Confocal Raman spectroscopy coupled with in vitro permeation testing to study the effects of formalin fixation on the skin barrier function of reconstructed human epidermis.

Confocal Raman Spectroscopy is recognised as a potent tool for molecular characterisation of biological specimens. There is a growing demand for In Vitro Permeation Tests (IVPT) in the pharmaceutical and cosmetic areas, increasingly conducted using Reconstructed Human Epidermis (RHE) skin models. In this study, chemical fixation of RHE in 10 % Neutral Buffered Formalin for 24 h has been examined for storing RHE samples at 4 °C for up to 21 days. Confocal Raman Spectroscopy (CRS), combined with Principal Components Analysis, revealed the molecular-level effects of fixation, notably in protein and lipid conformation within the stratum corneum and viable epidermis. IVPT by means of high-performance liquid chromatography, using caffeine as a model compound, showed minimal impact of formalin fixation on the cumulative amount, flux, and permeability coefficient after 12 h. While the biochemical architecture is altered, the function of the model as a barrier to maintain rate-limiting diffusion of active molecules within skin layers remains intact. This study opens avenues for enhanced flexibility and utility in skin model research, promising insights into mitigating the limited shelf life of RHE models by preserving performance in fixed samples for up to 21 days.

Strat-M® positioning for skin permeation studies: A comparative study including EpiSkin® RHE, and human skin.

In the development and optimization of dermatological products, In Vitro Permeation Testing (IVPT) is pivotal for controlled study of skin penetration. To enhance standardization and replicate human skin properties reconstructed human skin and synthetic membranes are explored as alternatives. Strat-M® is a membrane designed to mimic the multi-layered structure of human skin for IVPT. For instance, in Strat-M®, the steady-state fluxes (JSS) of resorcinol in formulations free of permeation enhancers were found to be 41 ± 5 µg/cm2·h for the aqueous solution, 42 ± 6 µg/cm2·h for the hydrogel, and 40 ± 6 µg/cm2·h for the oil-in-water emulsion. These results were closer to excised human skin (5 ± 3, 9 ± 2, 13 ± 6 µg/cm2·h) and surpassed the performance of EpiSkin® RHE (138 ± 5, 142 ± 6, and 162 ± 11 µg/cm2·h). While mass spectrometry and Raman microscopy demonstrated the qualitative molecular similarity of EpiSkin® RHE to human skin, it was the porous and hydrophobic polymer nature of Strat-M® that more faithfully reproduced the skin's diffusion-limiting barrier. Further validation through similarity factor analysis (∼80-85%) underscored Strat-M®'s significance as a reliable substitute for human skin, offering a promising approach to enhance realism and reproducibility in dermatological product development.

Line-field confocal optical coherence tomography coupled with artificial intelligence algorithms to identify quantitative biomarkers of facial skin ageing.

Quantitative biomarkers of facial skin ageing were studied from one hundred healthy Caucasian female volunteers, aged 20-70 years, using in vivo 3D Line-field Confocal Optical Coherence Tomography (LC-OCT) imaging coupled with Artificial Intelligence (AI)-based quantification algorithms. Layer metrics, i.e. stratum corneum thickness (SC), viable epidermal thickness and Dermal-Epidermal Junction (DEJ) undulation, as well as cellular metrics were measured for the temple, cheekbone and mandible. For all three investigated facial areas, minimal age-related variations were observed in the thickness of the SC and viable epidermis layers. A flatter and more homogeneous epidermis (decrease in the standard deviation of the number of layers means), a less dense cellular network with fewer cells per layer (decrease in cell surface density), and larger and more heterogeneous nuclei within each layer (increase in nuclei volume and their standard deviation) were found with significant variations with age. The higher atypia scores further reflected the heterogeneity of nuclei throughout the viable epidermis. The 3D visualisation of fine structures in the skin at the micrometric resolution and the 1200 µm × 500 µm field of view achieved with LC-OCT imaging enabled to compute relevant quantitative biomarkers for a better understanding of skin biology and the ageing process in vivo.

Label-Free Quantification of Nanoencapsulated Piperonyl Esters in Cosmetic Hydrogels Using Raman Spectroscopy.

