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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.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.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.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.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.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.

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.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.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.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 β sheets and α helices, respectively.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 β 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.

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

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. our aim was to study the impact of suberythemal doses of UV

International audience; 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.

International audience; 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.

International audience; 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.

In a clinical setting, analytical quality control of the administration of chemotherapeutic preparations is required to ensure patient safety with respect to the dose and more importantly the correct anticancer drug. As such, analytical tools enabling both qualitative and quantitative verification of the composition of prepared solutions can greatly benefit the hospital workflow, reducing cost and ultimately ensuring optimum patient care and outcome. Raman and infrared spectroscopy are rapid and cost-effective techniques, delivering label free molecular characterisation of samples. A comparative study has been conducted using four commercial intravenous formulations, DOXORUBICINE TEVA®, CERUBIDINE®, HOLOXAN® and METHOTREXATE MYLAN®, respectively containing doxorubicin, daunorubicin, ifosfamide and methotrexate as active drugs. Using clinically relevant concentration ranges prepared in 0.9 % NaCl or 5% glucose solutions, it is demonstrated that 100 % discrimination can be achieved for all formulations using either Raman or IR spectroscopic techniques, combined with multivariate discriminant analysis. Employing a partial least square regression analysis, Raman spectroscopy performed on liquid samples delivers a better accuracy compared to infrared for quantification, based on the mean squared error of cross validation. However, it is demonstrated that, despite the strong contribution of glucose and mannitol, an excipient found in CERUBIDINE®, infrared spectroscopy remains an equally viable option for translation into clinics.

International audience; This work relies on the use of dip-coating at various withdrawal speeds to form nanocomposite films, with a detailed analysis of the influence of the mode of deposition on the nanoparticle (NP) concentration in the dried film. While the deposition of polymer solutions on the one hand and colloidal suspensions of NPs on the other hand have been separately studied by dip coating, their combination is far being a simple superposition of the two separate behaviors. The formation of nanocomposite thin films composed of model water-soluble biocompatible polymers (polyvinyl alcohol - PVA and polyvinylpyrrolidone - PVP) loaded with poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) was studied through a dip coating process. Flat silicon substrates were removed at a controlled withdrawal speed from an aqueous colloidal suspension. Thin films of PLGA NPs with concentrations ranging from 1 to 10 % wt./wt. in PVA or PVP matrices were prepared. The presence of nanoparticles on the well-established process of thin film deposition was examined, as well as the influence of the deposition regime on the nanoparticle concentration in the deposited coating. We demonstrate that the presence of colloidal dispersion of PLGA nanoparticles in water solution of PVA and PVP does not modify the process of film deposition by dip coating. A typical “V” shaped curve was observed, with two well-known deposition regimes: capillary and draining modes respectively obtained at low and high withdrawal speeds. Due to crystallization at low withdrawal speed (favored by slow evaporation of the solvent) it was not possible to identify individual PLGA nanoparticles by AFM in the case of the PVA matrice. Amorphous PVP nanocomposite films were successfully prepared by dip coating, and allowed us to identify individual PLGA nanoparticles with AFM. Because of the prevalence of an evaporation-driven phenomenon at low withdrawal speed, incorporation of NPs was observed over the whole range of withdrawal speeds, showing original behavior compared to recent studies relying on a pure Landau-Levich regime (i.e. non-evaporative systems). Our results indicate that the nanoparticles were not equally retrieved from the solution in the capillary and the draining regimes. This suggests that the balance between the viscous drag and the interfacial effects depends on the deposition mode and calls for a more detailed analysis of the physical processes involved in both regimes.

Graphical abstract, Highlights • State of the art of quantitative Vibrational Spectroscopic analysis of human blood serum is reviewed. • Technical considerations for infrared absorption and Raman analysis are discussed. • Quantitative analyses of Endogenous and Exogenous constituents are presented. • The potential for clinical translation of spectroscopic serology is argued., Analysis of bodily fluids using vibrational spectroscopy has attracted increasing attention in recent years. In particular, infrared spectroscopic screening of blood products, particularly blood serum, for disease diagnostics has been advanced considerably, attracting commercial interests. However, analyses requiring quantification of endogenous constituents or exogenous agents in blood are less well advanced. Recent advances towards this end are reviewed, focussing on infrared and Raman spectroscopic analyses of human blood serum. The importance of spectroscopic analysis in the native aqueous environment is highlighted, and the relative merits of infrared absorption versus Raman spectroscopy are considered, in this context. It is argued that Raman spectroscopic analysis is more suitable to quantitative analysis in liquid samples, and superior performance for quantification of high and low molecular weight components, is demonstrated. Applications for quantitation of viral loads, and therapeutic drug monitoring are also discussed.

