Validation of Fast Spectrochemical Screening Methods for the Identification of Counterfeit Pharmaceutical Packaging

Counterfeit pharmaceuticals are an actively developing health and economic threat worldwide. Particularly prevalent are counterfeit pharmaceuticals distributed in emerging nations and through internet pharmacies or e-pharmacies. Although technology has been developed that discourages anti-counterfeiting practices (such as optically variable devices, invisible ink, and track-and-trace technology), it remains somewhat novel and expensive to implement on a widespread scale. In this study, Laser Induced Breakdown Spectroscopy (LIBS) and Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR) were proposed as fast and non-invasive tools for the identification of counterfeit pharmaceutical packages. The main objective of this research was to develop and evaluate the capabilities of LIBS and ATR-FTIR to determine chemical differences between counterfeit and authentic pharmaceutical packaging samples. LIBS and ATR-FTIR possess several characteristics that render them suitable for rapid on-site detection. They produce analytical results in less than one minute per sample, with high sensitivity and selectivity, limited sample preparation, and minimal destructivity. The methods were evaluated through the analysis of a dataset of 166 packages (112 counterfeits and 54 authentic sources). The dataset was divided into two main subsets. The first subset was evaluated to identify the informative value of LIBS for fast screening of black barcodes and the carton substrate (100 counterfeit and 35 authentic). The multi-color inks and paper of the second subset was investigated for variation of chemical profiles within and between sources, and the method’s capabilities to distinguish between counterfeits (112) and authentic samples (12). One hundred and twelve counterfeit pharmaceutical cartons were printed from five different sources, mimicking six authentic counterparts. The authentic subset consisted of twelve secondary packages of six common medical products, including packages from the same and different manufacturing lots. The selected products consisted of vasodilators, antivirals, steroids, and other commonly counterfeited pharmaceuticals. Intra-source variation of the counterfeit subset was investigated; it was determined to be sufficiently lower than inter-source variation. False exclusion rates were calculated to be less than 20% for samples originating from the same source (e.g., same package, intra-lots, replicate printouts). Using LIBS, a two-class classification system was used for the combined black barcode ink and paperboard carton spectra (n = 135, 100 counterfeit, 35 authentic packages). As black barcode ink is very common on pharmaceutical packaging, this system was used as a general screening technique to quickly identify a sample as authentic or counterfeit, regardless of counterfeit printing source. In general, the correct classification rates for this set were over 92%. The classification models were established using six machine learning methods: Random Forest, Naïve Bayes, Neural Networks, k-Nearest Neighbors, Quadratic Discriminant Analysis, and Linear Discriminant Analysis. A random split of 60% and 40% of the dataset was applied for training and testing of the classifier algorithms. Principal Component Analysis (PCA) was utilized on the LIBS and ATR-FTIR data for variable reduction purposes. The principal components for each ink type were combined prior to classification. Also, a six-class system was also used to classify the dataset using LIBS, ATR-FTIR, and combined data from both techniques (n = 124, 112 counterfeit, 12 authentic packages). The machine learning methods classified the samples as belonging to one of five counterfeit printing sources or their corresponding authentic counterpart. Seven ink colors (red, blue, yellow, green, brown, pink, black) were analyzed; additionally, in ATR-FTIR, the paperboard substrate was also analyzed. In most comparisons, LIBS had a successful classification rate of over 70% and ATR-FTIR had a successful classification rate of over 85%. When the data from both techniques were combined, the discrimination power of the system increased to 93% correct classification. Although LIBS and ATR-FTIR had a low misclassification rate when used in isolation, the misclassification rate could be reduced even further through data combination. The results of this study are encouraging for the inclusion of LIBS and ATR-FTIR as a screening method for the detection of counterfeit pharmaceutical packaging. The utilization of combined data to discover chemical signatures addresses an urgent need in the investigation of counterfeit pharmaceuticals. Also, the classification of counterfeit samples into their specific counterfeit source may benefit investigators as they make determinations in the counterfeit pharmaceutical packaging supply chain. This study is anticipated to offer relevant tools to both government and pharmaceutical industry in the detection and fight against counterfeit pharmaceuticals

Non-destructive detection of counterfeit and substandard medicines using X-ray diffraction

