The analytical methods are directly linked to the quality of pharmaceutical products, since they ensure the identity, purity, physical characteristics and potency of the drugs.

According to the International Conference on Harmonization (ICH), the chemical methods may belong (but not only) to the following: (i) identification tests, (ii) quantitative tests of the active moiety in samples of API or drug product or other selected component(s) in the drug product, (iii) quantitative tests for impurities’ content, (iv) limits tests for the control of impurities.

Methods are developed to support drug testing against specifications during manufacturing and quality release operations, as well as during long-term stability studies. Methods may also support the product development, safety and characterization studies or evaluations of drug performance.

Effective method development ensures that laboratory resources are optimized, while methods meet the objectives according to the intended use. The selectivity of the method is of paramount importance, while increased robustness characteristics reassure the stable performance of the method over time, and the efficient transfer worldwide. Apart from the scientific goals, the simplification of the samples’ treatment and reduction of time and consumables (e.g. simultaneous determinations for combination drugs) are also an important part of the method development.

On top of the scientific drivers and appropriate software, the development team owns deep knowledge and indispensable experience in the field through hundreds of products and several publications in the research scientific literature and international conferences.

According to Article 10 (1) of Directive 2001/83/EC the applicant is not required to provide the results of pre-clinical tests and clinical trials if he can demonstrate that the medicinal product is a generic of a reference medicinal product. A generic medicinal product is defined as a medicinal product that has:

Based on the above regulatory provisions the knowledge of the reference products quantitative composition is not mandatory in order to develop a generic product however, it could be a key issue in several cases, such as:

The development of a product specific dissolution method is a significant element of the pharmaceutical products documentation. Dissolution testing serves not only as a routine quality control test of production batches but also as a guide during the early stages of a successful formulation development.

The dissolution method development study includes the following:

Extractables are compounds that are forced out of container closure system and manufacturing materials under laboratory experimental conditions. All extractables from a given pharmaceutical container closure system and manufacturing materials are, therefore, potential leachables in a drug product.

Since the pharmaceutical packaging/delivery system is the primary source of potential leachables, it is generally appropriate that any leachables assessment be preceded by an extractables assessment performed on the packaging/delivery system, its primary and certain critical secondary packaging components (that are non-contacting but potentially interacting) and/or packaging and delivery system materials of construction; consistent with regulatory guidelines and best-practice recommendations. Such an extractables assessment can also be performed on particular components and/or materials of construction of manufacturing and packaging equipment, as well as certain tertiary packaging components that are deemed of high leaching potential or have been implicated in an identified leachables problem with a particular drug product.

“Extractables” assessments can be used to:

In 2018, nitrosamine impurities, including N-nitrosodimethylamine (NDMA), were found in a number of blood pressure medicines known as ‘sartans’. This led to some product recalls and to a regulatory review, which set strict new manufacturing requirements for these medicines. Subsequently, a nitrosamine impurity has been detected in batches of ranitidine, a medicine used to treat heartburn and stomach ulcers, and the Agency’s Committee for Medicinal Products for Human Use (CHMP) has started a review.

Nitrosamines are chemical compounds classified as probable human carcinogens on the basis of animal studies. However, there is a very low risk that nitrosamine impurities at the levels found could cause cancer in humans. The European Medicines Agency (EMA) has asked marketing authorisation holders to take precautionary measures to mitigate the risk of nitrosamine formation or presence during the manufacture of all medicines containing chemically synthesised active substances. Moreover, the EMA has provided guidance to marketing authorization holders (MAHs) on how to avoid the presence of nitrosamine impurities in human medicines, while the CHMP has requested MAHs for human medicines containing chemically synthesized active substances to review their medicines for the possible presence of nitrosamines and test all products at risk.