Raman spectroscopy is a well-established technique for the molecular characterisation of samples and does not require extensive pre-analytical processing for complex cosmetic products. As an illustration of its potential, this study investigates the quantitative performance of Raman spectroscopy coupled with partial least squares regression (PLSR) for the analysis of Alginate nanoencapsulated Piperonyl Esters (ANC-PE) incorporated into a hydrogel. A total of 96 ANC-PE samples covering a 0.4% w/w-8.3% w/w PE concentration range have been prepared and analysed. Despite the complex formulation of the sample, the spectral features of the PE can be detected and used to quantify the concentrations. Using a leave-K-out cross-validation approach, samples were divided into a training set (n = 64) and a test set, samples that were previously unknown to the PLSR model (n = 32). The root mean square error of cross-validation (RMSECV) and prediction (RMSEP) was evaluated to be 0.142% (w/w PE) and 0.148% (w/w PE), respectively. The accuracy of the prediction model was further evaluated by the percent relative error calculated from the predicted concentration compared to the true value, yielding values of 3.58% for the training set and 3.67% for the test set. The outcome of the analysis demonstrated the analytical power of Raman to obtain label-free, non-destructive quantification of the active cosmetic ingredient, presently PE, in complex formulations, holding promise for future analytical quality control (AQC) applications in the cosmetics industry with rapid and consumable-free analysis.

Efficiency of emulsifier-free emulsions in delivering caffeine and α-tocopherol to human skin.

OBJECTIVE: Increasing consumer demand for natural and environmentally friendly products is driving the cosmetic industry to seek greener and safer processes. High-frequency ultrasound technology (HFUT) stabilizes emulsions without adding emulsifying surfactants (ES). In this work, the formulation characteristics of an HFUT-treated emulsion and a Reference emulsion were compared for both caffeine and α-tocopherol. METHODS: A comparison was made between ES-free emulsions and the Reference emulsions based on droplet size, viscosity, pH and rheology behaviour for both active cosmetic ingredients. The permeation of caffeine and the skin retention of α -tocopherol were studied in vitro using Franz diffusion cells on human skin biopsies, considered the gold standard for permeation assays. RESULTS: The formulations developed were stable and showed suitable droplet size distribution. In the case of ES-free emulsions, the average droplet size was inferior to 1.5 µm regardless of the polarity of the active. All formulations presented a shear-thinning pseudoplastic behaviour, an attribute usually desired for cosmetic products. The skin permeation studies showed that in the case of caffeine (model hydrophilic molecule), the ES-free emulsion presented a delivery capacity similar to that of the Reference emulsion. However, for α-tocopherol (highly lipophilic model molecule), differences were observed in the distribution of the active in the stratum corneum with an advantage for the Reference emulsion, probably due to the impact of surfactants on the SC lipids. CONCLUSION: This work demonstrates that HFUT is a reliable tool that is able to prepare stable ES-free emulsions loaded with hydrophilic or lipophilic active ingredients. Skin permeation studies confirm that the emulsions produced by HFUT promote the delivery of the actives to the human skin. In the case of α-tocopherol, the delivery efficiency was lower than with the Reference emulsion, especially in the SC layers, due to the absence of surfactants. Nevertheless, the ES-free emulsion still represents a good compromise between efficacy and the need for green cosmetics in the market.

OBJECTIF: La demande croissante des consommateurs pour des produits naturels et respectueux de l'environnement encourage l'industrie cosmétique à développer des procédés plus écologiques et plus sûrs. La technologie des ultrasons à haute fréquence (HFUT) permet de stabilizer les émulsions sans ajouter de tensioactifs émulsionnants (ES). Dans ce travail, les caractéristiques d'une émulsion traitée par HFUT et d'une émulsion de référence ont été comparées. La caféine et l'α-tocophérol ont été utilisés comme actifs modèles. MÉTHODES: Les émulsions sans ES et les émulsions de référence on été comparées en termes de taille des gouttelettes, de viscosité, de pH et de comportement rhéologique pour les deux actifs. La perméation de la caféine et la rétention cutanée de l'α-tocophérol ont été étudiées in vitro sur des biopsies de peau humaine, en utilisant des cellules de diffusion de Franz, le 'gold standard' des tests de perméation. RÉSULTATS: Les formulations développées sont stables et présentent une distribution appropriée de la taille des gouttelettes. La taille moyenne des gouttelettes des émulsions sans ES est inférieure à 1.5 µm, quelle que soit la polarité de l'actif. Toutes les formulations présentent un comportement rhéofluidifiant adapté à un usage cosmétique. Les études de perméation cutanée montrent que l'émulsion sans ES contenant de la caféine (molécule modèle hydrophile) présente une capacité de délivrance similaire à celle de l'émulsion de référence. Dans le cas de l'α-tocophérol (molécule modèle lipophile), des différences ont été observées dans la distribution de l'actif dans le stratum corneum (SC) avec un avantage pour l'émulsion de référence, probablement lié à l'interaction entre les tensioactifs et les lipides du SC. CONCLUSION: Ce travail démontre que le traitement par HFUT permet de préparer des émulsions stables sans ES, quelle que soit la polarité des actifs cosmétiques à véhiculer. Les études de perméation cutanée confirment que les émulsions produites par HFUT permettent la diffusion cutanée des actifs dans la peau humaine. Même si dans le cas de l'α-tocophérol la quantité accumulée était plus faible, l'émulsion traitée par HFUT propose un bon compromis entre efficacité et éco-responsabilité.