International audience; Confocal Raman mapping (CRM) is a powerful, label free, non‐destructive tool, enabling molecular characterization of human skin with applications in the dermo‐cosmetic field. Coupling CRM to multivariate analysis can be used to monitor the penetration and permeation of active cosmetic ingredients (ACI) after topical application. It is presently illustrated how multivariate curve resolution alternating least squares (MCR‐ALS) can be applied to detect and semi‐quantitatively describe the diffusion profile of Delipidol, a commercially available slimming ACI, from Raman spectral maps. Although the analysis outcome can be critically dependent on the a priori selection of the number of regression components, it is demonstrated that profiling of the kinetics of diffusion into the skin can be established with or without additionnal spectral equality constraints in the multivariate analysis, with similar results. Ultimately, MCR‐ALS, applied without spectral equality contraints, specifically identifies the ACI as one of main spectral components enabling to investigate its distribution and penetration into the stratum corneum and underlying epidermis layers.

International audience; 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.

Natural Deep Eutectic solvents (NADES) are green, ionic solutions prepared by mixing vegetal cellular constituents. They are renewable and biodegradable solvents composed of hydrogen bonded donor and acceptor compounds. NADES have a wide range of applications, mainly as solvents for extraction of plants, biocatalysis, and nanoparticle synthesis. Water content is very critical, impacting significantly on NADES properties. Therefore, controlling H2O concentration appears essential for optimal use of NADES. In the present study, Attenuated Total Reflectance – infrared spectroscopy (ATR-IR) coupled to multivariate analysis, namely Partial Least Square s regression (PLSR), was investigated as a rapid, label free and cost-effective tool for measuring the water concentration of NADES. Betaine_Glycerol (BG), Choline Chloride_Glycerol (CCG) and Glucose_Glycerol (GG) were the selected model systems, each over a range of concentrations between 0% (w/w) and 40% (w/w). PLSR results demonstrate the robustness of the analysis, yielding R2 values of 0.99 and Root Mean Square Error of Cross Validation (RMSECV) of respectively 0.2602% w/w, 0.6883% w/w and 0.7034% w/w. Moreover, the % relative error achieved is below a 5% threshold, further highlighting the suitability of ATR-IR for water content monitoring. This preliminary work demonstrates the appropriateness of developing easily accessible analytical tools to support the development of green chemistry solvents, which currently represent a major focus of interest in both research and industrial environments.

Cosmetic films and patches are interesting forms to promote skin penetration of active ingredients as they ensure their long stay on the treated zone of the skin. Nevertheless, currently developed films and patches are most of all hydrophilic and are not adapted to the hydrophobic molecules. The aim of this study was to establish whether nanodispersion of fatty acid-based active cosmetic ingredients (ACI) could be a manner to introduce high concentrations of those ACI in hydrophilic films.Punica granatum seed oil hydroxyphenethyl esters (PHE) constitute a commercialized lipolytic cosmetic ingredient obtained by enzymatic conjugation of tyrosol to long-chain fatty acids and to enhance its skin diffusion. Nanodispersions of PHE were prepared by a green emulsion-solvent evaporation process and dispersed in polyvinyl alcohol films. Raman imaging coupled to multivariate analysis was used to study the distribution of PHE in the films.Nanodispersions of PHE combined with antioxidant vitamin E and stabilized by Pluronic® F127 were successfully prepared. The nanodispersions show a spherical shape and a hydrodynamic diameter close to 100 nm. Raman images analysis with multivariate approaches showed a very homogeneous distribution of PHE nanodispersions in the films compared to free PHE introduced as an ethanol solution.Nanodispersions of hydrophobic fatty acid-based ingredients seem to be relevant method to introduce this type of ingredient in hydrophilic film matrix. The co-suspension with vitamin E limits their degradation in time.Les films et patchs cosmétiques sont des formes intéressantes pour augmenter la pénétration cutanée des actifs cosmétiques car ils assurent une exposition prolongée de la zone de peau traitée ce qui favorise la diffusion. Néanmoins, les films et patchs actuellement développés sont majoritairement de nature hydrophile et ne sont pas adaptés aux molécules hydrophobes. Le but de cette étude est d'établir si la nanodispersion d'actifs cosmétiques à base d'acides gras peut être un moyen d'introduire des concentrations élevées de ces actifs dans des films hydrophiles. MÉTHODES: Les esters hydroxyphénéthyliques de l'huile de graines de grenade Punica granatum (PHE) sont commercialisés comme un agent lipolytique. Cet actif obtenu par conjugaison enzymatique du tyrosol à des acides gras à longue chaîne ce qui favorise sa pénétration cutanée. Des nanodispersions de PHE ont été préparées par un procédé d'émulsion- évaporation développé avec un solvant vert. Ces nanosystèmes sont ensuite dispersées dans des films d'alcool polyvinylique. L'imagerie Raman couplée à une méthode d’analyse statistique multivariée a été utilisée pour étudier la distribution des PHE dans les films. RÉSULTATS: Des nanodispersions de PHE associées à de la vitamine E antioxydante et stabilisées par Pluronic® F127 ont été préparées avec succès. Les nanodispersions présentent une forme sphérique et un diamètre hydrodynamique proche de 100 nm. L'analyse d'images Raman au moyen d’une approche multivariée a montré une distribution très homogène des nanodispersions dans les films par rapport aux PHE libres introduits sous forme de solution éthanolique.Les nanodispersions d'ingrédients hydrophobes à base d'acides gras semblent être une méthode pertinente pour introduire ce type d'ingrédient dans la matrice de film hydrophile. L'introduction de vitamine E dans les nanodispersion ralentit leur dégradation.