The prevalence of counterfeit and substandard medicines has been growing rapidly over the past decade, and fast, non-destructive techniques for their detection are urgently needed to counter this trend. In this thesis, both energy-dispersive X-ray diffraction (EDXRD) and pixelated diffraction (“PixD”) combined with chemometric methods were assessed for their effectiveness in detecting poor-quality medicines within their packaging. Firstly, a series of caffeine, paracetamol and cellulose mixtures of known concentrations were pressed into tablets. EDXRD spectra of each tablet were collected both with and without packaging. Principal component analysis (PCA) and partial least-squares regression (PLSR) were used to study the data and construct calibration models for quantitative analysis. The concentration prediction errors for the packaged data were found to be very similar to those obtained in the unpackaged case, and were also on a par with reported values in the literature using higher-resolution angular-dispersive X-ray diffraction (ADXRD). Following this, soft independent modelling by class analogy (SIMCA) classification was used to compare EDXRD spectra from a test set of over-the-counter (OTC) medicines containing various combinations of active pharmaceutical ingredients (APIs) against PCA models constructed using spectra collected for paracetamol and ibuprofen samples. The test samples were selected to emulate different levels of difficulty in authenticating medicines correctly, ranging from completely different APIs (easy) to those with a small quantity of additional API (difficult). This classification study found that the sensitivity and specificity were optimal at data acquisition times on the order of 75~150s, and regardless of whether layers of blister and card packaging surrounded the tablet in question. This experiment was repeated on a novel, compact system incorporating a pixellated detector, which was found to reduce the required data acquisition times for optimal classification by a factor of five

Anti-malarial medicine quality field studies and surveys: a systematic review of screening technologies used and reporting of findings.

BACKGROUND: Assessing the quality of medicines in low-middle income countries (LMICs) relies primarily on human inspection and screening technologies, where available. Field studies and surveys have frequently utilized screening tests to analyse medicines sampled at the point of care, such as health care facilities and medicine outlets, to provide a snap shot of medicine quality in a specific geographical area. This review presents an overview of the screening tests typically employed in surveys to assess anti-malarial medicine quality, summarizes the analytical methods used, how findings have been reported and proposes a reporting template for future studies. METHODS: A systematic search of the peer-reviewed and grey literature available in the public domain (including national and multi-national medicine quality surveys) covering the period 1990-2016 was undertaken. Studies were included if they had used screening techniques to assess the quality of anti-malarial medicines. As no standardized set of guidelines for the methodology and reporting of medicine quality surveys exist, the included studies were assessed for their standard against a newly proposed list of criteria. RESULTS: The titles and abstracts of 4621 records were screened and only 39 were found to meet the eligibility criteria. These 39 studies utilized visual inspection, disintegration, colorimetry and Thin Layer Chromatography (TLC) either as components of the Global Pharma Health Fund (GPHF) MiniLab® or as individual tests. Overall, 30/39 studies reported employing confirmatory testing described in international pharmacopeia to verify the quality of anti-malarials post assessment by a screening test. The authors assigned scores for the 23 criteria for the standard of reporting of each study. CONCLUSIONS: There is considerable heterogeneity in study design and inconsistency in reporting of field surveys of medicine quality. A lack of standardization in the design and reporting of studies of medicine quality increases the risk of bias and error, impacting on the generalizability and reliability of study results. The criteria proposed for reporting on the standard of studies in this review can be used in conjunction with existing medicine quality survey guidelines as a checklist for designing and reporting findings of studies. The review protocol has been registered with PROSPERO (CRD42015026782)

Raman spectroscopy, a non-invasive mesurement technique for the detection of counterfeit medicines

