Raman Chemical Imaging Research Articles

Detection of low dose of piroxicam polymorph in pharmaceutical tablets by surface-enhanced Raman chemical imaging (SER-CI) and multivariate analysis.

This study demonstrates, for the first time, the ability of surface-enhanced Raman chemical imaging (SER-CI) combined with multivariate analysis to detect low levels (0.1% (w/w)) of a polymorphic form in a pharmaceutical mixture. In the studied formulation, piroxicam was used as a model molecule to develop this approach. Piroxicam is a widely available non-steroidal anti-inflammatory drug, exhibiting an interesting case of polymorphism, with two most commonly observed forms (β and α2). In this work, the SERS spectra of piroxicam polymorphic forms β and α2 are presented. These forms showed clear spectral differences in terms of band position and intensity. From a crystallographic point of view, the difference of exaltation between both forms was correlated with a preferred orientation of crystallites of form α2 making its SERS detection difficult compared to form β. A preferred orientation of the (1k0) crystallographic planes of α2 was demonstrated in samples, not promoting an appropriate molecular orientation onto the metallic surface. Additionally, a semi-quantitative approach using SER-CI combined with chemometric tools was developed enabling to detect crystallites of form β below the detection limit of conventional Raman microscopy. The exaltation of the Raman signal in the presence of silver nanoparticles allowed a higher sensitivity and a reduction of the acquisition time by a factor of 6.

Fast Raman chemical imaging of tablets with non-flat surfaces.

A curved area with embossment on an analgesic tablet with three APIs is imaged with a Raman instrument equipped with hardware options that allow for continuous focus adjustment and very fast acquisition of micro-Raman spectra, about sixty spectra per second in this study. Univariate, self-modelling curve resolution, and regression derived images via the pure component spectra are obtained for all three APIs. The quality of the images and reasons for thresholding are discussed, as well as the relation between the histograms of the images and respective thresholds. The univariate image of the API of lowest concentration (caffeine) is found to be most affected by the Raman signal of the more abundant components, which is much less noticeable in the multivariate images. Particle size analysis is conducted on the thresholded caffeine images only, as the other two components are too abundant for such type of analysis. Depending on the method used for imaging, the area coverage for caffeine varies from 1% in univariate to 4% in regression images.

Label-free Raman spectroscopy monitoring of cytotoxic response induced by a telomerase inhibitor

We proposed a new approach to directly and quantitatively inspect cytotoxicity response at the unicellular scale based on Raman micro-spectroscopy. Variations of intracellular multicomponent caused by the telomerase inhibitor could be revealed by high-throughput Raman spectral screening simultaneously. The changes and modifications of the biochemical compositions including proteins and nucleic acids responded to the telomerase inhibitor were observed through Raman fingerprint assignment analysis of nucleic acids (570, 785, 1093, 1337 and 1484 cm−1) and proteins (570, 1001, 1231, 1337, 1484, 1662 and 2873 cm−1). HeLa cells stressed by different doses of telomerase inhibitor could be distinguished by unsupervised clustering analysis based on their Raman spectroscopy. Four Raman band ratios—I1093/1001, I1093/1032, I1093/1055, and I1093/1066—which were significantly correlated with the inhibitory rate of HeLa cells were identified, and nonlinear quantitative model for cell cytotoxicity was then established based on these Raman ratios. Furthermore, spatial distribution of intracellular important bio-chemical components was showed by Raman chemical imaging with these characteristics Raman ratios, indicating that the granules representing the corresponding substances first exhibited a significant degree of agglomeration (0.4 and 0.8 μM groups), which subsequently declined, becoming smaller and more divergent (1.6 and 4 μM groups) as cytotoxicity increased. This study provides a rapid and quantitative assessment for cytotoxicity induced by chemicals at the single-cell level. The ability to directly monitor chemically-induced cell cytotoxicity in situ promotes understanding of molecular level changes and improves knowledge of the molecular mechanisms involved, which has great application potential in pharmacological mechanism exploration and drug development.

