Interest of Raman spectroscopy for the detection and analysis of poor-quality medicines

editorial reviewedAccess to quality medicines is an essential right of the patients. However, in 2017, the World Health Organization estimated that 1 in 10 medical products circulating in low- and middle-income countries is either substandard or falsified. This reinforces the fact that post-marketing surveillance (PMS) of medical products by strong national regulatory authorities (NRA) is crucial. To achieve an efficient PMS, the NRA need analytical tools at the inspection, screening, confirmatory and forensics levels to control the physicochemical properties of the samples. Among the analytical tools available, Raman spectroscopy is particularly interesting because of its spectral specificity and the wide variety of acquisition modes available. Handheld devices may be used directly on the field to confirm the presence of a specific active pharmaceutical ingredient (API) in a formulation [1]. Thanks to databases of pure ingredients, it is also possible to identify the compound present when a wrong API is present [2]. Recent developments have extended the applicability of handheld devices to the analysis of fluorescent chemicals and the analysis through barriers [3]. The detection of substandard medicines is also made possible with the construction of regression chemometrics models [4]. Benchtop systems and among them imaging systems are particularly useful in the confirmatory and forensic steps. Indeed, the imaging systems enable the visualization and identification of a large range of both organic and inorganic compounds used as API or excipients [5]. In addition, thanks to the high spatial resolution, it allows the detection of trace contaminants. This information may be of particular interest during prosecutions and the clustering of criminal cases. Nevertheless, the extraction of the relevant information from the raw measurements requires once again intensive work by highly trained staff. In conclusion, Raman spectroscopy have particularly interesting features for the PMS of medicines

Applications of vibrational spectroscopy and hyperspectral imaging for the analysis of substandard and falsified medicines.

peer reviewedAccess to quality medicines is an essential right of the patients. However, in 2017, the World Health Organization estimated that 1 in 10 medical products circulating in low- and middle-income countries is either substandard or falsified. This reinforces the fact that post-marketing surveillance (PMS) of medical products by strong national regulatory authorities (NRA) is crucial. To achieve an efficient PMS, the NRA need analytical tools at the inspection, screening, confirmatory and forensics levels to control the physicochemical properties of the samples. Because of their fast, non-destructive, and relatively affordable character, vibrational spectroscopy tools are unavoidably present at each step. Handheld devices are particularly useful during inspection and screening phases since these tools can identify and/or quantify active pharmaceutical ingredients (API) even through opaque packaging in seconds. However, they generally need exhaustive and up-to-date databases for each specific product. Another limitation is the work and time needed before going into the field to develop and validate the chemometric models. Indeed, this mandatory step requires highly skilled scientists and a prior collection of certified references of the medicines to analyse. Benchtop systems and among them imaging systems are particularly useful in the confirmatory and forensic steps. Indeed, the imaging systems enable the visualization and identification of a large range of both organic and inorganic compounds used as API or excipients. In addition, thanks to the high spatial resolution, it allows the detection of trace contaminants. This information may be of particular interest during prosecutions and the clustering of criminal cases. Nevertheless, the extraction of the relevant information from the raw measurements requires once again intensive work by highly trained staff. In conclusion, vibrational spectroscopy tools have particularly interesting features for the PMS of medicines, but research is still needed to make them easier to set up and use by NRA inspectors and non-specialists

Development of a SERS strategy to overcome the nanoparticle stabilisation effect in serum-containing samples: Application to the quantification of dopamine in the culture medium of PC-12 cells

The analysis of serum samples by surface-enhanced Raman spectroscopy (SERS) has gained ground over the last years. However, the stabilisation of colloids by the proteins contained in these samples has restricted their use in common practice, unless antibodies or aptamers are used. Therefore, this work was dedicated to the development of a SERS methodology allowing the analysis of serum samples in a simple and easy-to-implement way. This approach was based on the pre-aggregation of the colloid with a salt solution. Gold nanoparticles (AuNPs) were used as the SERS substrate and, owing to its physiopathological importance, dopamine was chosen as a model to implement the SERS approach. The presence of this neurotransmitter could be determined in the concentration range 0.5 to 50 ppm (2.64 – 264 μM) in the culture medium of PC-12 cells, with a R2 of 0.9874, and even at lower concentrations (0.25 ppm, 1.32 μM) in another matrix containing fewer proteins. Moreover, the effect of calcium and potassium on the dopamine exocytosis from PC-12 cells was studied. Calcium was shown to have a predominant and dose-dependent effect. Finally, PC-12 cells were exposed to dexamethasone in order to increase their biosynthesis and release of dopamine. This increase was monitored with the developed SERS approach