International audience; 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.

International audience; Changes in protein concentrations within human blood are used as an indicator for nutritional state, hydration and underlying illnesses. They are often measured at regular clinical appointments and the current analytical process can result in long waiting times for results and the need for return patient visits. Attenuated total reflectance – Fourier transform infrared (ATR-FTIR) spectroscopy has the ability to detect minor molecular differences, qualitatively and quantitatively, in biofluid samples, without extensive sample preparation. ATR-FTIR can return an analytical measurement almost instantaneously and therefore could be deemed as an ideal technique for monitoring molecular alterations in blood within the clinic.To determine the suitability of using ATR-FTIR spectroscopy to enable protein quantification in a clinical setting, pooled human serum samples spiked with varying concentrations of human serum albumin (HSA) and immunoglobulin G (IgG) were analysed, before analysing patient clinical samples. Using a validated partial least squares method, the spiked samples (IgG) produced a linearity as high as 0.998 and a RMSEV of 0.49 ± 0.05 mg mL−1, with the patient samples producing R2 values of 0.992 and a corresponding RMSEV of 0.66 ± 0.05 mg mL−1. This claim was validated using two blind testing models, leave one patient out cross validation and k-fold cross validation, achieving optimum linearity and RMSEV values of 0.934 and 1.99 ± 0.79 mg mL−1, respectively.This demonstrates that ATR-FTIR is able to quantify protein within clinically relevant complex matrices and concentrations, such as serum samples, rapidly and with simple sample preparation. The ability to provide a quantification step, along with rapid disease classification, from a spectroscopic signature will aid clinical translation of vibrational spectroscopy to assist with problems currently faced with patient diagnostic pathways.

Infection with the dengue virus is currently clinically detected according to different biomarkers in human blood plasma, commonly measured by enzyme linked immunosorbent assays, including non-structural proteins (Ns1), immunoglobulin M (IgM) and immunoglobulin G (IgG). However, there is little or no mutual correlation between the biomarkers, as demonstrated in this study by a comparison of their levels in samples from 17 patients. As an alternative, the label free, rapid screening technique, Raman spectroscopy has been used for the characterisation/diagnosis of healthy and dengue infected human blood plasma samples. In dengue positive samples, changes in specific Raman spectral bands associated with lipidic and amino acid/protein content are observed and assigned based on literature and these features can be considered as markers associated with dengue development. Based on the spectroscopic analysis of the current, albeit limited, cohort of samples, Principal Components Analysis (PCA) coupled Factorial Discriminant Analysis, yielded values of 97.95% sensitivity and 95.40% specificity for identification of dengue infection. Furthermore, in a comparison of the normal samples to the patient samples which scored low for only one of the biomarker tests, but high or medium for either or both of the other two, PCA-FDA demonstrated a sensitivity of 97.38 % and specificity of 86.18 %, thus providing an unambiguous screening technology.

International audience; Calcium supplements are used as an aid in the prevention of osteopenia and osteoporosis and also for the treatment of patients when used along with medication. Many of these supplements are calcium carbonate based. This study compared a calcium-rich, marine multi-mineral complex (Aquamin) to calcium carbonate in an ovariectomised rat model of osteoporosis in order to assess Aquamin’s efficacy in preventing the onset of bone loss. Animals were randomly assigned to either non-ovariectomy control (Control), ovariectomy (OVX) plus calcium carbonate, ovariectomy plus Aquamin or ovariectomy plus Aquamin delay where Aquamin treatment started 8 weeks post OVX. At the end of the 20-week study, the trabecular architecture was measured using micro computed tomography, bone composition was assessed using Fourier transform infrared spectroscopy and the mechanical properties were assessed using nanoindentation and three-point bend testing. The study demonstrates that oral ingestion of Aquamin results in less deterioration of trabecular bone structure, mineral composition and tissue level biomechanical properties in the tibia of rats following ovariectomy than calcium carbonate. This study has shown that in an animal model of osteoporosis, Aquamin is superior to calcium carbonate at slowing down the onset of bone loss.