Quantitative visualization of intracellular lipids concentration in a microalgae cell based on Raman micro-spectroscopy coupled with chemometrics

An in vivo, label-free, nondestructive, and quantitative visualization of the intracellular lipids concentration in individual microalgae cells under nitrogen stress was obtained by integrating Raman chemical imaging and multiple regression analysis. Lipids and biomass were significantly affected by nitrogen stress and induction time. Especially, in the first four days under stress, the higher the stress intensity, the faster the increase in the lipid concentration. A quantitative linear relationship between the Raman spectral response and the lipid concentration of colonial microalgae was established with satisfactory results (Rp2 = 0.925). Characteristic Raman bands for quantitative determination of lipids were then extracted based on competitive adaptive reweighted sampling (CARS). Furthermore, the visualization of intracellular lipid concentration derived from the quantitative linear regression model outperformed that by integration of Raman intensity at a fingerprint band (1266 or 1445 cm−1). This quantitative visualization of intracellular lipids concentration could effectively provide the spatial and temporal dynamic characteristics of intracellular lipids in microalgae cells under nitrogen stress, which should be helpful in revealing intracellular lipid metabolism and accumulation for the utilization of microalgae biomass in biofuel production.

Mapping and Profiling Lipid Distribution in a 3D Model of Breast Cancer Progression.

Aberrant lipid accumulation and marked changes in cellular lipid profiles are related to breast cancer metabolism and disease progression. In vitro, these phenomena are primarily studied using cells cultured in monolayers (2D). Here, we employ multicellular spheroids, generated using the MCF10A cell line series of increasing malignancy potential, to better recapitulate the 3D microenvironmental conditions that cells experience in vivo. Breast cancer cell lipid compositions were assessed in 2D and 3D culture models as a function of malignancy using liquid chromatography coupled with mass spectrometry. Further, the spatial distribution of lipids was examined using Raman chemical imaging and lipid staining. We show that with changes in the cellular microenvironment when moving from 2D to 3D cell cultures, total lipid amounts decrease significantly, while the ratio of acylglycerols to membrane lipids increases. This ratio increase could be associated with the formation of large lipid droplets (>10 μm) that are spatially evident throughout the spheroids but absent in 2D cultures. Additionally, we found a significant difference in lipid profiles between the more and less malignant spheroids, including changes that support de novo sphingolipid production and a reduction in ether-linked lipid fractions in the invasive spheroids. These differences in lipid profiles as a function of cell malignancy and microenvironment highlight the importance of coupled spatial and lipidomic studies to better understand the connections between lipid metabolism and cancer.

UV Raman chemical imaging using compressed sensing.

Different chemical (hyperspectral) imaging techniques have proven to be powerful tools to provide and illustrate insightful data within a broad range of research areas. The present communication includes proof-of-principle results of UV Raman hyperspectral imaging, achieved via compressed sensing measurements using coded apertures (CA) and a reconstruction algorithm. The simple and cheap CA set up, obtained by a 50% overall transmissive random binary mask (chromium on fused silica with 100 μm × 100 μm pixel size) positioned at the entrance plane of an imaging spectrograph, resulted in an overall high throughput for the UV region of interest. The mask was mounted on a translation stage, allowing reproducible switching to different CA, thus making possible for multi frame CA imaging. Results from a scene containing liquid droplets are shown as examples and, as expected, qualitative improvements in resolution and contrast could be observed in both the spatial and spectral domain as the number of CA frames was increased.