Studying the effects of suberythemal UV doses on the human stratum corneum by in vivo confocal Raman spectroscopy

Background: The stratum corneum (SC) plays an important role in skin barrier function. It acts as a protective barrier against water loss, eliminates foreign substances and micro-organisms and acts against harmful effects of UVR. Objectives: Our aim was to study the impact of suberythemal doses of UVA and UVB exposure on the molecular structure, organization and barrier function of the SC by following different Raman descriptors. Materials & Methods: Twenty female volunteers, aged 20-30 years, with healthy skin were enrolled. Doses of 95 mJ/cm² UVA and 15 mJ/cm² UVB were applied to volunteers' forearms. In vivo Raman measurements were performed at irradiated and control regions. Results: The impact of UVA and UVB irradiation was observed following several Raman descriptors, i.e. the ratio of vasymCH2/vsymCH2 (2885 cm-1/2850 cm-1) corresponding to the organizational order of the lipid bilayer. Water content and mobility descriptors were obtained by calculating vOH/vCH ratio. Finally, protein secondary structure was evaluated based on the 1670 cm-1/1650 cm-1 ratio related to ß sheets and α helices, respectively. Conclusion: UVA induced a loosening of the lateral packing of lipids immediately after irradiation. In contrast, delayed impact caused a tightening of the lipid barrier, an increase in water content -mainly in the unbound water fraction and a higher relative amount of ß sheets in SC proteins. Overall, these observations may explain the thickening of the SC observed in previous studies. A UVB dose of 15 mJ/cm² was apparently below the threshold necessary to induce significant changes despite the trends observed in this study.

Comparison of Vibrational Spectroscopic Techniques for Quantification of Water in Natural Deep Eutectic Solvents.

Vibrational spectroscopic techniques, i.e., attenuated total reflectance infrared (ATR-IR), near infrared spectroscopy (NIRS) and Raman spectroscopy (RS), coupled with Partial Least Squares Regression (PLSR), were evaluated as cost-effective label-free and reagent-free tools to monitor water content in Levulinic Acid/L-Proline (LALP) (2:1, mol/mol) Natural Deep Eutectic Solvent (NADES). ATR-IR delivered the best outcome of Root Mean Squared Error (RMSE) of Cross-Validation (CV) = 0.27% added water concentration, RMSE of Prediction (P) = 0.27% added water concentration and mean % relative error = 2.59%. Two NIRS instruments (benchtop and handheld) were also compared during the study, respectively yielding RMSECV = 0.35% added water concentration, RMSEP = 0.56% added water concentration and mean % relative error = 5.13% added water concentration, and RMECV = 0.36% added water concentration, RMSEP = 0.68% added water concentration and mean % relative error = 6.23%. RS analysis performed in quartz cuvettes enabled accurate water quantification with RMECV = 0.43% added water concentration, RMSEP = 0.67% added water concentration and mean % relative error = 6.75%. While the vibrational spectroscopic techniques studied have shown high performance in relation to reliable determination of water concentration, their accuracy is most likely related to their sensitivity to detect the LALP compounds in the NADES. For instance, whereas ATR-IR spectra display strong features from water, Levulinic Acid and L-Proline that contribute to the PLSR predictive models constructed, NIRS and RS spectra are respectively dominated by either water or LALP compounds, representing partial molecular information and moderate accuracy compared to ATR-IR. However, while ATR-IR instruments are common in chemistry and physics laboratories, making the technique readily transferable to water quantification in NADES, Raman spectroscopy offers promising potential for future development for in situ, sample withdrawal-free analysis for high throughput and online monitoring.

Quantification of clinical mAb solutions using Raman spectroscopy: Macroscopic vs microscopic analysis.