Core-shell nanocarriers are increasingly being adapted in cosmetic and dermatological fields, aiming to provide an increased penetration of the active pharmaceutical or cosmetic ingredients (API and ACI) through the skin. In the final form, the nanocarriers (NC) are usually prepared in hydrogels, conferring desired viscous properties for topical application. Combined with the high chemical complexity of the encapsulating system itself, involving numerous ingredients to form a stable core and quantifying the NC and/or the encapsulated active without labor-intensive and destructive methods remains challenging. In this respect, the specific molecular fingerprint obtained from vibrational spectroscopy analysis could unambiguously overcome current obstacles in the development of fast and cost-effective quality control tools for NC-based products. The present study demonstrates the feasibility to deliver accurate quantification of the concentrations of curcumin (ACI)-loaded alginate nanocarriers in hydrogel matrices, coupling partial least square regression (PLSR) to infrared (IR) absorption and Raman spectroscopic analyses. With respective root mean square errors of 0.1469 ± 0.0175% w/w and 0.4462 ± 0.0631% w/w, both approaches offer acceptable precision. Further investigation of the PLSR results allowed to highlight the different selectivity of each approach, indicating only IR analysis delivers direct monitoring of the NC through the quantification of the Labrafac®, the main NC ingredient. Raman analyses are rather dominated by the contribution of the ACI which opens numerous perspectives to quantify the active molecules without interferences from the complex core-shell encapsulating systems thus positioning the technique as a powerful analytical tool for industrial screening of cosmetic and pharmaceutical products. Graphical abstract Quantitative analysis of encapuslated active molecules in hydrogel-based samples by means of infrared and Raman spectroscopy.

Alterations in biomolecular components in human blood are commonly used as an indication of disease states, namely differences in protein concentration. Unfortunately, conventional test kits currently employed in hospitals suffer from long time delays, meaning patients often have to wait anxiously for their test results. Vibrational spectroscopic techniques, such as infrared and Raman, have the ability to replace current practices, as they are label-free, cost-effective, easy to operate, and require minimal sample preparation. The sensitivity to subtle changes in biochemical composition makes them ideal diagnostic tools, and recent advances in technology and data analytics means bodily fluids can be analyzed rapidly and noninvasively to detect disease-related fluctuations in protein concentration. In this chapter, we outline the current clinical procedures for blood tests, examine the capability of biomedical vibrational spectroscopy for disease diagnostics and monitoring, and discuss the potentiality for the techniques to be successfully translated into the clinic.

International audience; Attenuated total reflectance-infrared (ATR-IR) spectroscopy is a robust tool for molecular characterisation of matter. Applied to semi-solid formulations, it enables rapid and reliable data collection without pre-analytical requirements. Based on nano-encapsulated Omegalight®, a skin-lightening active cosmetic ingredient (ACI), incorporated in a hydrogel, it is first demonstrated that, despite the high water content and the chemical complexity of the samples (i.e. number of ingredients), the spectral features of the ACI can be detected and monitored. Secondly, with a total of 105 samples divided into a training set (n = 60) and an unknown set (n = 45) covering a 0.5% w/w-5% w/w concentration range, the study further investigates the quantitative performance of ATR-IR coupled with partial least squares regression (PLSR). Through a step-by-step approach in testing different cross-validation protocols, accuracy (root mean square error of cross-validation (RMSECV)) and linearity between the experimental and predicted concentrations (R2) of ATR-IR are consistently evaluated to be respectively 0.097% (w/w) and 0.995 with a lower LOD = 0.067% (w/w). Subsequently, further evaluation of the accuracy (relative error of the predicted concentration compared with the true value, expressed as %) of the analysis was undertaken with the 45 unknown samples that were defined as unknown and analysed by PLSR. The outcome of the analysis demonstrates the ruggedness and the consistency of the determination performed using the ATR-IR data. With an average relative error of 2.5% w/w and only 5 samples out of 45 blind samples exhibiting a relative error above the 5% threshold, high accuracy quantification of the nano-encapsulated ACI can be unambiguously achieved by means of the label-free and non-destructive technique of ATR-IR spectroscopy. Ultimately, the study demonstrates that the analytical capabilities of ATR-IR hold significant potential for applications in the cosmetics industry, and although the path remains long, the present study is one step further to support validation of the technique, albeit for the specific case of Omegalight®.