Tese de mestrado, Engenharia Farmacêutica, Universidade de Lisboa, Faculdade de Farmácia, 2016Perhaps no greater challenge exists for public health, patient safety, and shared global health security, than fake, falsified, fraudulent or poor quality unregulated medicines - also commonly known as “counterfeit medicines” - now endemic in the global drug supply chain (Tim K Mackey & Liang, 2013). Counterfeit medicines pose a serious risk to public health around the world. It is low- and middle-income countries and those in areas of conflict, or civil unrest, with very weak or non-existent health systems that bear the greatest burden of SSFFC (Substandard, Spurious, Falsely labelled, Falsified and Counterfeit) medical products because the cost of legitimate drugs is beyond the reach of much of the population and legal controls are often weak (World Health Organization, 2010). For this purpose, further scientific research and development is called for to design user-friendly, low-cost and robust portable (hand-held) devices and techniques for detecting and identifying counterfeit medicines in real settings (Karunamoorthi, 2014). This dissertation promotes a technique called Raman spectroscopy that has the potential to optimize quality testing for a broad range of medicines. The aim of this dissertation was to study de detection of medicines through its package, for which it was relevant to investigate if there were differences within the package material and how these differences could affect the spectrums of the tablets under analysis. The results revealed that in fact there are variations in package materials that influence the spectrums acquired. These variations can be due to thickness or density variations. Nevertheless, these variations can be easily overcome using preprocessing methods. For this study, calibration tablets of paracetamol, were also made on purpose for experiencing the detection of different concentrations of an API through blister packages. Furthermore, in this investigation, three different probes were used for the measurements: PhAT probe, MKII probe attached to Raman Workstation microscope, and a green laser, to compare results. Results show that it is possible to detect different concentrations of an active ingredient of tablets through a white blister package using Raman spectroscopy, namely the PhAT probe. This equipment can be put in place analysis enabling faster detection of counterfeit medicines, in a non-invasive and non-destructive way that requires no sample preparation and does not need highly specialized personnel for its use. It provides reliable and useful data and it is portable. This is a promising technique for detecting counterfeit medicines and it is relatively low cost.Talvez não exista maior desafio para a saúde pública global do que a existência de medicamentos não regulamentados, de fraca qualidade, falsificados ou fraudulentos – também conhecidos como "medicamentos falsificados" - agora endémicos na cadeia global de abastecimento e distribuição de medicamentos (Tim K Mackey & Liang, 2013). A qualidade dos medicamentos disponíveis varia muito entre diferentes países devido à falta de regulamentos bem definidos e existência de práticas de controlo de qualidade deficientes. Os medicamentos falsificados incluem produtos com as substâncias adequadas ou com ingredientes errados, sem substância ativa, com quantidade insuficientes ou excessiva de substância ativa, ou com rotulagem errada e falsa. Por vezes, os medicamentos podem ser contaminados com outras substâncias ou podem até sofrer degradação química devido a fracas condições de armazenamento por exemplo em ambientes húmidos. Os medicamentos falsificados são amplamente distribuídos e podem ser bastante sofisticados, incluindo embalagens e estratégias de marketing muito convincentes. Os medicamentos falsificados representam um sério risco para a saúde pública em todo o mundo, sendo que a maioria das denúncias estão relacionadas com antibióticos, anti protozoários, hormonas e esteroides. São os países em desenvolvimento e aqueles em áreas de conflito, ou agitação civil, com sistemas de saúde muito fracos ou inexistentes que mais sofrem com produtos médicos “SSFFC” (Substandard, Spurious, Falsely labelled, Falsified and Counterfeit - denominação atribuída pela Organização Mundial de Saúde, para a definição de medicamentos falsificados), uma vez que o custo dos medicamentos legítimos está fora do alcance de grande parte da população e o controlo legal dos medicamentos é muitas vezes fraco (World Health Organization, 2010). Os medicamentos falsificados nestes países aumentaram devido à existência de muitas doenças infecciosas como a malária e a tuberculose. Neste estudo, é dado o exemplo do sistema de vigilância e monitorização da Organização Mundial de Saúde, que utiliza um sistema de inspeção constituído por três níveis: um primeiro nível realizado no local, que inclui uma inspeção visual da embalagem do medicamento em análise e a sua comparação com embalagens de medicamentos originais; um segundo nível, também este realizado no local, que se segue quando existem dúvidas relativamente aos testes realizados no primeiro nível e que inclui uma validação laboratorial utilizando métodos relativamente simples; se ainda assim os resultados forem inconclusivos segue-se o terceiro nível onde o medicamento é enviado para um laboratório forense, fora deste local, onde são realizados testes mais específicos de confirmação. Trata-se de um processe demorado pelo que mais investigação e desenvolvimento científico são requeridos para projetar técnicas de deteção e equipamentos fáceis de utilizar, de baixo custo e que sejam robustos e portáteis permitindo a