Development and optimisation of methods to synthesise sol-gel nanoparticles and sol-gel core-shell nanoparticles to produce nanosensors

The aim of this project was to synthesise nanosensors. It can be defined as an inert, biofriendly matrix in which sensing molecules are entrapped, allowing determination of analytes concentration. Two types of nanosensors were synthesised according to the Stöber method and characterised by a scanning electron microscope and a CPS disc centrifuge. The nanosensors were calibrated with succes using a fluorimeter.Synthesis of nanosensor

Diffusion Raman exaltée de surface appliquée à des petites molécules: Etude des performances quantitatives

During the last decade, Raman spectroscopy has taken an important place in the pharmaceutical and biological fields. However, this technique presents a main limitation, which is its lack of sensitivity. When the target analyte is adsorbed or in the vicinity of metallic nanostructure, acting as antennas, the Raman scattering is dramatically exalted, enabling to solve the main issue of normal Raman spectroscopy. This effect is commonly reported as surface-enhanced Raman scattering (SERS). The exaltation factor can vary from 103 to 1014 depending on the structure of the studied molecule and the kind of metallic nanostructure used to perform the SERS analyses. Despite the dramatic advances in SERS research, a few numbers of applications dealing with quantitative SERS method development have been published. In this context, the major aim of this thesis was to investigate the use of SERS mainly as quantitative analytical tool considering various applications. In a first step, the analytical performances of SERS were evaluated through the development and validation of a quantitative method. A simple pharmaceutical model based on paracetamol/4-aminophenol (4-AP) has been used to perform this study aiming to detect quantitatively 4-AP in a pharmaceutical formulation based on paracetamol taking account of its specification limit of 1000 ppm. Silver nanoparticles were used as SERS substrate after having tested the repeatability of their syntheses in terms of size and size dispersion. The SERS sample preparation was further optimized in order to maximize the SERS enhancement before performing SERS quantitative analyses. A standard addition method was then selected as calibration method to take account of the matrix effect coming from the pharmaceutical excipients. Solutions comprising the pharmaceutical formulation were spiked with different concentrations of 4-AP and analyzed using SERS. Finally, the SERS method developed was validated in a range of concentrations from 3 to 15 µg L-1 using the accuracy profile as decision tool. The accuracy profile allowed to have a visual representation of the future analytical performances of the developed method. Based on these results, a first estimation of the true analytical performances of SERS was carried out in the pharmaceutical field. Moreover, that was, to the best of our knowledge, the first SERS quantitative method fully validated. A feasibility study of carrying out quantitative analyses using Surface-enhanced Raman chemical imaging (SER-CI) was also performed keeping the pharmaceutical model previously used but presented as a solid form. The main issue for this project was the covering of the tablet surface, based on paracetamol, by the silver nanoparticles suspension. After having tested two ways to cover the tablets surface with the SERS substrate and evaluated the homogeneity of the covering, the SER-CI method was developed using tablets comprising different concentrations of 4-AP. Two different approaches were used to normalize the acquired data and the precision profile allowed to select the best normalization approach. Finally, using the capillarity as covering way and the most appropriate normalization approach which was the BT band intensity normalization, SER-CI quantitative analyses of 4-AP from concentrations of 0.025% to 0.2% in the pharmaceutical tablets were successfully carried out. These preliminary results were very promising and this study was the first published semi-quantitative application of SER-CI in the pharmaceutical field. According to our objectives, the following study was focused on the development of a SERS multiplexed quantitative approach. The molecules of interest selected were bisphenols. Bisphenol A (BPA) is well known for its use in plastic manufacture and thermal paper production despite its health toxicity as an endocrine disruptor. Since the publication of new legislations regarding the use of BPA, manufacturers have begun to replace BPA with other phenolic molecules such as bisphenol B (BPB) and bisphenol F (BPF) to produce plastic material without any guarantee regarding their health safety. In this context, the development of a SERS method allowing the detection of these molecules simultaneously at low concentrations was interesting. Silver nanoparticles were used as SERS substrate. To solve the affinity issue of bisphenols for metallic surface, the surface of the silver nanoparticles was first modified with pyridine and the resulting bisphenols solutions were then exposed to ultrasounds to generate its monohydroxylated form. These strategies allowed to reach a limit of detection of 250 ng L-1 for BPA and BPB and of 5 µg L-1 for BPF. Semi-quantitative detection of these molecules were performed in tap water solutions at a concentration range of 0.25 to 20 µg L-1 for BPA and BPB and from 5 to 