Raman Spectroscopy is well emerged in the field of Analytical Quality Control (AQC) as a rapid and cost-effective technique useful in many applications. The advantage of Raman spectroscopy is the non-invasiveness of measurements that enablesto analyse samples directly in its container. In this study, the potential of Raman spectroscopy was investigated for analysis of clinical preparations of mAbs. Three commercial formulations of monoclonal antibodies (mAbs) Avastin®, Ontruzant® and Tecentriq® corresponding to Bevacizumab (BVC), Trastuzumab (TRS) and Atezolizumab (ATZ) respectively, were analysed in quartz cuvette in macroscopic analysis and through the wall of perfusion bags in microscopic analysis. The spectra have been compared to those of excipients (trehalose and sucrose) and of γ-Globulin, in order to investigate the origin of Raman bands. As expected, Raman spectra were a combination of bands from monoclonal antibodies and correspoding excipients found in formulas. For quantitative analysis of the solutions, models have been constructed using Partial Least Square Regression (PLSR) with Leave K-Out Cross Validation (LKOCV). The quantification performance was comparable for both macroscopic and microscopic analysis, in terms of error and linearity. The results are thus promising for future AQC in situ, in perfusion bags.

Monitoring the water content in NADES extracts from spirulina biomass by means of ATR-IR spectroscopy.

Attenuated total reflectance-infrared spectroscopy (ATR-IR) coupled with partial least squares regression (PLSR) was evaluated as a rapid, label free and cost-effective tool to quantify water content in extracts obtained from spirulina wet biomass using a glucose glycerol natural deep eutectic solvent (NADES). NADESs are green, renewable and biodegradable solvents with unique properties outcompeting existing organic solvents, for instance, for plant or biomass extraction. The properties of NADESs depend critically on their water concentration, and therefore, it is essential to develop methods to monitor it, to ensure optimal extraction efficiency and experimental repeatability to achieve a better standardization of extraction protocols. First, Karl Fischer titration was performed on a set of 20 NADES extracts in order to obtain reference water concentrations. Secondly, ATR-IR spectra were collected and subjected to datamining to construct PLSR predictive models. An R2 value of 0.9996, a mean root mean square error of cross validation of 0.136% w/w and a root mean square error of prediction of 0.130% w/w highlight the feasibility and reliability to perform quantitative analysis using ATR-IR. Moreover, the mean relative error percentage achieved, ∼0.5%, confirms the high accuracy of water concentration determination in NADES extracts. This work demonstrates that powerful alternatives are available to provide more environmentally responsible analytical protocols. ATR-IR spectroscopy applied to NADES extracts does not require any sample preparation, reagents or solvents and has minimal requirements for single use consumables. The technique is consistent with current concerns to develop greener chemistry, especially in the field of extraction of natural compounds from plants which currently represents a major focus of interest in both research and industry.

Estimating the Analytical Performance of Raman Spectroscopy for Quantification of Active Ingredients in Human Stratum Corneum.

Confocal Raman microscopy (CRM) has become a versatile technique that can be applied routinely to monitor skin penetration of active molecules. In the present study, CRM coupled to multivariate analysis (namely PLSR-partial least squares regression) is used for the quantitative measurement of an active ingredient (AI) applied to isolated (ex vivo) human stratum corneum (SC), using systematically varied doses of resorcinol, as model compound, and the performance is quantified according to key figures of merit defined by regulatory bodies (ICH, FDA, and EMA). A methodology is thus demonstrated to establish the limit of detection (LOD), precision, accuracy, sensitivity (SEN), and selectivity (SEL) of the technique, and the performance according to these key figures of merit is compared to that of similar established methodologies, based on studies available in literature. First, principal components analysis (PCA) was used to examine the variability within the spectral data set collected. Second, ratios calculated from the area under the curve (AUC) of characteristic resorcinol and proteins/lipids bands (1400-1500 cm-1) were used to perform linear regression analysis of the Raman spectra. Third, cross-validated PLSR analysis was applied to perform quantitative analysis in the fingerprint region. The AUC results show clearly that the intensities of Raman features in the spectra collected are linearly correlated to resorcinol concentrations in the SC (R2 = 0.999) despite a heterogeneity in the distribution of the active molecule in the samples. The Root Mean Square Error of Cross-Validation (RMSECV) (0.017 mg resorcinol/mg SC), The Root Mean Square of Prediction (RMSEP) (0.015 mg resorcinol/mg SC), and R2 (0.971) demonstrate the reliability of the linear regression constructed, enabling accurate quantification of resorcinol. Furthermore, the results have enabled the determination, for the first time, of numerical criteria to estimate analytical performances of CRM, including LOD, precision using bias corrected mean square error prediction (BCMSEP), sensitivity, and selectivity, for quantification of the performance of the analytical technique. This is one step further towards demonstrating that Raman spectroscopy complies with international guidelines and to establishing the technique as a reference and approved tool for permeation studies.

Highlighting the efficiency of ultrasound-based emulsifier-free emulsions to penetrate reconstructed human skin.

