Assessment of hand-held Raman instrumentation for in situ screening for potentially counterfeit artesunate antimalarial tablets by FT-Raman spectroscopy and direct ionization mass spectrometry

Pharmaceutical counterfeiting has become a significant public health problem worldwide and new, rapid, user-friendly, reliable and inexpensive methods for drug quality screening are needed. This work illustrates the chemical characterization of genuine and fake artesunate antimalarial tablets by portable Raman spectroscopy and validation by FT-Raman spectroscopy and ambient mass spectrometry. The applicability of a compact and robust portable Raman spectrometer (TruScan™) for the in situ chemical identification of counterfeit tablets is reported.     

Combined Fourier-transform infrared imaging and desorption electrospray-ionization linear ion-trap mass spectrometry for analysis of counterfeit antimalarial tablets

This paper reports use of a combination of Fourier-transform infrared (FTIR) spectroscopic imaging and desorption electrospray ionization linear ion-trap mass spectrometry (DESI MS) for characterization of counterfeit pharmaceutical tablets. The counterfeit artesunate antimalarial tablets were analyzed by both techniques. The results obtained revealed the ability of FTIR imaging in non-destructive micro-attenuated total reflection (ATR) mode to detect the distribution of all components in the tablet, the identities of which were confirmed by DESI MS. Chemical images of the tablets were obtained with high spatial resolution. The FTIR spectroscopic imaging method affords inherent chemical specificity with rapid acquisition of data. DESI MS enables high-sensitivity detection of trace organic compounds. Combination of these two orthogonal surface-characterization methods has great potential for detection and analysis of counterfeit tablets in the open air and without sample preparation.     

Testing the performance of pure spectrum resolution from Raman hyperspectral images of differently manufactured pharmaceutical tablets

Chemical imaging is a rapidly emerging analytical method in pharmaceutical technology. Due to the numerous chemometric solutions available, characterization of pharmaceutical samples with unknown components present has also become possible. This study compares the performance of current state-of-the-art curve resolution methods (multivariate curve resolution-alternating least squares, positive matrix factorization, simplex identification via split augmented Lagrangian and self-modelling mixture analysis) in the estimation of pure component spectra from Raman maps of differently manufactured pharmaceutical tablets. The batches of different technologies differ in the homogeneity level of the active ingredient, thus, the curve resolution methods are tested under different conditions. An empirical approach is shown to determine the number of components present in a sample. The chemometric algorithms are compared regarding the number of detected components, the quality of the resolved spectra and the accuracy of scores (spectral concentrations) compared to those calculated with classical least squares, using the true pure component (reference) spectra. It is demonstrated that using appropriate multivariate methods, Raman chemical imaging can be a useful tool in the non-invasive characterization of unknown (e.g. illegal or counterfeit) pharmaceutical products.     

Subsurface probing of calcifications with spatially offset Raman spectroscopy (SORS): future possibilities for the diagnosis of breast cancer

Breast calcifications are often the only mammographic features indicating the presence of a cancerous lesion. Calcium oxalate (type I) may be found in and around benign lesions, however calcium hydroxyapatite (type II) is usually found within proliferative lesions, which can include both benign and malignant pathologies. However, the composition of type II calcifications has been demonstrated to vary between benign and malignant proliferative lesions, and could be an indicator for the possible disease state. Raman spectroscopy has previously been demonstrated as a powerful tool for non-destructive analysis of tissues, utilising laser light to probe chemical composition. Raman spectroscopy is traditionally a surface technique. However, we have recently developed methods that permit its application for obtaining sample composition to clinically relevant depths of many mm. We report the first demonstration of spatially offset Raman spectroscopy (SORS) for potential in vivo breast analysis. This study evaluates the possibility of utilising SORS for measuring calcification composition through varying thicknesses of tissues (2 to 10 mm), which is about one to two orders of magnitude deeper than has been possible with conventional Raman approaches. SORS can be used to distinguish non-invasively between calcification types I and II (and carbonate substitution of phosphate in calcium hydroxyapatite) within tissue of up to 10 mm deep. This result secures the first step in taking this technique forward for clinical applications seeking to use Raman spectroscopy as an adjunct to mammography for early diagnosis of breast cancer, by utilising both soft tissue and calcification signals. Non-invasive elucidation of calcification composition, and hence type, associated with benign or malignant lesions, could eliminate the requirement for biopsy in many patients.     