identificação de medicamentos falsificados rapidamente e no local (Karunamoorthi, 2014). Esta dissertação promove uma técnica chamada espectroscopia Raman, que tem o potencial para otimizar testes de qualidade para uma ampla gama de medicamentos. Quando um feixe de luz interage com matéria, este pode ser transmitido, absorvido ou espalhado. Quando a luz é espalhada a partir de uma molécula, a maioria dos fotões é espalhada elasticamente, sendo que os fotões espalhados têm a mesma energia, frequência e comprimento de onda que os fotões incidentes. Contudo, uma pequena quantidade de luz é espalhada inelasticamente ou seja, a frequências diferentes, e normalmente mais baixas, do que os fotões incidentes. Este é chamado efeito Raman e foi descoberto por Krishna e Raman. Um espectro Raman contém bandas que são características e proporcionais a concentrações específicas de moléculas numa amostra, pelo que a espectroscopia Raman fornece uma boa análise qualitativa e quantitativa. A intensidade do espalhamento Raman é proporcional ao número de moléculas que produzem este espalhamento Raman. Como resultado, a intensidade do espalhamento Raman pode ser utilizada para medir quanto de um material está presente na amostra em análise (análise quantitativa). A forma de um espectro Raman pode ser utilizada para determinar que tipos de vibrações moleculares existem na amostra em análise. Esta informação vibracional pode ser utilizada para identificar materiais numa amostra (análise qualitativa). Diferenças em termos de stress, temperatura, estrutura cristalina, micro-heterogeneidade etc, podem, portanto, frequentemente ser medidas usando a espectroscopia Raman. A espectroscopia Raman é benéfica para diversos tipos de análise quantitativa e qualitativa num vasto número de campos, incluindo investigação química fundamental, ciências da vida (por exemplo, estudos biomédicos in vivo), controlo de processos, ciências forenses e na área farmacêutica. A espectroscopia Raman pode, deste modo, ser utilizada como uma tecnologia não destrutiva, não invasiva e ainda, como uma tecnologia de monitorização à distância. De um modo breve, as vantagens conhecidas da espectroscopia Raman incluem: elevada especificidade química, a capacidade de quantificar múltiplos constituintes numa forma farmacêutica sólida, a capacidade de analisar diferentes polimorfos e formas cristalinas; a elevada velocidade de análise; a ausência de necessidade de preparação da amostra; a ausência de necessidade de utilização de solventes e/ou consumíveis e a natureza de análise não destrutiva em comparação com outras técnicas de análise tradicionais. Portanto, a espectroscopia Raman é uma tecnologia estabelecida para assegurar a qualidade de produtos farmacêuticos permitindo identificar substâncias ativas e dar informação adicional sobre os excipientes, assim como a concentração relativa das substâncias ativas para os excipientes. Estes rácios podem ser a chave para detetar medicamentos falsificados uma vez que os indivíduos que produzem este tipo de produtos frequentemente têm em conta a quantidade da substância ativa mas não são tão precisos com as quantidades exatas de excipientes. Os instrumentos baseados na tecnologia do efeito Raman têm vindo a evoluir ao longo dos anos, passando de espectrómetros tradicionais de laboratório para ferramentas de menores dimensões, mais acessíveis em termos de custo, mais rápidas e eficientes, pelo que a análise de amostras em situações reais (em campo) ou até dentro de embalagens ou contentores, passou de um conceito ideal para uma tecnologia bem estabelecida para diferentes produtos farmacêuticos. O objetivo desta dissertação é explorar a deteção de medicamentos falsificados utilizando técnicas de espectroscopia Raman para uma análise não destrutiva e não invasiva, tendo em vista a sua aplicabilidade em campo (ou seja, em locais como fronteiras ou locais onde se realiza a inspeção e distribuição de medicamentos) e não, em laboratórios com boas condições e equipamentos assim como pessoal especializado. Esta investigação procura estudar a deteção de medicamentos falsificados através da sua embalagem, para a qual foi relevante investigar possíveis diferenças no próprio material de embalagem e o quanto essas diferenças poderiam interferir com os espectros obtidos dos comprimidos em análise. Os resultados revelaram que, de facto, existem variações em materiais de embalagem que influenciam os espectros adquiridos. Estas variações podem dever-se a diferenças de espessura ou densidade no material. Ainda assim, concluiu-se que estas variações podem ser facilmente ultrapassadas através da utilização de métodos de pré-processamento dos espectros. Para este estudo, foram também produzidos comprimidos de paracetamol, no sentido de experimentar a deteção de diferentes concentrações de um princípio ativo, através da sua embalagem. Além disto, nesta dissertação, foram ainda utilizadas três sondas para as mesmas medições: a sonda PhAT, a sonda MKII conectada ao microscópio Raman Workstation, e um laser verde, para comparar os resultados. Os resultados mostram que é possível detetar concentrações diferentes de um princípio ativo em comprimidos, através de um blister branco, utilizando espectroscopia de Raman, mais precisamente, a sonda PhAT. Este equipamento pode ser colocado em qualquer local de análise, permitindo uma deteção rápida dos medicamentos falsificados, de modo não-invasivo e não-destrutivo sem necessitar de preparação prévia da amostra nem de pessoal altamente especializado para a sua utilização. Este equipamento fornece dados confiáveis e é portátil. Esta é uma técnica promissora para a deteção de medicamentos falsificados e apresenta um custo relativamente baixo