OBJECTIVE: The cosmetic industry endeavours to strengthen the greener and safer claims of processes to respond to the high demand from customers for natural and environmentally friendly products. High-frequency ultrasonication technology (HFUT) is a physical process enabling the stabilization of emulsions without requiring additional ingredients, such as emulsifying surfactants (ES) to be introduced into the formulations. In this study, key formulation characteristics of an emulsion synthesized by HFUT and a reference emulsion (RE) were compared, as well as the permeation kinetics of caffeine, used as a model active cosmetic ingredient, from both types of emulsions. METHODS: The pH, droplet size and viscosity of emulsions prepared by the HFUT and the RE were determined and compared. The permeation of caffeine from the HFUT emulsion and the RE applied to the surface of reconstructed human epidermis (RHE) models was compared. RESULTS: The ES-free formulations prepared by HFUT displayed a nearly 2-fold lower average droplet size and over 3-fold greater viscosity, compared to the RE. Despite these differences, the absence of ES in the HFUT emulsion did not significantly alter the permeation kinetics of caffeine through RHE. The caffeine steady-state flux, lag time and permeability coefficients differed by 20%-30% only. CONCLUSION: This study demonstrates the potential of the HFUT to yield topical cosmetic products with lower requirements ingredients-wise, without losing efficacy, supporting the possible implementation of the technology in the cosmetic industry.

OBJECTIF: l'industrie cosmétique œuvre à renforcer les revendications plus écologiques et plus sûres des processus pour répondre à la forte demande des clients de produits naturels et plus respectueux de l'environnement. La technologie d'ultrasons à haute fréquence (High-Frequency Ultrasonication Technology, HFUT) est un processus physique permettant de stabiliser les émulsions sans qu'il soit nécessaire d'ajouter des ingrédients supplémentaires, tels que des surfactants émulsifiants, aux formulations. Dans cette étude, les principales caractéristiques de formulation d'une émulsion synthétisée par HFUT et d'une émulsion de référence ont été comparées, ainsi que la cinétique de perméation de la caféine, utilisée comme ingrédient cosmétique actif modèle, dans les deux types d'émulsion. MÉTHODES: le pH, la taille des gouttelettes, et la viscosité de l'émulsion préparée par HFUT et de l'émulsion de référence ont été déterminés et comparés. La perméation de la caféine de l'émulsion HFUT et de l'émulsion de référence appliquées à la surface de modèles d'épiderme humain reconstruit a été comparée. RÉSULTATS: la formulation sans surfactants émulsifiants préparée par HFUT présentait une taille moyenne de gouttelettes presque 2 fois plus faible et une viscosité plus de 3 fois supérieure comparée à l'émulsion de référence. Malgré ces différences, l'absence de surfactants émulsifiants dans l'émulsion HFUT n'a pas significativement modifié la cinétique de perméation de la caféine dans l'épiderme humain reconstruit. Le flux à l'état d'équilibre de la caféine, le temps de latence et les coefficients de perméabilité différaient de 20 à 30 % uniquement. CONCLUSION: cette étude démontre le potentiel de la technologie HFUT à générer des produits cosmétiques topiques possédant des exigences plus faibles en termes d'ingrédients, sans perte d'efficacité, soutenant la mise en œuvre éventuelle de la technologie dans l'industrie cosmétique.

Confocal Raman Spectroscopic Imaging for Evaluation of Distribution of Nano-Formulated Hydrophobic Active Cosmetic Ingredients in Hydrophilic Films.

Film-forming systems are highly relevant to the topical administration of active ingredients (AI) to the body. Enhanced contact with the skin can increase the efficacy of delivery and penetration during prolonged exposure. However, after the evaporation of volatile solvents to form a thin film, the distribution of the ingredient should remain homogenous in order to ensure the effectiveness of the formula. This is especially critical for the use of hydrophobic molecules that have poor solubility in hydrophilic films. In order to address this concern, hydroxyphenethyl esters (PHE) of Punica granatum seed oil were prepared as a nanosuspension stabilised by poloxamers (NanoPHE). NanoPHE was then added to a formulation containing polyvinyl alcohol (PVA) as a film forming agent, Glycerol as a plasticiser and an antimicrobial agent, SepicideTM HB. Despite their reliability, reference methods such as high-performance liquid chromatography are increasingly challenged due to the need for consumables and solvents, which is contrary to current concerns about green industry in the cosmetics field. Moreover, such methods fail to provide spatially resolved chemical information. In order to investigate the distribution of ingredients in the dried film, Confocal Raman imaging (CRI) coupled to Non-negatively Constrained Least Squares (NCLS) analysis was used. The reconstructed heat maps from a range of films containing systematically varying PHE concentrations highlighted the changes in spectral contribution from each of the ingredients. First, using NCLS scores it was demonstrated that the distributions of PVA, Glycerol, SepicideTM HB and PHE were homogenous, with respective relative standard deviations (RSD) of 3.33%, 2.48%, 2.72% and 6.27%. Second, the respective relationships between ingredient concentrations in the films and their Raman responses, and the spectral abundance were established. Finally, a model for absolute quantification for PHE was be constructed using the percentage of spectral abundance. The prepared %w/w concentrations regressed against predicted %w/w concentrations, displaying high correlation (R2 = 0.995), while the Root Mean Squared Error (0.0869% w/w PHE) confirmed the precision of the analysis. The mean percent relative error of 3.75% indicates the accuracy to which the concentration in dried films could be determined, further supporting the suitability of CRI for analysis of composite solid film matrix. Ultimately, it was demonstrated that nanoformulation of hydrophobic PHE provides homogenous distribution in PVA based film-forming systems independent of the concentration of NanoPHE used in the formula.