A novel approach to investigate vascular wall in 3D: Combined Raman spectroscopy and atomic force microscopy for aorta en face imaging

A novel approach based on the combination of Raman confocal 3D imaging with atomic force microscopy (AFM) for analysis of the murine vessel wall en face is described. The approach is based on subsequent Raman and AFM imaging of the same areas of the sample. This methodology allows for direct correlation of the chemical structure (Raman data) with morphology of the surface (AFM). The sub-cellular structures of the tissue e.g., cell nuclei, heme, or lipid-rich species are visualized and localized by the application of Raman imaging, while AFM complements these data with high-resolution information about the surface topography and size of lipid-rich structures. Overall, the applied approach enables detailed characterization of the inner layer of the vessel wall.     

Detection and chemical profiling of medicine counterfeits by Raman spectroscopy and chemometrics

Raman spectroscopy combined with chemometrics has recently become a widespread technique for the analysis of pharmaceutical solid forms. The application presented in this paper is the investigation of counterfeit medicines. This increasingly serious issue involves networks that are an integral part of industrialized organized crime. Efficient analytical tools are consequently required to fight against it. Quick and reliable authentication means are needed to allow the deployment of measures from the company and the authorities. For this purpose a method in two steps has been implemented here. The first step enables the identification of pharmaceutical tablets and capsules and the detection of their counterfeits. A nonlinear classification method, the Support Vector Machines (SVM), is computed together with a correlation with the database and the detection of Active Pharmaceutical Ingredient (API) peaks in the suspect product. If a counterfeit is detected, the second step allows its chemical profiling among former counterfeits in a forensic intelligence perspective. For this second step a classification based on Principal Component Analysis (PCA) and correlation distance measurements is applied to the Raman spectra of the counterfeits.     

In situ Raman spectroscopy of surfaces modified by ion soft landing

The design and characterization of a system for in situ Raman analysis of surfaces prepared by ion soft landing (SL) is described. The performance of the new high vacuum compatible, low cost, surface analysis capability is demonstrated with surface enhanced Raman spectroscopy (SERS) of surfaces prepared by soft landing of ions of crystal violet, Rhodamine 6G, methyl orange and copper phthalocyanine. Complementary in situ mass spectrometric information is recorded for the same surfaces using a previously implemented secondary ion mass spectrometer (SIMS). Imaging of the modified surfaces is achieved using 2D Raman imaging as demonstrated for the case of Rhodamine 6G soft landing. The combination of the powerful molecular characterization tools of SERS and SIMS in a single instrument fitted with in-vacuum sample transport capabilities, facilitates in situ analysis of surfaces prepared by ion SL. In particular, information is provided on the charge state of the soft landed species. In the case of crystal violet the SERS data suggest that the positively charged ions being landed retain their charge state on the surface under vacuum. By contrast, in the case of methyl orange which is landed as an anion, the SERS spectra suggest that the SL species has been neutralized.     

Time-resolved spatially offset Raman spectroscopy for depth analysis of diffusely scattering layers

The objective of this study is to use time-resolved (TR) Raman spectroscopy, spatially offset Raman spectroscopy (SORS), and a combination of these approaches to obtain high quality Raman spectra from materials hidden underneath an opaque layer. Both TR Raman and SORS are advanced techniques that allow for an increased relative selectivity of photons from deeper layers within a sample. Time-resolved detection reduces fluorescence background, and the selectivity for the second layer is improved. By combining this with spatially offset excitation we additionally increased selectivity for deeper layers. Test samples were opaque white polymer blocks of several mm thicknesses. Excitation was carried out with a frequency-doubled Ti:sapphire laser at 460 nm, 3 ps pulse width and 76 MHz repetition rate. Detection was either with a continuous-wave CCD camera or in time-resolved mode using an intensified CCD camera with a 250 ps gate width. The Raman photons were collected in backscatter mode, with or without lateral offset. By measuring the delay of the Raman signal from the second layer (polyethylene terephthalate/PET/Arnite), the net photon migration speeds through Teflon, polythene, Delrin and Nylon were determined. Raman spectra could be obtained from a second layer of PET through Teflon layers up to 7 mm of thickness. The ability to obtain chemical information through layers of diffusely scattering materials has powerful potential for biomedical applications.     

Resonance Raman and surface- and tip-enhanced Raman spectroscopy methods to study solid catalysts and heterogeneous catalytic reactions

Resonance Raman (RR) spectroscopy has several advantages over the normal Raman spectroscopy (RS) widely used for in situ characterization of solid catalysts and catalytic reactions. Compared with RS, RR can provide much higher sensitivity and selectivity in detecting catalytically-significant surface metal oxides. RR can potentially give useful information on the nature of excited states relevant to photocatalysis and on the anharmonic potential of the ground state. In this critical review a detailed discussion is presented on several types of RR experimental systems, three distinct sources of so-called Raman (fluorescence) background, detection limits for RR compared to other techniques (EXAFS, PM-IRAS, SFG), and three well-known methods to assign UV-vis absorption bands and a band-specific unified method that is derived mainly from RR results. In addition, the virtues and challenges of surface-enhanced Raman spectroscopy (SERS) are discussed for detecting molecular adsorbates at catalytically relevant interfaces. Tip-enhanced Raman spectroscopy (TERS), which is a combination of SERS and near-field scanning probe microscopy and has the capability of probing molecular adsorbates at specific catalytic sites with an enormous surface sensitivity and nanometre spatial resolution, is also reviewed (300 references).     

