articles/2023-02-08/how-are-toxins-like-lead-arsenic-getting-into-baby-foods 
  8. https://www.consumerreports.org/health/food-safety/lead-and-cadmium-in-dark-chocolate-a8480295550/ 
  9. https://www.usp.org/sites/default/files/usp/document/our-work/chemical-medicines/key-issues/c232-usp-39.pdf 
  10. https://www.fda.gov/safety/recalls-market-withdrawals-safety-alerts/recall-resources 
  11. https://www.ema.europa.eu/en/documents/scientific-guideline/international-conference-harmonisation-technical-requirements-registration-pharmaceuticals-human-use_en-16.pdf 
  12. https://www.fda.gov/media/148474/download 
  13. https://www.ema.europa.eu/en/documents/scientific-guideline/international-conference-harmonisation-technical-requirements-registration-pharmaceuticals-human-use_en-32.pdf 
  14. https://www.pharmtech.com/view/approaching-elemental-impurity-analysis 

Resources, Related Topics, and Further Reading 

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. 

 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 


General Information on Nitrosamines 

Nitrosamine and the Diet