International audience; Raman spectroscopy was employed for the characterization of blood plasma samples from patients at different stages of breast cancer. Blood plasma samples taken from clinically diagnosed breast cancer patients were compared with healthy controls using multivariate data analysis techniques (principal components analysis – PCA) to establish Raman spectral features which can be considered spectral markers of breast cancer development. All the stages of the disease can be differentiated from normal samples. It is also found that stage 2 and 3 are biochemically similar, but can be differentiated from each other by PCA. The Raman spectral data of the stage 4 is found to be biochemically distinct, but very variable between patients. Raman spectral features associated with DNA and proteins were identified, which are exclusive to patient plasma samples. Moreover, there are several other spectral features which are strikingly different in the blood plasma samples of different stages of breast cancer. In order to further explore the potential of Raman spectroscopy as the basis of a minimally invasive screening technique for breast cancer diagnosis and staging, PCA-Factorial Discriminant Analysis (FDA) was employed to classify the Raman spectral datasets of the blood plasma samples of the breast cancer patients, according to different stages of the disease, yielding promisingly high values of sensitivity and specificity for all stages.

International audience; A methodology is proposed, based on Raman spectroscopy coupled with multivariate analysis, to determine the Limit of Detection (LOD) and Limit of Quantification (LOQ) for therapeutic drug monitoring in human serum, using the examples of Busulfan, a cell cycle non-specific alkylating antineoplastic agent, and, Methotrexate, a chemotherapeutic agent and immune system suppressant. In this study, ultrafiltration is employed to fractionate spiked human pooled serum to efficiently recover the drug in the filtrate prior to performing Raman analysis. The drug concentration ranges were chosen to encompass the recommended therapeutic ranges and toxic levels in patients. Raman spectra were collected from the filtrates in the liquid form, using an inverted backscattering microscopic geometry, using 532 nm as source. Finally, prediction models were built by using Partial Least Squares Regression (PLSR) and LOD and LOQ were calculated directly from the linear prediction models. The LOD calculated for Busulfan is 0.0002 ± 0.0001 mg mL−1, 30–40 times lower than the level of toxicity, enabling the application of this method in target dose adjustment of Busulfan for patients undergoing, for example, bone marrow transplantation. The LOD and LOQ calculated for Methotrexate are 7.8 ± 5 μM and 26 ± 5 μM, respectively, potentially enabling high dose monitoring. The promising results obtained from this study suggest the potential of Raman spectroscopy for therapeutic drug monitoring of drugs in bodily fluids.

International audience; This study explores the suitability of Raman spectroscopy as a bioanalytical tool, when coupled with ultra-filtration and multivariate analysis, to detect imbalances in both high molecular weight (total protein content, γ globulins and albumin) and low molecular weight (urea and glucose) fractions of the same samples of human patient serum, in the native liquid form. Ultracentrifugation was employed to separate and concentrate the high and low molecular weight fractions of the serum. Initially, aqueous solutions of the respective molecular species, covering physiologically relevant concentration ranges, were analysed to optimise the measurement protocols. An adapted Extended Multiplicative Signal Correction (EMSC) algorithm was applied to raw spectra to remove water background signal and spectral interferents (β-carotene). Using a validated partial least squares regression modelling method, R2 values, Root Mean Square Error of Cross Validation (RMSECV) and standard deviations were established for the quantification of γ globulin, total protein, albumin, urea and glucose content of the patient serum samples. The study demonstrates that Raman spectroscopy in the liquid form is a viable alternative and/or adjunct to current clinical practice for the parallel analysis of high and low molecular weight fractions, and simultaneous analysis of multiple analytes in the low molecular weight fraction, of human serum for diagnostic applications.

International audience; Analysis of biomarkers present in the blood stream can potentially deliver crucial information on patient health and indicate the presence of numerous pathologies. The potential of vibrational spectroscopic analysis of human serum for diagnostic purposes has been widely investigated and, in recent times, infrared absorption spectroscopy, coupled with ultra-filtration and multivariate analysis techniques, has attracted increasing attention, both clinical and commercial. However, such methods commonly employ a drying step, which may hinder the clinical work flow and thus hamper their clinical deployment. As an alternative, this study explores the use of Raman spectroscopy, similarly coupled with ultra-filtration and multivariate analysis techniques, to quantitatively monitor diagnostically relevant changes of glucose in liquid serum samples, and compares the results with similar analysis protocols using infrared spectroscopy of dried samples. The analysis protocols to detect the imbalances in glucose using Raman spectroscopy are first demonstrated for aqueous solutions and spiked serum samples. As in the case of infrared absorption studies, centrifugal filtration is utilised to deplete abundant analytes and to reveal the spectral features of Low Molecular Weight Fraction analytes in order to improve spectral sensitivity and detection limits. Improved Root Mean Square Error of Cross Validation (RMSECV) was observed for Raman prediction models, whereas slightly higher R2 values were reported for infrared absorption prediction models. Summarising, it is demonstrated that the Raman analysis protocol can yield accuracies which are comparable with those reported using infrared absorption based measurements of dried serum, without the need for additional drying steps.