A novel ATR-FTIR approach for characterisation and identification of ex situ immobilised species.

We demonstrate a novel method to analyse ex situ prepared protein chips by attenuated total reflection Fourier IR spectroscopy (ATR-FTIR), which circumvents tedious functionalisation steps of internal reflection elements (IREs), and simultaneously allows for complementary measurements by other analytical techniques. This concept is proven by utilising immobilised metal affinity capture (IMAC) chips containing about 10 mum thick films of copolymers coated with nitrilotriacetic acid (NTA) groups, which originally was manufactured for surface enhanced laser desorption ionisation (SELDI) spectrometry. Three immobilisation steps were analysed by ATR-FTIR spectroscopy: 1) NTA complexation with nickel(II) ions 2) binding of two histidine (His)-tagged synthetic peptides of 25 (25-His6) and 48 (48-His6) amino acids to the NTA-groups and 3) attachment of a ligand, mesyl amide, to the surface-bound 48-His6. Despite interference from H(2)O, both amide I and II were well resolved. Utilising peptide adsorption in the thick copolymer matrix yields a high saturation peptide concentration of approximately 100 mg mL(-1) and a dissociation constant of 116+/-11 muM, as determined by a detailed analysis of the Langmuir adsorption isotherm. The mesyl amide ligand was directly seen in the raw ATR-FTIR spectrum with specific peaks in the fingerprint region at 1172 and 1350 cm(-1). Several aspects of the fine structure of the amide I band of the peptide were analysed: influences from secondary structure, amino side chains and competing contamination product. We believe that this approach has great potential as a stand-alone or complementary analytical tool for determination of the chemical composition of functionalised surfaces. We emphasise further that with this approach no chemical treatment of IREs is needed; the chips can be regenerated and reused, and applied in other experimental set-ups.     

Detection of counterfeit stevia products using a handheld Raman spectrometer

Stevia is a highly appreciated natural sweetener because it can be consumed by diabetic patients. Due to the increasing popularity of stevia during the last years, counterfeit products have been making their way into the market. Raman spectroscopy is a versatile analytical technique that can be used for control tasks and handheld modern devices expand its possible applications to instant in situ measurements. The Raman spectra of six commercial stevia products (five purchased in Bolivia and one in Germany) were recorded and compared to the spectra of standards of rebaudioside A and stevioside as well as the spectra of the artificial sweeteners sodium cyclamate and sodium saccharin. Based on the Raman spectroscopic data, it was verified that three of the Bolivian products were counterfeit products and another one was rich in maltodextrin. The Raman spectra of one Bolivian product and the German one revealed rebaudioside A and stevioside as major components. Raman spectroscopy was capable of detecting contents as low as 5% (w/w) of sodium cyclamate during measurements of stevia-sodium cyclamate mixtures. The results show that Raman spectroscopy can successfully be used to detect counterfeit stevia and underline its high potential for the detection of food adulteration.     

Raman Imaging: An Impending Approach Towards Cancer Diagnosis

In accordance with the recent studies, Raman spectroscopy is well experimented as a highly sensitive analytical and imaging technique in biomedical research, mainly for various disease diagnosis including cancer. In comparison with other imaging modalities, Raman spectroscopy facilitate numerous assistances owing to its low background signal, immense spatial resolution, high chemical specificity, multiplexing capability, excellent photo stability and non-invasive detection capability. In cancer diagnosis Raman imaging intervened as a promising investigative tool to provide molecular level information to differentiate the cancerous vs non-cancerous cells, tissues and even in body fluids. Anciently, spontaneous Raman scattering is very feeble due to its low signal intensity and long acquisition time but new advanced techniques like coherent Raman scattering (CRS) and surface enhanced Raman scattering (SERS) gradually superseded these issues. So, the present review focuses on the recent developments and applications of Raman spectroscopy-based imaging techniques for cancer diagnosis.     