A Nondestructive Detection Method for Mixed Veterinary Drugs in Pork Using Line-Scan Raman Chemical Imaging Technology

This study reports a nondestructive detection method using Raman chemical imaging (RCI) technology for the simultaneous determination of multiple veterinary drugs, such as ofloxacin, chloramphenicol, and sulfadimidine, in pork. A line-scan Raman imaging system was employed to acquire images of pure veterinary drugs and pork samples containing single and mixed drugs. Raman characteristic peaks at 1623, 1353, and 1147 cm−1 were identified for ofloxacin, chloramphenicol, and sulfadimidine, respectively. An image processing method was proposed to calculate the “pixel-ratio” values in each feature image, and linear regression models for a single veterinary drug were then established between the “pixel-ratio” values and the actual concentrations. By applying the models to samples with mixed veterinary drugs, the concentrations of the three drugs were predicted with correlation coefficients of 0.978, 0.986, and 0.984. The satisfactory results indicated that along with the proposed “pixel-ratio” method, RCI technology enables the nondestructive quantitation and spatial distribution visualization of multiple veterinary drug residues.

Applications of Raman Spectroscopy on Quality Control of Hospital Formulations

Hospital formulation has several advantages, including the flexibility of customization as per the disease state or the patients' precise requirements. However, compared with commercial formulations, hospital formulations are usually not under the same level of quality check. In the present study, we tested mixed powder formulations prepared in a hospital pharmacy using Raman spectroscopy to investigate the feasibility of applying Raman spectroscopy as a quality-control tool of hospital formulations. For this purpose, we first established a numerical evaluation method to determine the uniformity of a powder mixture using Raman chemical imaging data with atropine sulfate/lactose mixture samples and revealed that the mixing uniformity correlated to the experience level of the pharmacist. Next, we developed a content quantification method in a one-dose packaged powder formulation by measuring the Raman spectra from the outside of the package. Because this method allows for quantification of the components inside the package in a non-destructive and non-contact manner, it can be applied for content confirmation after one-dose packaging. Using this method, the content uniformity of the mixed powder formulation in the one-dose package was compared between the formulations prepared by the pharmacists and those prepared by a pharmacy robot. Our study indicates the possibility of applying Raman spectroscopy as a quality-control tool of hospital formulations. Studies on further applications of Raman spectroscopy in the field of clinical pharmacology are expected.

Towards a spray-coating method for the detection of low-dose compounds in pharmaceutical tablets using surface-enhanced Raman chemical imaging (SER-CI)

Surface-enhanced Raman chemical imaging (SER-CI) is a highly sensitive analytical tool recently used in the pharmaceutical field owing to the possibility to obtain high sensitivity along with spatial information. However, the covering method of the pharmaceutical samples such as tablets with metallic nanoparticles is a major issue for SER-CI analyses due to the difficulty to obtain a homogeneous covering of tablet surface with the SERS substrates. In this context, a spray-coating method was proposed in order to fully exploit the potential of SER-CI. A homemade apparatus has been developed from an electrospray ionization (ESI) probe in order to cover the pharmaceutical tablets with the colloidal suspension in a homogeneous way. The silver substrate was pulled through the airbrush by a syringe pump which was then nebulized into small droplets due to the contact of the solution with the gas flow turbulence. A robust optimization of the process was carried out by adjusting experimental parameters such as the liquid flow rate and the spraying time. Besides, the performances of this spraying technique were compared with two others covering methods found in the literature which are drop casting and absorption coating. A homogeneity study, conducted by SER-CI and matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) applied to the different covering techniques was performed. The influence of the metallic nanoparticles deposit on soluble compounds was also investigated in order to highlight the advantages of using this new spray coating approach.

Quantification and visualization of α-tocopherol in oil-in-water emulsion based delivery systems by Raman microspectroscopy

Emulsion is a commonly investigated bioactive loaded delivery system. The bioactive content and its location in oil phase primarily determine the quality and chemical stability of emulsion. In this study, Raman microspectroscopy was used to quantify α-tocopherol and to visualize its distribution in oil-in-water emulsion stabilized by whey protein isolates. Results suggested that α-tocopherol contents (25–300 g/kg) in corn oil with the integrating Raman intensity at 481.9 and 588.6 cm−1 showed determination coefficient (R2) of 0.98 and 0.99, and limit of detection of 5.1 and 21.2 g/kg, respectively. For detecting α-tocopherol in emulsions, the relative standard deviation values from Raman method using intensities at 481.9 cm−1 and 588.6 cm−1 were in the ranges of 4%–16% and 2%–6%, respectively. The developed Raman method provided correlative results with those of HPLC method (R2 = 0.99). Moreover, Raman chemical imaging depicted the non-homogeneous TOC distribution within oil droplets, where TOC had the trend of migrating to the interface of oil and water. This study provided a novel approach for functional emulsion analysis, which may serve the basis for designing stable and controllable release of emulsion systems in future.