Multiplexed, affordable, and portable platform for real time quantification of counterfeit and substandard medicines

The World Health Organization estimates that about 10-30% of pharmaceuticals in the world are either substandard or counterfeit. The number is even higher in the developing countries. From a public health perspective, a key contributor to the development and proliferation drug resistant strains of infections, including tuberculosis (TB), malaria and other infections that are leading killers in resource limited settings is poor quality medicines. Most of the main causes are profit driven corruption in many pharmaceutical companies, the poor manufacture and quality control, and/or the inappropriate storage conditions. Poor quality drugs lead to loss of life, create morbidity, strain the financial structure of the health system and lead to long-term drug resistance that affects us all. The current technology for screening poor quality drugs can be divided into 2 categories: the high end, precise and high cost technologies (such as High Performance Liquid Chromatography) and lower cost and qualitative technologies (such as Thin-Layered Chromatography). The high-end methods can give a precise measurement of active pharmaceutical ingredient (API) concentration and the presence of impurities in the tablets, but require trained personnel, advanced machine and lab set up, not suitable for field testing where most of poor quality pharmaceuticals have been found. The lower cost techniques require little training and simple equipment to operate at a relatively inexpensive price, but only gives qualitative results. In addition, most of current methods do not look at the dissolution profile of the tablets simultaneously with the concentration of API. Therefore, we propose to develop an assay that can quantify the concentrations of multiple APIs simultaneously and measure dissolution rates. In order to address current gaps in knowledge, my research proposal has three main parts in the assay development: 1) Development of an fluorescent/luminescent assay for detection of counterfeit/substandard antimalarial using small-molecules-based methods and field testing in Ghana; 2) Development of a fluorescent assay for detection of water-soluble pharmaceuticals using SELEX; and 3) Design a detection platform using microfluidic chips for real time quantification of multiple active pharmaceutical ingredients. For proof-of-concept, an antimalarial drug (artesunate and amodiaquine) and antibacterial antibiotics (sulfamethoxazole and trimethoprim) are selected to demonstrate the probe development and test the chip performance. Overall, the assay will be rapid, robust, portable, inexpensive, multiplexed, quantitative, specific, and sensitive. At a big picture level, emphasizing drug quality and creating robust mechanisms of drug testing will improve health outcomes and enhance treatment efficacy in resource limited settings