To this end, MAHs should follow the below steps:

Nitrosamines include but are not limited to:

Qualimetrix can assist MAHs as well as other parties involved in the supply chain of a pharmaceutical product in properly addressing the “nitrosamine issue” by fully undertaking the:

Qualimetrix has extensive knowledge and experience in the determination of nitrosamines
in APIs, finished products of solid (tabs, caps) and liquid dosage forms (e.g. syrup, solutions) as well as water or excipients.

The determination of nitrosamines is based on published methods, proposed by the authorities (EDQM, FDA, Taiwan FDA) and on extensive literature research, which are finely tuned according to the needs of every matrix. The analysis is in line with the interim limits set, based upon the maximum daily intake, however the current methodologies are able to ensure absence (i.e. “not quantifiable” levels) of nitrosamine impurities (< 0.03 ppm). Qualimetrix is equipped with state-of-the-art instrumentation, dedicated for the analysis of nitrosamines, operating under GMP environment. Methodologies based on LC-(APCI)-MS/MS and (HS)-GC-MS are included in the portfolio of nitrosamine testing, with various extraction techniques, in order to ensure confident identification and accurate quantitation of the impurities. Nitrosamine testing is performed by our technically competent and experienced personnel. Method development and validation are designed in order to cover the needs, as per the demanding limits, challenging matrix and urgent requests.

Elemental impurities in drug products may arise from several sources (e.g. residual catalysts employed during the synthetic process of the drug substance, interaction of the final product with manufacturing equipment or the container closure system, etc.). Since elemental impurities do not provide any therapeutic benefit to the patient, their levels in the drug product should be controlled within acceptable limits. To this end, ICH Q3D presents a process to assess and control elemental impurities in the drug product using the principles of risk management.

The risk assessment process can be described in three steps:

The first step of the risk assessment process comprises of the identification of known and potential sources of elemental impurities that may find their way into the final product. The figure below illustrates potential sources that will be considered during the evaluation.

A Failure mode and effect analysis (FMEA) is conducted in order to identify and assess the risk associated with each potential source of elemental impurities. FMEA is a highly structured, systematic technique for failure analysis. It involves the review of all components depicted in the Figure above in order to identify failure modes and their effects. For each component, their failure modes and their resulting effects are recorded in a FMEA sheet. Each failure mode is associated with a material, process or parameter that could serve as a source of elemental impurities. The failure effects correspond to the metals that could emanate from each source. The degree of severity of the effects, their respective probabilities of occurrence, and their detectability are assessed by assigning appropriate numerical values. A Risk ranking and filtering approach is subsequently adopted in order to compare and rank risks. The hybrid FMEA and Risk ranking and filtering methodology aims at facilitating the investigation and the subsequent establishment of additional measures / controls  in case the elemental impurity levels exceed the control threshold ( 30% of the PDE). The identification process is based on the review of the API, excipient and drug product manufacturing process to identify known and potential sources of Elemental Impurities. The elements that have to be considered are Class 1 and 2A metals, 2B in case they have been intentionally added, and class 3, depending on the route of administration. In all cases, all intentionally added elements have to be assessed.

The next step is the evaluation of the presence of particular elemental impurities in the drug product. The latter is based on the collection of predicted and/or observed levels of elemental impurities and the subsequent comparison of data with the established Permitted Daily Exposure. The data to support the evaluation may emanate from published literature, data generated from similar processes, supplier information data (e.g Certificates of Analysis), testing of the components of the drug product or the final product itself.

The final step comprises of the documentation and summary of the risk assessment and the identification of additional control requirements if required.

The proper implementation of the guideline requires a combination of a scientifically sound risk-assessment and testing of the final product and/or components. Based on the amount and quality of available data one could minimize the required testing. However, the level and variability of an elemental impurity should be well established. The latter means that in the absence of other justification, data from testing 3 representative production scale lots or 6 representative pilot scale lots of the component(s) or drug product would be required.