Quantitative analysis of the distribution and mixing of cellulose nanocrystals in thermoplastic composites using Raman chemical imaging.

Cellulose nanofibers hold much promise for enhancing the mechanical properties of composites owing to their uniquely high stiffness and strength. One major issue limiting this performance however is the dispersion and mixing of cellulose nanofibers within thermoplastic resins. A combination of Raman imaging and chemical analysis has been used to quantify the distribution and mixing of cellulose nanocrystals (CNCs) in a polyethylene-matrix composite. Large area spectral imaging provides information about the effect of a compatibilizer – namely poly(ethylene oxide) (PEO) and maleated polyethylene (MAPE) – on the distribution of CNCs in the thermoplastic matrix. High-resolution images enable quantification of the degree of mixing between the CNCs and HDPE. Lower resolution images, but with greater spatial spread, allow quantification of the distribution of the CNCs. It is shown that the CNCs tend to agglomerate, with little increase in distribution even with the use of the compatibilizer. A shift in the position of characteristic Raman bands indicates the formation of hydrogen bonding between the PEO compatibilizer and the CNCs, which in turn is thought to affect the distribution of aggregates of the reinforcing phase.

Inspection of maleic anhydride in starch powder using line-scan hyperspectral Raman chemical imaging technique

Inspection of maleic anhydride in starch powder using line-scan hyperspectral Raman chemical imaging technique

Analysis of Fiber-Enhanced Raman Gas Sensing Based on Raman Chemical Imaging.

Fiber enhanced Raman spectroscopy (FERS) is an arising new technique for versatile highly sensitive and selective multigas analysis in various applications, such as environmental monitoring and medical breath diagnosis. In this study, the performance of FERS was thoroughly studied with the help of a specially designed multichannel Raman chemical imaging. Several types of hollow core photonic crystal fibers were thoroughly analyzed in terms of their performance in light confinement and sensitive gas sensing. The optimal fiber length for Raman gas sensing was found to be 15 cm in our spectroscopic system. To separate the Raman scattering of the target gas molecules from the background generated by the silica microstructure of the fiber, the optimal diameter of a spatial filter was analyzed and quantified as Ø3.9 μm, which balances the suppression of the silica background and the attenuation of the gas signal, originating from different regions in the plane of the fiber end-face. To achieve an easy-to-use gas monitoring system with stable performance, an automated coupling-method was developed, to simplify the alignment of the FERS setup. The optimized design of the FERS setup has remarkable potential for highly sensitive, miniaturized, easy-to-use, and versatile gas sensing.

Raman chemical imaging for spectroscopic screening and direct quantification of falsified drugs

Falsified drugs are a threat to the health of patients. The analytical control of such products contributes to the fight against this global issue. Raman chemical imaging is a method that relies on consecutive measurements at the surface of a sample, combining spectroscopy, microscopy and chemometrics. This article explores the capabilities of this analytical technique proposing an innovative methodology with spectroscopic screening for the identification of chemical compounds and the direct quantification of the active substance (without prior calibration). Two chemometric methods were used: Multivariate Curve Analysis − Alternate Least Squares for the qualitative analysis and Direct Classical Least Squares for the quantitative analysis. The methodology was optimized with samples prepared in the laboratory and validation parameters were studied. The methodology was then applied to real (authentic and falsified) samples of Viagra® and Plavix®. Despite the presence of fluorescence emission in some samples, the methodology succeeded in the detection of active pharmaceutical ingredients, and in the discrimination of three salts of clopidogrel (in generic formulations of Plavix®). The quantitative deviation from the reference method ranged from −15% to +24% of the active substance content. This deviation may be considered to be acceptable since it is sufficient for assessing the risk to the health of patients and for quickly alerting the health authorities.