100 µg L-1 for BPF. A feasibility study of performing a multiplexed SERS detection of these molecules was further successfully carried out before implementing this developed SERS method on real samples. In order to complete our research work, SERS technology was implemented on biological samples. A SERS method enabling to monitor the release of anatabine by methyl jasmonate (MeJa) elicited Bright Yellow-2 cells (BY-2) in the culture medium was successfully developed. The first step of this project was to study the SERS activity of anatabine in the culture medium. Anatabine was SERS detected at low concentration despite a matrix effect coming from the several compounds of the medium, mainly sugar and salts. A calibration was then carried out by spiking the culture medium in the presence of the BY-2cells with different concentrations of anatabine ranging from 250 to 5000 µg L-1. Finally, the monitoring study was performed after the elicitation of BY-2 cells by collecting samples from the culture medium after 0, 24, 48, 72 and 96 h. These samples were analyzed by SERS and the amounts of anatabine released were determined using the calibration linear regression (y = 0.0054x – 0.8754; R2 = 0.9951) previously obtained. The release of anatabine reached a plateau after 72 h of 82 µg/g of fresh weight of elicited BY-2 cells. This additional study enabled to confirm the interest of SERS technology in a more complex matrix while proposing a very original application of SERS in the plant material analysis. The last part of this present thesis was focused on a SERS study of a new SERS substrate synthesized by the Centre Spatial de Liège (CSL) using the laser induced forward transfer (LIFT) technique. In order to open the way towards new SERS developments, a CSL substrate presenting areas with different nanoparticles properties in terms of size and density was tested in SERS with the aim to highlight the area presenting the higher enhancement. These interesting results will be used to improve the CSL SERS substrate characteristics and to optimize the LIFT parameters for coming applications studies. To conclude, based on these results, despite the need to improve the analytical performances of the technique, we are convinced that SERS is an amazing and challenging analytical tool offering the possibility to perform quantitative analyses and semi-quantitative analyses in complex matrices and considering various applications. SERS could clearly be used as a promising alternative to separative techniques in routine, reducing the cost, the time and the solvents consumptions during analyses./Durant ces dix dernières années, la spectroscopie Raman a pris une place importante dans les domaines pharmaceutique et biologique. Cependant, cette technique présente un grand désavantage, à savoir sa faible sensibilité. Lorsque l’analyte d’intérêt est adsorbé ou placé à proximité d’une nanostructure métallique, agissant comme une antenne, la diffusion Raman est considérablement exaltée, ce qui permet de contourner le principal inconvénient de la spectroscopie Raman normale. Cet effet est communément appelé diffusion Raman exaltée de surface (SERS). Le facteur d’exaltation peut varier d’un facteur allant de 103 à 1014 suivant, d’une part, la structure de la molécule étudiée, et d’autre part, le type de nanostructure métallique utilisé. Malgré les avancées considérables dans le domaine du SERS, les publications traitant du développement de méthodes SERS quantitatives demeurent rares. Pour cette raison, l’objectif principal de cette thèse était d’examiner en profondeur l’utilisation du SERS principalement comme outil analytique quantitatif à travers des applications variées. Dans un premier temps, les performances analytiques du SERS ont été évaluées au travers du développement et de la validation d’une méthode quantitative. Un modèle pharmaceutique basé sur le paracétamol/4-aminophénol (4-AP) a été utilisé pour réaliser cette étude. Celle-ci avait pour objectif de doser le 4-AP dans une formulation pharmaceutique à base de paracétamol compte tenu de sa teneur maximale tolérée de 1000 ppm. Des nanoparticules d’argent ont été utilisées comme substrat SERS après avoir testé la répétabilité de leurs synthèses quant à la taille et la dispersion de la taille. La préparation des échantillons SERS a été par la suite optimisée afin de maximiser l’exaltation du signal avant de réaliser les analyses SERS quantitatives. La méthode d’ajouts dosés a été utilisée comme outil de calibration afin de tenir compte de l’effet de matrice important provenant des excipients. Des solutions comprenant la formulation pharmaceutique ont été dopées avec différentes concentrations en 4-AP avant d’être analysées en SERS. Finalement, la méthode SERS développée a été validée sur une gamme de concentrations allant de 3 à 15 µg L-1 en utilisant le profil d’exactitude comme outil de décision. Ledit profil nous a permis d’obtenir une représentation visuelle des futures performances analytiques de la méthode développée. En se basant sur ces résultats, une première estimation des vraies performances analytiques du SERS a pu être réalisée dans le domaine pharmaceutique. De plus, à notre connaissance, il s’agit de la première méthode quantitative SERS complètement validée. Une étude de faisabilité concernant l’analyse quantitative en imagerie SERS (SER-CI) a été réalisée en gardant le modèle pharmaceutique précédemment étudié mais utilisé sous une forme solide. La principale difficulté de ce projet a été le