Percutaneous absorption actually infers permeation of the drug through the epidermis and into the deep layers of skin and general circulation in vivo, a total process that includes transport through the skin and local clearance. Skin permeation relates to the first part of the process, diffusion across the skin. In percutaneous absorption either diffusion or clearance factors can, in principle, be rate controlling; however, with few exceptions skin permeation is the kinetically determining event. Thus, skin permeation observed in vitro is believed to reflect accurately the rate determining aspects of drug delivery in most instances and is, therefore, projected as a means of determining relative availability from dosage forms (Skelly et al 1987). Based on these principles, in the “Critical Opportunities Pathway,” the FDA identifies the in vitro diffusion study combined with rheological testing to demonstrate bioequivalence of qualitatively and quantitatively (Q1Q2) equivalent drug products.

The primary permeability barrier of the skin, the stratum corneum, is a rugged, non-living membrane like hair or nails, and it retains its barrier properties following excision from the body. The in vitro permeation test involves the use of a diffusion cell that maintains excised human skin at a physiological hydration and temperature. The excised skin is routinely dermatomed to a thickness that includes the epidermis (with its stratum corneum) and part of the dermis. The in vitro diffusion study set up for topical products consists of excised human skin between donor and receptor chambers. Test formulations are applied to the skin membrane surface facing the donor chamber. A physiologically based receptor solution bathes the underside of the skin and accepts drug diffusing through the skin with adequate solubility to maintain sink conditions. It is important to maintain the sink conditions by including solubilising agents in the receptor fluid, if needed. Minimal effect on the skin barrier properties from these receptor solutions is desired and should be demonstrated. The receptor solution beneath is sampled through a side-arm sampling port at various time points frequent enough to maintain sink conditions that would adequately mimic the clearance created by the dermal microcirculation. Sampling to measure dermal drug flux in the in vitro permeation test model is performed in a manner analogous to pharmacokinetic sampling by blood draws (Narkar 2010, Raney et al 2015).

Permeation studies are a useful tool for Systemically as well as Locally Acting – Locally Aplied products, not only dermal, but also ocular (ophthalmic), rectal, vaginal, nasal and mouth (through oromucosal) products.

Structural Elucidation of an unknown compound is a challenging task, evolving rapidly in the field of pharmaceutical and forensic analysis. This critical analytical activity often emerges when unknown impurities are found to be present above the prescribed stringent identification threshold set by ICH Q3A/B, during routine control of an API or a final product (e.g. batch testing, stability study). The focus on impurities in pharmaceutical products closely relates to patient safety considerations associated with the induction of toxic or even carcinogenic effects. Furthermore, the identification / structural characterization of impurities serves the following purposes:

Structural elucidation of unknowns requires high throughput techniques and sophisticated chemometric tools. Mass spectrometry (MS) is the technique of choice, combined with a separation technique, mainly Liquid and Gas Chromatography.

The structural elucidation is based on information over the physicochemical properties of the molecule, as well as its chemical formula, derived from information of its molecular weight and fragmentation pattern. Sophisticated software combines the available information and based on advanced algorithms, a probable structure is proposed, with a certain degree of confidence. Moreover, the chromatographic retention time plausibility of the proposed structure is evaluated through chemometric models.

Accurate mass measurements, through high resolving power mass analyzers (HRMS) can contribute to the confidence degree of the proposed structure. Complementary techniques, regarding separation or detection, such as HILIC chromatography or NMR can boost the identification, by providing additional evidence for the characterization. Statistical evaluation and high scientific know-how, are necessary, as a final step for the identification.

MS techniques, mainly HRMS, together with statistical models and chemometric methods can maximize the retrieved information and combined with scientific expertise can frame the scheme of the identification of unknown impurities.

Qualimetrix combines the range of the required hyphenated techniques, mentioned above, with “cutting edge” software and scientific expertise in order to facilitate and successfully complete the challenging task of structural elucidation. A tailor-made approach and methodology is applied according to the purpose of the study and the customer’s requirements that may include, but is not limited to, the following:

All of the above aim at both the identification of the unknown impurity and the determination of its source so that the most effective control strategy can be defined and implemented.