Quantitative Detection of Benzoyl Peroxide in Wheat Flour Using Line-Scan Macroscale Raman Chemical Imaging.

A high-throughput Raman chemical imaging method was developed for direct inspection of benzoyl peroxide (BPO) mixed in wheat flour. A 5 W, 785 nm line laser (240 mm long and 1 mm wide) was used as a Raman excitation source in a push-broom Raman imaging system. Hyperspectral Raman images were collected in a wavenumber range of 103-2881 cm-1 from dry wheat flour mixed with BPO at eight concentrations (w/w) from 50 to 6400 ppm. A sample holder with a sampling volume of 150 × 100 × 2 mm3 was used to present a thin layer (2 mm thick) of the powdered sample for line-scan image acquisition with a spatial resolution of 0.2 mm. A baseline correction method based on adaptive iteratively reweighted penalized least squares was used to remove the fluctuating fluorescence signals from the wheat flour. To isolate BPO particles from the flour background, a simple thresholding method was applied to the single-band fluorescence-free images at a unique Raman peak wavenumber (i.e., 1001 cm-1) preselected for the BPO detection. Chemical images were created to detect and map the BPO particles. Limit of detection for the BPO was estimated in the order of 50 ppm, which is on the same level with regulatory standards. Pixel concentrations were calculated from the percentages of the BPO pixels in the chemical images. High correlation was found between the pixel concentrations and the mass concentrations of the BPO, indicating that the Raman chemical imaging method can be used for quantitative detection of the BPO mixed in the wheat flour.

Visualization and Non-Destructive Quantification of Inkjet-Printed Pharmaceuticals on Different Substrates Using Raman Spectroscopy and Raman Chemical Imaging.

The purpose of this study was to investigate the applicability of Raman spectroscopy for visualization and quantification of inkjet-printed pharmaceuticals. Haloperidol was used as a model active pharmaceutical ingredient (API), and a printable ink base containing lactic acid and ethanol was developed. Inkjet printing technology was used to apply haloperidol ink onto three different substrates. Custom-made inorganic compacts and dry foam, as well as marketed paracetamol tablets were used as the substrates. Therapeutic personalized doses were printed by using one to ten printing rounds on the substrates. The haloperidol content in the finished dosage forms were determined by high-performance liquid chromatography (HPLC). The distribution of the haloperidol on the dosage forms were visualized using Raman chemical imaging combined with principal components analysis (PCA). Raman spectroscopy combined with modeling by partial least squares (PLS) regression was used for establishment of a quantitative model of the haloperidol content in the printed dosage forms. A good prediction of the haloperidol content was achieved for the inorganic compacts, while a slightly poorer prediction was observed for the paracetamol tablets. It was not possible to quantify haloperidol on the dry foam due to the low and varying density of the substrate. Raman spectroscopy is a useful tool for visualization and quality control of inkjet printed personalized medicine.

Detection of Chemical Additives in Food Using Raman Chemical Imaging System

Detection of Chemical Additives in Food Using Raman Chemical Imaging System

Characterization of ferruginous cements related with weathering of slag in a temperate anthropogenic beachrock.