In Situ Water Quantification in Natural Deep Eutectic Solvents Using Portable Raman Spectroscopy.

Raman spectroscopy is a label-free, non-destructive, non-invasive analytical tool that provides insight into the molecular composition of samples with minimum or no sample preparation. The increased availability of commercial portable Raman devices presents a potentially easy and convenient analytical solution for day-to-day analysis in laboratories and production lines. However, their performance for highly specific and sensitive analysis applications has not been extensively evaluated. This study performs a direct comparison of such a commercially available, portable Raman system, with a research grade Raman microscope system for the analysis of water content of Natural Deep Eutectic Solvents (NADES). NADES are renewable, biodegradable and easily tunable "green" solvents, outcompeting existing organic solvents for applications in extraction from biomass, biocatalysis, and nanoparticle synthesis. Water content in NADES is, however, a critical parameter, affecting their properties, optimal use and extraction efficiency. In the present study, portable Raman spectroscopy coupled with Partial Least Squares Regression (PLSR) is investigated for rapid determination of water content in NADES samples in situ, i.e., directly in glassware. Three NADES systems, namely Betaine Glycerol (BG), Choline Chloride Glycerol (CCG) and Glucose Glycerol (GG), containing a range of water concentrations between 0% (w/w) and 28.5% (w/w), were studied. The results are directly compared with previously published studies of the same systems, using a research grade Raman microscope. PLSR results demonstrate the reliability of the analysis, surrendering R2 values above 0.99. Root Mean Square Errors Prediction (RMSEP) of 0.6805%, 0.9859% and 1.2907% w/w were found for respectively unknown CCG, BG and GG samples using the portable device compared to 0.4715%, 0.3437% and 0.7409% w/w previously obtained by analysis in quartz cuvettes with a Raman confocal microscope. Despite the relatively higher values of RMSEP observed, the comparison of the percentage of relative errors in the predicted concentration highlights that, overall, the portable device delivers accuracy below 5%. Ultimately, it has been demonstrated that portable Raman spectroscopy enables accurate quantification of water in NADES directly through glass vials without the requirement for sample withdrawal. Such compact instruments provide solvent and consumable free analysis for rapid analysis directly in laboratories and for non-expert users. Portable Raman is a promising approach for high throughput monitoring of water content in NADES that can support the development of new analytical protocols in the field of green chemistry in research and development laboratories but also in the industry as a routine quality control tool.

Raman spectral cytopathology for cancer diagnostic applications.

Raman spectroscopy can provide a rapid, label-free, nondestructive measurement of the chemical fingerprint of a sample and has shown potential for cancer screening and diagnosis. Here we report a protocol for Raman microspectroscopic analysis of different exfoliative cytology samples (cervical, oral and lung), covering sample preparation, spectral acquisition, preprocessing and data analysis. The protocol takes 2 h 20 min for sample preparation, measurement and data preprocessing and up to 8 h for a complete analysis. A key feature of the protocol is that it uses the same sample preparation procedure as commonly used in diagnostic cytology laboratories (i.e., liquid-based cytology on glass slides), ensuring compatibility with clinical workflows. Our protocol also covers methods to correct for the spectral contribution of glass and sample pretreatment methods to remove contaminants (such as blood and mucus) that can obscure spectral features in the exfoliated cells and lead to variability. The protocol establishes a standardized clinical routine allowing the collection of highly reproducible data for Raman spectral cytopathology for cancer diagnostic applications for cervical and lung cancer and for monitoring suspicious lesions for oral cancer.

Comparison of Raman and attenuated total reflectance (ATR) infrared spectroscopy for water quantification in natural deep eutectic solvent.