recouvrement de la surface du comprimé de paracétamol par les nanoparticules d’argent en suspension. Après avoir testé deux voies différentes permettant de couvrir les comprimés par le substrat SERS et évalué l’homogénéité du dépôt, la méthode SER-CI a été développée en utilisant des comprimés contenant plusieurs concentrations en 4-AP. Deux approches ont également été utilisées pour normaliser les données collectées et l’utilisation du profil de précision a permis de sélectionner l’approche de normalisation la plus adéquate. Sur base de ces résultats, en utilisant la capillarité comme méthode de recouvrement et le normalisation via l’intensité de la bande du BT, des analyses quantitatives en SER-CI du 4-AP pour une gamme de concentrations allant de 0,025% à 0,2% ont pu être réalisées dans la matrice pharmaceutique. Ces résultats préliminaires sont très encourageants dans la mesure où il s’agissait d’une première utilisation du SER-CI dans le cadre d’analyses semi-quantitatives dans une matrice pharmaceutique solide.Compte tenu de nos objectifs, l’étude suivante a été focalisée sur le développement d’une approche SERS multiplex et quantitative. Les molécules d’intérêt sélectionnées étaient les bisphénols. Le bisphénol A (BPA) est bien connu pour son utilisation dans la fabrication de plastique et la production de papiers thermiques malgré sa toxicité en tant que perturbateur endocrinien. Depuis la publication de nouvelles législations concernant l’utilisation du BPA, les industriels ont commencé à remplacer le BPA par d’autres molécules phénoliques comme le bisphénol B (BPB) et le bisphenol F (BPF) pour produire des matériaux en plastique, et ce, sans avoir de garantie sur leur absence de toxicité. En l’espèce, le développement d’une approche SERS permettant de détecter ces molécules simultanément et à faible concentration était intéressant. Des nanoparticules d’argent ont été utilisées comme substrat SERS. La surface de ces nanoparticules a été modifiée dans un premier temps par l’ajout de pyridine. Quant aux solutions de bisphénols, elles ont été exposées par la suite aux ultrasons afin de générer leur forme mono-hydroxylée. Cette stratégie a permis de résoudre le manque de sensibilité et d’atteindre des limites de détection de 250 ng L-1 pour le BPA et le BPB et de 5 µg L-1 pour le BPF. Par la suite, le dosage semi-quantitatif de ces molécules dans l’eau alimentaire a été réalisé pour une gamme de concentrations allant de 0,25 à 20 µg L-1 pour le BPA et le BPB et allant de 5 à 100 µg L-1 pour le BPF. A partir de ces données, une étude de faisabilité visant à réaliser une détection multiplex de ces molécules a été menée avec succès avant d’utiliser cette méthode dans le cadre de l’analyse d’échantillons réels.Dans une dernière étape, le SERS a été envisagé pour l’analyse d’échantillons biologiques. Une méthode permettant de monitorer la libération d’anatabine dans le milieu de culture par des cellules Bright Yellow-2 (BY-2) élicitées au jasmonate de méthyle a été développée. Des nanoparticules d’or, étant d’avantage biocompatibles que l’argent, ont été utilisées comme substrat SERS. Dans un premier temps, l’activité SERS de l’anatabine a été étudiée dans le milieu de culture. Celle-ci fut détectée à de faibles concentrations malgré l’effet de matrice provenant des différents composants du milieu, principalement du sucre et des sels. Ensuite, une calibration a été réalisée en dopant le milieu de culture en présence des cellules BY-2 avec différentes concentrations en anatabine en allant de 250 à 5000 µg L-1. Finalement, l’étude de monitoring a été réalisée après l’exposition des cellules BY-2 au jasmonate de méthyle en prélevant des échantillons à partir du milieu de culture après 0, 24, 48, 72, 96h. Ces échantillons ont été analysés en SERS et les quantités d’anatabine libérées en fonction du temps ont été déterminées en utilisant la régression linéaire (y = 0.0054x-0.8754 ; R2 =0.9951) obtenue précédemment lors de la calibration. La libération d’anatabine a atteint un plateau après 72h de 82 µg d’anatabine libérée pour 1g de BY-2 cellules fraîchement pesées. Cette analyse supplémentaire a permis de confirmer l’intérêt de la technologie du SERS dans des milieux complexes tout en proposant une application originale dans le domaine de l’analyse de matériel végétal.La dernière partie de cette thèse s’est focalisée sur l’examen d’un nouveau substrat SERS synthétisé par le Centre Spatial de Liège (CSL) en utilisant la technique du LIFT dans le but de développer de nouvelles applications du SERS. Ce substrat présentait différentes zones avec des propriétés variables quant à la taille et la densité de nanoparticules. Ces dernières ont été testées afin de mettre en évidence la zone présentant la plus grande exaltation du signal. Ces résultats intéressants pourront être utilisés pour améliorer les propriétés du substrat SERS du CSL et optimiser les paramètres du LIFT pour des applications futures.En conclusion, eu égard aux résultats obtenus durant ces recherches, malgré la nécessité d’améliorer les performances analytiques de cette technique, il apparait clairement que le SERS est un outil analytique à haut potentiel. Il offre la possibilité de réaliser des analyses quantitatives ou semi-quantitatives dans des matrices complexes à travers des approches très variées. Le SERS pourrait devenir une alternative très prometteuse aux méthodes séparatives en routine permettant de réduire les coûts, le temps et la consommation de solvants lors des analyses