Raman spectroscopy has been used to identify the biochemical changes associated with the presence of the Hepatitis C virus (HCV) in infected human blood plasma samples as compared with healthy samples, as control. The aim of the study was to establish the Raman spectral markers of hepatitis infection, which could be used for diagnostic purposes. Moreover, multivariate data analysis techniques, including Principal Component Analysis (PCA), coupled with Linear Discriminant Analysis (LDA), and Partial Least Square Regression (PLSR) are employed to further demonstrate the diagnostic capability of the technique. The PLSR model is developed to predict the viral loads of the HCV infected plasma on the basis of the biochemical changes caused by the viral infection. Specific Raman spectral features are observed in the mean spectra of HCV plasma samples which are not observed in the control mean spectra. PCA differentiated the ‘normal’ and ‘HCV’ groups of the Raman spectra and PCA-LDA was employed to increase the efficiency of prediction of the presence of HCV infection, resulting in a sensitivity and specificity 98.8% and 98.6%, with corresponding Positive Predictive Value of 99.2%, and Negative Predictive Value of 98%. PLSR modelling was found to be 99% accurate in predicting the actual viral loads of the HCV samples, as determined clinically using the Polymerase Chain Reaction (PCR) technique, on the basis of the Raman spectral changes caused by the virus during the process of the development of Hepatitis C. Copyright © 2017 John Wiley & Sons, Ltd.

International audience; The potential of Raman micro spectroscopy as an in vitro, non-invasive tool for clinical applications has been demonstrated in recent years, specifically for cancer research. To further illustrate its potential as a high content and label free technique, it is important to show its capability to elucidate drug mechanisms of action and cellular resistances. In this study, cytotoxicity assays were employed to establish the toxicity profiles for 24 hr exposure of lung cancer cell lines, A549 and Calu-1, to the commercially available drug, doxorubicin (DOX). Raman spectroscopy, coupled with Confocal Laser Scanning Microscopy and Flow Cytometry, was used to track the DOX mechanism of action, at a subcellular level, and to study the mechanisms of cellular resistance to DOX. Biomarkers related to the drug mechanism of action and cellular resistance to apoptosis, namely reactive oxygen species (ROS) and bcl-2 protein expression, respectively, were also measured and correlated to Raman spectral profiles. Calu-1 cells are shown to exhibit spectroscopic signatures of both direct DNA damage due to intercalation in the nucleus and indirect damage due to oxidative stress in the cytoplasm, whereas the A549 cell line only exhibits signatures of the former mechanism of action. PCA of nucleolar, nuclear and cytoplasmic regions of A549 and Calu-1 with corresponding loadings of PC1 and PC2.

Diabetes is a chronic disease that affects millions of people every year worldwide. Patients with diabetes have high levels of glucose in their blood, since their bodies cannot produce or adequately use the insulin produced. Identification of diabetes is usually performed by tests of glucose in the blood by means of colorimetric reactions, which are time consuming and use a considerable amount of reagents. Attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy has been used in clinical research as a potential tool to obtain spectrochemical information of biological materials. The infrared spectra can be used as source of information for classiciation models and biomarker extraction by using specific computational tools. In this paper, a semi-portable Bruker Alpha ATR-FTIR was employed to analyse urine samples of 7 patients (3 normal, 2 diabetics and 2 pre-diabetics) in order to distinguish these three groups based on their spectrochemical information. Cross-validated principal component analysis, coupled with linear discriminant analysis was applied to the spectral dataset, resulting in 94% total accuracy. Sensitivities were observed to be 95%, 96% and 100% for normal, pre-diabetics and diabetics patients, respectively, with specificities of 93%, 91% and 100%. These findings show the potential of ATR-FTIR as a new possible tool for identification of diabetics in clinical environments, whereby the diagnosis can be performed in a rapid, non-invasive and automated way.

International audience; Two dimensional correlation analysis is explored to data mine the time evolution of the characteristic Raman microspectroscopic signatures of the subcellular responses of the nucleoli of human lung cancer cells to the uptake of doxorubicin. A simulated dataset of experimental control spectra, perturbed with systematically time-dependent spectral changes, constituted by a short term response which represents the initial binding of the drug in the nucleolus, followed by a longer term response of the organelle metabolism, is used to validate the analysis protocol. Applying two dimensional correlation analysis, the in phase, synchronous correlation co-efficients are seen to contain contributions of both response profiles, whereas they can be independently extracted from the out of phase, asynchronous correlation co-efficients. The methodology is applied to experimental data of the uptake of doxorubicin in human lung cell lines to differentiate the signatures of chemical binding and subsequent cellular response.