Natural deep eutectic solvents (NADES) are ionic solutions, of great interest for extraction from biomass, biocatalysis, and nanoparticle synthesis. They are easily synthesised and eco-friendly, have low volatility and high dissolution power, and are biodegradable. However, water content in NADES is a critical parameter, affecting their optimal use and extraction efficiency. Vibrational spectroscopic techniques are rapid, label-free, non-destructive, non-invasive, and cost-effective analytical tools that can probe the molecular composition of samples. A direct comparison between a previous study using attenuated total reflectance infrared (ATR-IR) spectroscopy for water quantification in NADES and the same investigation performed with Raman spectroscopy is presently reported. Three NADES systems, namely betaine-glycerol (BG), choline chloride-glycerol (CCG), and glucose-glycerol (GG), containing a range of water concentrations between 0% (w/w) and 40% (w/w), have been analysed with Raman spectroscopy coupled to partial least squares regression multivariate analysis. The values of root mean square error of cross-validation (RMSECV) obtained from analysis performed on the pre-processed spectra over the full spectral range (150-3750 cm-1) are respectively 0.2966% (w/w), 0.4703% (w/w), and 0.2351% (w/w) for BG, GG, and CCG. While the direct comparison to previous ATR-IR results shows essentially similar outcomes for BG, the RMSECV is 33.14% lower and 65.84% lower for CG and CCG. Furthermore, mean relative errors obtained with Raman spectroscopy, and calculated from a set of samples used as independent samples, were 1.452% (w/w), 1.175% (w/w), and 1.188% (w/w). Ultimately, Raman spectroscopy delivered performances for quantification of water in NADES with similar accuracy to ATR-IR. The present demonstration clearly highlights the potential of Raman spectroscopy to support the development of new analytical protocols in the field of green chemistry.

In situ Analytical Quality Control of chemotherapeutic solutions in infusion bags by Raman spectroscopy.

Analytical Quality Control (AQC) in centralised preparation units of oncology centers is a common procedure relying on the identification and quantification of the prepared chemotherapeutic solutions for safe intravenous administration to patients. Although the use of Raman spectroscopy for AQC has gained much interest, in most applications it remains coupled to a flow injection analyser (FIA) requiring withdrawal of the solution for analysis. In addition to current needs for more rapid and cost-effective analysis, the risk of exposure of clinical staff to the toxic molecules during daily handling is a serious concern to address. Raman spectroscopic analysis, for instance by Confocal Raman Microscopy (CRM), could enable direct analysis (non-invasive) for AQC directly in infusion bags. In this study, 3 anticancer drugs, methotrexate (MTX), 5-fluorouracil (5-FU) and gemcitabine (GEM) have been selected to highlight the potential of CRM for withdrawal free analysis. Solutions corresponding to the clinical range of each drug were prepared in 5% glucose and data was collected from infusion bags placed under the Raman microscope. Firstly, 100% discrimination has been obtained by Partial Least Squares Discriminant Analysis (PLS-DA) confirming that the identification of drugs can be performed. Secondly, using Partial Least Squares Regression (PLSR), quantitative analysis was performed with mean % error of predicted concentrations of respectively 3.31%, 5.54% and 8.60% for MTX, 5-FU and GEM. These results are in accordance with the 15% acceptance criteria used for the current clinical standard technique, FIA, and the Limits of Detection for all drugs were determined to be substantially lower than the administered range, thus highlighting the potential of confocal Raman spectroscopy for direct analysis of chemotherapeutic solutions.

Raman spectroscopy for the qualitative and quantitative analysis of solid dosage forms of Sitagliptin.

To demonstrate the potential of Raman spectroscopy for the qualitative and quantitative analysis of solid dosage pharmacological formulations, different concentrations of Sitagliptin, an Active Pharmaceutical Ingredient (API) currently prescribed as an anti-diabetic drug, are characterised. Increase of the API concentrations induces changes in the Raman spectral features specifically associated with the drug and excipients. Principal Component Analysis (PCA) and Partial Least Squares Regression (PLSR), were used for the qualitative and quantitative analysis of the spectral responses. A PLSR model is constructed which enables the prediction of different concentrations of drug in the complex excipient matrices. During the development of the prediction model, the Root Mean Square Error of Cross Validation (RMSECV) was found to be 0.36 mg and the variability explained by the model, according to the (R2) value, was found to be 0.99. Moreover, the concentration of the API in the unknown sample was determined. This concentration was predicted to be 64.28/180 mg (w/w), compared to the 65/180 mg (w/w). These findings demonstrate Raman spectroscopy coupled to PLSR analysis to be a reliable tool to verify Sitagliptin contents in the pharmaceutical samples based on calibration models prepared under laboratory conditions.