This work outlines a temperate latitude beachrock occurrence, which represents the legacy of heavy anthropogenic environmental disturbance. The units contain high amounts of slag and iron-rich wastes derived from metallurgical activities that attest the impact of the past industrial development on such coastal systems. The exposition of the anthropogenic wastes to weathering processes, such as the influence of marine aerosols and the chemical attack of acid gases like the SOx coming from the nearby urban-industrial atmosphere, gave rise to the formation of early diagenetic ferruginous cements. A new analytical methodology based on the combination of micro-Raman spectroscopy (MRS), Raman chemical imaging, SEM-EDS and the Structural and Chemical Analyzer (SCA, an emerging system that hyphenates micro-Raman and SEM-EDS), was applied for the first time to characterize the ferruginous cements. The MRS analyses revealed Fe2+/Fe3+ oxides and oxyhydroxides, CaCO3 polymorphs and less frequently silicates. The Fe mineral species detected were hydrated goethite, hematite, magnetite, magnesioferrite, lepidocrocite and goethite. Complementary Raman imaging, SEM-EDS and SCA analyses unraveled the preferential distribution of hydrated goethite. The identified iron mineral phases are weathering sub-products of hematite commonly derived from atmospheric/aqueous leaching processes triggered by the chemical attack of the acid gases. EDS showed the existence of other elements such as Si, Mg, Cl, Na, Al, K and sporadically S that indicated the importance of permeability, atmospheric deposition and the acid attack. Additionally, calcite and gypsum minerals also evidenced the action of meteoric waters, dry deposition processes or the attack of SOx acid gases. The presence of such compounds is modifying the cement stratigraphy and suggests that the dissolution of carbonates is currently taking place. Those facts influence the erosive susceptibility and the release of the anthropogenic materials trapped originally in the beachrocks, which could act as potential secondary sources of contaminants to the coastal environment.

Simple, Fast and Economic Way for Estimation of Calcipotriene Crystal Size and Particle Distribution in its Drug Product. Raman Microscope Particle Size Verses Pre-Calci Content - An Approximation by HPLC Analysis

Pharmaceutical development has become more expensive and time taking affair to achieve required quality standards. Calcipotriol/ Calcipotriene (Calci) and Betamethasone dipropionate (BD) is a combined drug product popular in tropical treatment for Psoriasis. BD and Calci crystals are formed in manufacturing drug product 1. The suspended particles of Calci are focused for its size and distribution in ointment as it is clinically important 2. Drug particles are identified and differentiated by Raman dispersive spectroscopy, shape of particles are recorded with the help of Raman Chemical Imaging (RCI) combined with high resolution optical microscope, Particles size and number are statically measured to derive distribution histograms. Pre-Calci content of ointment is determined by High performance liquid chromatography (HPLC). It is found Pre-Calci content is inversely proportional to size of Calci crystals formed. Less than 0.21% Pre-Calci in ointment ensure Calci crystal D90 > 120 μm and more than 0.58% of Pre-Calci formation indicate smaller crystals of Calci with D90 < 25 μm. Hence fast and economic HPLC analysis can be an alternate approach to RCI in estimating Calci crystals.

Detection and quantification of adulterants in milk powder using a high-throughput Raman chemical imaging technique

ABSTRACTMilk is a vulnerable target for economically motivated adulteration. In this study, a line-scan high-throughput Raman imaging system was used to authenticate milk powder. A 5 W 785 nm line laser (240 mm long and 1 mm wide) was used as a Raman excitation source. The system was used to acquire hyperspectral Raman images in a wave number range of 103–2881 cm–1 from the skimmed milk powder mixed with two nitrogen-rich adulterants (i.e., melamine and urea) at eight concentrations (w/w) from 50 to 10,000 ppm. The powdered samples were put in sample holders with a surface area of 150 ×100 mm and a depth of 2 mm for push-broom image acquisition. Varying fluorescence signals from the milk powder were removed using a correction method based on adaptive iteratively reweighted penalised least squares. Image classifications were conducted using a simple thresholding method applied to single-band fluorescence-corrected images at unique Raman peaks selected for melamine (673 cm–1) and urea (1009 cm–1). Chemical images were generated by combining individual binary images of melamine and urea to visualise identification, spatial distribution and morphological features of the two adulterant particles in the milk powder. Limits of detection for both melamine and urea were estimated in the order of 50 ppm. High correlations were found between pixel concentrations (i.e., percentages of the adulterant pixels in the chemical images) and mass concentrations of melamine and urea, demonstrating the potential of the high-throughput Raman chemical imaging method for the detection and quantification of adulterants in the milk powder.