International audience; Anticancer drugs are prescribed and administrated to an increasing number of patients on a daily basis. As a consequence, a number of concerns have been raised about the patient health and safety in the case that the drugs administered are not at the required concentration or even worse not the correct ones. Quality control of therapeutic solutions has therefore been extensively implemented in hospital environments, in order to avoid any failure in the intense workflow faced by administering pharmacists. In the present study, infrared (IR) and Raman spectroscopy have been employed for the analysis of 3 commercially available therapeutic solutions TEVA®, MYLAN®, CERUBIDINE®, respectively containing doxorubicin, epirubicin and daunorubicin. They perfectly illustrate the analytical difficulties encountered, as these 3 chemotherapeutic drugs are isomers, hardly distinguishable with conventional approaches such as UV/VIS spectrometry. Any analytical failure to identify these molecules can lead to delays in patient treatment. While Partial Least Squares Regression analysis demonstrates that both Raman and IR can deliver satisfactory quantitative analysis in the clinical range, with respective Root Mean Square Error of Cross Validation (RMSECV) between 0.0127 – 0.0220 g·L−1 and 0.0573 – 0.0759 g·L−1, the identification rate between the 2 techniques differs substantially. Indeed, Principal Component Analysis – Factorial Discriminant Analysis (PCA-FDA) highlights that, depending on the data preprocessing applied to Raman spectra, the discrimination between the 3 drugs is decreased, with in some cases specificity and sensitivity below 50%. However, IR analysis displays encouraging results with an overall specificity and sensitivity between 99 and 100%, suggesting that reliable validation of the therapeutic solution for administration to patients can be achieved. IR and Raman spectroscopy could assist and support quality control of chemotherapeutic solutions prepared in personalised concentrations for each patient. The effective and reliable characterisation of therapeutic solutions could have a lot to offer to improve current practices in a near future.

The hyperspectral imaging is commonly used in the cosmetic area. Indeed, spectroscopic imaging techniques are usually employed to study the molecular composition of a cosmetic product, notably the Raman imaging. For this matter, Nonnegative Constrained Least Square (NCLS), has been studied previously and has provided accurate distribution maps of Active Cosmetic Ingredient (ACI) with its associated penetration profile. However, it remains a supervised method since it requires an a priori knowledge of the Raman fingerprint of the ACI to track it and the availability of a large number of spectra from control data affects its performance. This work presents the comparison of a Minimum Volume Constrained Non-negative Matrix Factorization (MVC-NMF) with the NCLS and a popular method in the chemometry community, Multivariate Curve Resolution Alternating Least Square (MCR-ALS) for hyperspectral image analysis. MVC-NMF proposes an unsupervised geometric approach to better fit a linear model to the data that provides lower modelling residuals. We also evaluate the parameter selection of the right number of constituent of Raman imaging from skin samples. It is shown that the MVC-NMF was able to accurately estimate the Raman spectrum of the ACI without supervision.

Made available in DSpace on 2018-12-11T17:20:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-04-03 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Fourier transform infrared (FTIR) spectroscopy is a powerful diagnosis technique and has been used to identify patterns of molecular changes based on vibration modes. The objective of this study was to evaluate inflammatory fibrous hyperplasia (IFH) lesions and oral normal mucosa (NM) initially with histopathological exam and then using micro-FTIR spectroscopy to analyse the samples. Eleven IFH and 11 NM samples were analysed at five different points to cover the largest area possible by the micro-FTIR technique. Bands were observed between 970 and 1743 cm−1 which corresponded to different structural components like collagen, lipids, fatty acids, proteins and amino acids. Spectral bands were more intense mostly for IFH lesions, including collagen bands, which are an important component of inflammatory fibrous hyperplasia. This study demonstrated that differentiation in the inflammatory tissue was observed in FTIR spectral differences, in terms of biochemical composition. Department of Bioscience and Oral Diagnosis Institute of Science and Technology of São José dos Campos Univ Estadual Paulista-UNESP Departamento de Odontologia Universidade de Taubaté Graduação em Odontologia Universidade Braz Cubas Faculty of Pharmacy Université François-Rabelais de Tours Centro de Ciências Naturais e Humanas Universidade Federal do ABC Department of Bioscience and Oral Diagnosis Institute of Science and Technology of São José dos Campos Univ Estadual Paulista-UNESP

In the confocal mode, Raman microspectroscopy can profile the biochemical content of biological cells at a subcellular level, and any changes to it by exogenous agents, such as therapeutic drugs or toxicants. As an exploration of the potential of the technique as a high-content, label-free analysis technique, this report reviews work to monitor the spectroscopic signatures associated with the uptake and response pathways of commercial chemotherapeutic agents and polymeric nanoparticles by human lung cells. It is demonstrated that the signatures are reproducible and characteristic of the cellular event, and can be used, for example, to identify the mode of action of the agent as well as the subsequent cell death pathway, and even mechanisms of cellular resistance. Data mining approaches are discussed and a spectralomics approach is proposed.