Understanding the discrimination and quantification of monoclonal antibodies preparations using Raman spectroscopy.

The use of Raman spectroscopy for analytical quality control of anticancer drug preparations in clinical pharmaceutical dispensing units is increasing in popularity, notably supported by commercially available, purpose designed instruments. Although not legislatively compulsory, analytical methods are frequently used post-preparation to verify the accuracy of a preparation in terms of identity and quantity of the drug in solution. However, while the rapid, cost effective and label free analysis achieved with Raman spectroscopy is appealing, it is important to understand the molecular origin of the spectral contributions collected from the solution of actives and excipients, to evaluate the strength and limitation for the technique, which can be used to identify and quantify either the prescribed commercial formulation, and/or the active drug itself, in personalised solutions. In the current study, four commercial formulations, Erbitux®, Truxima®, Ontruzant® and Avastin® of monoclonal antibodies (mAbs), corresponding respectively to cetuximab, rituximab, trastuzumab and bevacizumab have been used to highlight the key role of excipients in discrimination and quantification of the formulations. It is demonstrated that protein based anticancer drugs such as mAbs have a relatively weak Raman response, while excipients such as glycine, trehalose or histidine contribute significantly to the spectra. Multivariate analysis (partial least square regression and partial least square discriminant analysis) further demonstrates that the signatures of the mAbs themselves are not prominent in mathematical models and that those of the excipients are solely responsible for the differentiation of formulation and accurate determination of concentrations. While Raman spectroscopy can successfully validate the conformity of mAbs intravenous infusion solutions, the basis for the analysis should be considered, and special caution should be given to excipient compositions in commercial formulations to ensure reliability and reproducibility of the analysis.

Comparability of Raman Spectroscopic Configurations: A Large Scale Cross-Laboratory Study.

The variable configuration of Raman spectroscopic platforms is one of the major obstacles in establishing Raman spectroscopy as a valuable physicochemical method within real-world scenarios such as clinical diagnostics. For such real world applications like diagnostic classification, the models should ideally be usable to predict data from different setups. Whether it is done by training a rugged model with data from many setups or by a primary-replica strategy where models are developed on a 'primary' setup and the test data are generated on 'replicate' setups, this is only possible if the Raman spectra from different setups are consistent, reproducible, and comparable. However, Raman spectra can be highly sensitive to the measurement conditions, and they change from setup to setup even if the same samples are measured. Although increasingly recognized as an issue, the dependence of the Raman spectra on the instrumental configuration is far from being fully understood and great effort is needed to address the resulting spectral variations and to correct for them. To make the severity of the situation clear, we present a round robin experiment investigating the comparability of 35 Raman spectroscopic devices with different configurations in 15 institutes within seven European countries from the COST (European Cooperation in Science and Technology) action Raman4clinics. The experiment was developed in a fashion that allows various instrumental configurations ranging from highly confocal setups to fibre-optic based systems with different excitation wavelengths. We illustrate the spectral variations caused by the instrumental configurations from the perspectives of peak shifts, intensity variations, peak widths, and noise levels. We conclude this contribution with recommendations that may help to improve the inter-laboratory studies.

Freezing Weakens the Barrier Function of Reconstructed Human Epidermis as Evidenced by Raman Spectroscopy and Percutaneous Permeation.

The development and characterization of reconstructed human epidermis (RHE) is an active area of R&D. RHE can replace animal tissues in pharmaceutical, toxicological and cosmetic sciences, yielding scientific and ethical advantages. RHEs remain costly, however, due to consumables and time required for their culture and a short shelf-life. Storing, i.e., freezing RHE could help reduce costs but to date, little is known on the effects of freezing on the barrier function of RHE. We studied such effects using commercial EpiSkin™ RHE stored at -20, -80 and -150 °C for 1 and 10 weeks. We acquired intrinsic Raman spectra in the stratum corneum (SC) of the RHEs as well as spectra obtained following topical application of resorcinol in an aqueous solution. In parallel, we quantified the effects of freezing on the permeation kinetics of resorcinol from time-dependent permeation experiments. Principal component analyses discriminated the intrinsic SC spectra and the spectra of resorcinol-containing RHEs, in each case on the basis of the freezing conditions. Permeation of resorcinol through the frozen RHE increased 3- to 6-fold compared to fresh RHE, with the strongest effect obtained from freezing at -20 °C for 10 weeks. Due to the extensive optimization and standardization of EpiSkin™ RHE, the effects observed in our work may be expected to be more pronounced with other RHEs.