Etude de l'intérêt de la diffusion Raman exaltée de surface (SERS) dans la détection de nicotine dans les e-liquides à l'aide d'un système Raman portable

peer reviewe

Development of near infrared spectroscopic methods using desirability indexes: How to select the most appropriate calibration model

In the last decade, considerable research and developments dealing with near infrared spectroscopy (NIRS) have taken place in industrial field, especially in pharmaceutical industry. This enthusiasm can be explained by the fact that NIRS is regarded as promising and attractive tool in Process Analytical Technology (PAT) and Green Chemistry frameworks. Taking into account its non-invasive, non-destructive character, fast data acquisition and the use of probes in on-line, in-line and at-lines, this technique is expected to reach the aims of the latters. However, the development of a NIR quantitative method is not straightforward in comparison with conventional analytical techniques. Its development requires time-consuming reference methods, chemometrics and iterative heuristic approaches to build a model allowing the prediction of the analyte of interest according to the acceptance criteria consistent with the intended use of the method. Facing to the lack of objective decision rule of the traditional chemometric criteria such as R2, RMSEC, RMSECV and RMSEP, it is essential to develop innovative approaches for the selection of the most appropriate calibration model from a models plurality. In this context, a methodology using desirability indexes, such as the Fitting Model Index (FMI), based on tolerance intervals was developed in order to increase significantly the objectivity of the decision process. This latter allows to reduce dramatically the development and the validation steps and thus could ease the implementation of NIR spectroscopy in pharmaceutical industry

Validation methodologies of near infrared spectroscopy methods in pharmaceutical applications

As any analytical methods, a mandatory step at the end of the development of a near infrared spectroscopy (NIRS) method is the validation. This step enables to give enough guarantees that each future results coming from the application of the method in routine will be closed enough to the true value. However, from the literature, a minority of NIRS methods are thoroughly validated despite of the guidelines published by different group and regulatory authorities to help analyst to adequately decide if his method can be considered as valid. In this context, the aim of this review is to offer a critical overview of the different validation methodologies applied to assess the validity of quantitative methods using near infrared spectroscopy used in the field of pharmacy