International audience; Attenuated Total Reflectance-Infrared (ATR-IR) spectroscopy holds great promise for industrial applications as a quality control tool for complex galenic formulations. Although the technique is often promoted for the molecular information it delivers in a label free and cost effective fashion, other advantages can emerge compared to the gold standard analytical tools such as liquid chromatography coupled to mass spectrometry. The present study demonstrates how ATR-IR measurements enable accurate quantitative analysis of an active cosmetic ingredient such as Omegalight® encapsulated in a complex alginate based nano-capsule. The study demonstrates how precise concentrations can be obtained without the requirement for fastidious extraction and separation protocols prior to ATR-IR analysis. However, data mining remains a crucial aspect with particular emphasis on the preprocessing of the data that will be subjected to Partial Least Squares Regression (PLSR) analysis. Therefore, different pre-processing methods have been evaluated to investigate the relationship between corrections applied and PLSR outcomes (i.e. precision, ratio of performance to deviation (RPD) and accuracy of the analysis). Ultimately, it has been found that, against all expectations, some of the preprocessing methods do not necessarily lead to improvements in the end result, while Extended Multiplicative Scattering Correction (EMSC) is the only one which delivers satisfying results, as defined by a Root Mean Square Error (RMSEV) of 0.07% (w/w) and a RPD greater than 30 when performing analysis over the range 0.4–8.2% (w/w). Despite the presence of large amounts of additives such as glycerol and preservatives in the formulation, implementing Leave One Out Cross Validation (LOOCV) further validates the method with a RPD of 18 and relative errors for the predicted concentrations below the 5% (w/w) threshold, hence demonstrating that ATR-IR has analytical capabilities for applications in the cosmetic industry.

International audience; Cervical cancer is the fourth most common cancer in women worldwide and mainly affects younger women. The mortality associated with cervical cancer can be reduced if the disease is detected at the pre-cancer stage. Current best-practice methods include cytopathology, HPV testing, and histopathology, but these methods are limited in terms of subjectivity, cost, and time. There is an unmet clinical need for new methods to aid clinicians in the early detection of cervical pre-cancer. These methods should be objective and rapid and require minimal sample preparation. Raman spectroscopy is a vibrational spectroscopic technique by which incident radiation is used to induce vibrations in the molecules of a sample and the scattered radiation may be used to characterise the sample in a rapid and non-destructive manner. Raman spectroscopy is sensitive to subtle biochemical changes occurring at the molecular level, enabling spectral variations corresponding to disease onset to be detected. Over the past 15 years, there have been numerous reports revealing the potential of Raman spectroscopy together with multivariate statistical analysis for the detection of a variety of cancers. This paper discusses the recent advances and challenges for cervical-cancer screening and diagnosis and offers some perspectives for the future.

International audience; Raman spectroscopy can provide a molecular-level fingerprint of the biochemical composition and structure of cells with excellent spatial resolution and could be useful to monitor changes in composition for dysplasia and early, non-invasive cancer diagnosis (carcinoma in situ), both ex-vivo and in vivo. In this study, we demonstrate this potential by collecting Raman spectra of the nucleoli, nuclei and cytoplasm from oral epithelial cancer (SCC-4) and dysplastic (pre-cancerous, DOK) cell lines and from normal oral epithelial primary cell cultures, in vitro, which were then analysed by principal component analysis (PCA) as a multivariate statistical method to discriminate the spectra. Results show significant discrimination between cancer and normal cell lines. Furthermore, the dysplastic and cancer cell lines could be discriminated based on the spectral profiles of the cytoplasmic regions. The principal component loading plot, which elucidates the biochemical features responsible for the discrimination, showed significant contributions of nucleic acid and proteins for nucleolar and nuclear sites and variation in features of lipids for the cytoplasmic area. This technique may provide a rapid screening method and have potential use in the diagnosis of dysplasia and early, non-invasive oral cancer, the treatment of which involves much less extensive and complex surgery and a reduction in associated co-morbidity for the patient.

This overview groups some of the recent studies highlighting the potential application of Raman micro-spectroscopy as an analytical technique in preclinical development to predict drug mechanism of action and in clinical application as a companion diagnostic and in personalised therapy due to its capacity to predict cellular resistance and therefore to optimise chemotherapeutic treatment efficacy. Notably, the anthracyclines, Doxorubicin and Actinomycin D, elicit similar spectroscopic signatures of subcellular interaction characteristic of the mode of action of intercalation. Although Cisplatin and Vincristine show markedly different signatures, at low exposure doses, their signatures at higher doses show marked similarities to those elicited by the intercalating anthracyclines, confirming that anticancer agents can have different modes of action with different spectroscopic signatures, depending on the dose. The study demonstrates that Raman micro-spectroscopy can elucidate subcellular transport and accumulation pathways of chemotherapeutic agents, characterise and fingerprint their mode of action, and potentially identify cell resistant strains. The consistency of the spectroscopic signatures for drugs of similar modes of action, in different cell lines, suggests that this fingerprint can be considered a “spectralome” of the drug-cell interaction suggesting a new paradigm of representing spectroscopic responses.