FORENSIC PHARMACEUTICAL ANALYSIS OF COUNTERFEIT MEDICINES

The World Health Organisation suggests that falsified and substandard medicines (FSMs) constitute approximately 10% of medicines globally with higher figures expected in low and middle income countries (LMICs). To combat the proliferation of FSMs, this study is aimed at developing simple and rapid instrumental methods for the identification and quantification of these medicines. Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopy, Raman spectroscopy and two probe Mass Spectrometry (MS) methods were assessed for the rapid screening of tablet dosage forms. These systems were chosen because NO solvent extraction of the sample was required. Comparison with analyses of the tablets by accepted but more time consuming methods (UV-Vis and LC-MS) assessed the quality of the data obtained. Analgesic/antipyretic and antimalarial medicines tablet dosage forms are commonly falsified and for this study tablets were obtained opportunistically from different countries around the world. Reference spectra of appropriate active pharmaceutical ingredients (APIs) and excipients were created, for each method, as part of the identification process. Currently only Raman and ATR-FTIR delivered quantitative results which were based on automated multivariate analysis. For tablets with a single API, Raman and ATR-FTIR provided the simplest route to API confirmation and for tablets with multiple APIs or APIs present at <10%w/w, in the tablet, probe MS methods were superior. Quantitative screening using ATR-FTIR required the samples to be weighed and crushed to produce reproducible data. Comparison of API confirmation tests between trial methods and LC-MS showed complete agreement and the quantitative results were within ±15% of the UV-Vis data. Each of the new tests can be completed in under five minutes and a survey of 69 paracetamol tablets, from around the world, showed that 10% were suspect. Subsequent probe MS showed the presence of a second undeclared API in different samples. More complex tablet formulations, for example the antimalarials were difficult to quantify rapidly. Raman and PCA methods provide a rapid approach to tablet identification within a limited range of possibilities. Factors that may affect Raman spectra of tablets include the expected API, the API levels, different excipients, colours or surface coatings for the tablets. The simplicity, speed and cost effectiveness of the proposed analytical methods make them suitable for use in LMICs. The potential use of these simple analytical methods in addition to already established pharmacopoeia approved (solvent extraction) techniques could help provide more comprehensive data about FSMs globally

Surveillance approaches to detect the quality of medicines in low-middle income countries with a focus on artemisinin combination therapies for malaria.

Introduction: Recent years have seen an increase in reports of poor quality antimalarials with estimates that up to 30% have failed chemical analysis, even though robust empirical evidence for their prevalence remains scarce. Several internal (associated with national systems) and external (not under the direct control of national authorities) risk factors may contribute to the circulation of poor quality medicines. This thesis will explore these factors with an overall aim of providing evidence to strengthen medicines quality surveillance systems (MQSS) in low-middle income countries (LMICs). Methods: Data collection was conducted in two phases in Senegal between March 2013 and April 2014. The first phase involved interviews with key stakeholders of the MQSS such as authority representatives as well as treatment providers and explored the system’s vulnerability to risk factors for poor quality medicines and their perceptions of the quality of medicines available in Senegal. The second phase comprised a series of laboratory-based studies with technicians at the national medicine quality control laboratory (MQCL) including an assessment of the practical utility, usefulness and acceptability of a specific test, to check the quality of artemisinin based medicines, namely the artemisinin derivative test (ADT). Finally, a systematic literature review assessing the study design and reporting of antimalarial medicine quality studies and surveys was conducted with the included studies assessed for quality against our newly proposed list of criteria. Findings: Overall, interviewees expressed confidence in the quality of medicines available in the public and regulated private sectors which was attributed to effective national medicines regulation and adequate technical capacity at the MQCL. In contrast, poor quality medicines were thought to be available in the unregulated (informal) sector as they were not subjected to national regulatory processes or stored appropriately, resulting in exposure to direct sunlight and high temperatures. Generic medicines were also perceived to be of inferior quality when compared to their brand versions as they were lower in cost and thought to be less effective in alleviating symptoms. The ADT demonstrated a promising level of accuracy to detect fake or grossly substandard artemisinin based medicines and laboratory technicians favoured its simplicity of use without the need for specific training. The literature review found that there is much heterogeneity in study design and inconsistency in reporting which has impacted on the generalisability of findings for antimalarial medicine quality studies. Conclusion: A major shift is required in the framing of medicine quality from a technical/legal to a clinical paradigm with evidence required to demonstrate the impact of poor quality medicines on public health. National governments need to invest in regulatory and technical capacity to strengthen MQSS to minimise the likelihood of poor quality medicines circulating in a country. Utilising simple, and portable (preferably handheld) tests like the ADT, in non-laboratory settings may enhance post-marketing surveillance, especially in resource constrained contexts. Nonetheless, comparative evaluation of all currently available screening technologies for their capability to distinguish poor quality antimalarials for confirmatory pharmacopeia testing and public health action is required. Suggestions that reduce the risk of bias and error have been proposed for conducting medicine quality studies to enable standardisation of study design and reporting, thereby increasing the reliability of findings and allowing comparison between studies