Raman imaging analysis of pharmaceutical tablets by two-dimensional (2D) correlation spectroscopy

Chemical properties of active substances and insoluble excipient within tablets such as crystalline structures can be seen as an important index for solubility of ingredients. Spectroscopic imaging can potentially be a solid solution to understanding mechanisms at the molecular level and it may bring useful insight in terms of process analytical technique. In the present study, generalized two-dimensional (2D) correlation spectroscopy is utilized for the Raman image analysis of pharmaceutical tablets to reveal molecular interactions between chemical components. By using a spatial distance as a perturbation variable in 2D correlation scheme, synchronous and asynchronous correlation analysis becomes possible. Two kinds of pharmaceutical tablets, pentoxifylline (PTX) as an active substance and palmitic acid (PA) as an insoluble excipient, are prepared with different grinding times, 0.5 and 45 min. The 2D correlation analysis of Raman images of the tablets clearly reveals both physical and chemical effects of grinding process on the properties of the tablets. Asynchronous correlations indicate that a specific molecular structural change of PTX related to the crystallinity is induced by the grinding process. Namely, the crystallinity of PTX based on CH₂ structure is a key factor to control the solubility of the tablets. Some properties of pharmaceutical tablets, i.e. solubility or distribution of components in turn may become possible by the simple grinding process. Detailed analysis of Raman images becomes possible by the 2D correlation spectroscopy.     

Multivariate classification of pigments and inks using combined Raman spectroscopy and LIBS

The authenticity of objects and artifacts is often the focus of forensic analytic chemistry. In document fraud cases, the most important objective is to determine the origin of a particular ink. Here, we introduce a new approach which utilizes the combination of two analytical methods, namely Raman spectroscopy and laser-induced breakdown spectroscopy (LIBS). The methods provide complementary information on both molecular and elemental composition of samples. The potential of this hyphenation of spectroscopic methods is demonstrated for ten blue and black ink samples on white paper. LIBS and Raman spectra from different inks were fused into a single data matrix, and the number of different groups of inks was determined through multivariate analysis, i.e., principal component analysis, soft independent modelling of class analogy, partial least-squares discriminant analysis, and support vector machine. In all cases, the results obtained with the combined LIBS and Raman spectra were found to be superior to those obtained with the individual Raman or LIBS data sets.     

Total internal reflection Raman spectroscopy

Total internal reflection (TIR) Raman spectroscopy is an experimentally straightforward, surface-sensitive technique for obtaining chemically specific spectroscopic information from a region within approximately 100-200 nm of a surface. While TIR Raman spectroscopy has long been overshadowed by surface-enhanced Raman scattering, with modern instrumentation TIR Raman spectra can be acquired from sub-nm thick films in only a few seconds. In this review, we describe the physical basis of TIR Raman spectroscopy and illustrate the performance of the technique in the diverse fields of surfactant adsorption, liquid crystals, lubrication, polymer films and biological interfaces, including both macroscopic structures such as the surfaces of leaves, and microscopic structures such as lipid bilayers. Progress, and challenges, in using TIR Raman to obtain depth profiles with sub-diffraction resolution are described.     

Short-wave infrared excited spatially offset Raman spectroscopy (SORS) for through-barrier detection

Spatially offset Raman spectroscopy (SORS) using 1064 nm excitation is demonstrated here to detect chemicals through a physical barrier such as a container. This excitation wavelength overcomes the issue of fluorescence from the target chemical, whilst retaining the benefits of the SORS technique for through-barrier detection. These advantages have a wide range of applications in both civilian and military environments.     

NMR and MRI studies of drug delivery systems

Nuclear magnetic resonance imaging and spectroscopy are now commonplace in most academic and industrial research environments. The ability of magnetic resonance techniques to provide the researcher with non-invasive, quantitative, physicochemical information in the disciplines of chemistry, biology, materials science, chemical engineering and medicine is widely known. In the last 10–15 years a variety of magnetic resonance methods have provided the pharmaceutical research community with valuable information, especially in the important area of drug delivery using solid dosage forms. This review will highlight recent advances in magnetic resonance techniques and its specific applications to further our understanding of pharmaceutical drug delivery systems. The review is aimed at non-clinical research and development, and will focus on the behaviour and characterisation of drug release from pellets, tablets and capsules, which are the most commonly used drug delivery systems. In addition to magnetic resonance techniques a number of complementary analytical techniques are mentioned to illustrate the importance of adopting a multi-modal analytical approach to gain a better scientific understanding of the behaviour of drug delivery devices.     

Towards nanometric resolution in multilayer depth profiling: a comparative study of RBS, SIMS, XPS and GDOES

An increasing amount of effort is currently being directed towards the development of new functionalized nanostructured materials (i.e., multilayers and nanocomposites). Using an appropriate combination of composition and microstructure, it is possible to optimize and tailor the final properties of the material to its final application. The analytical characterization of these new complex nanostructures requires high-resolution analytical techniques that are able to provide information about surface and depth composition at the nanometric level. In this work, we comparatively review the state of the art in four different depth-profiling characterization techniques: Rutherford backscattering spectroscopy (RBS), secondary ion mass spectrometry (SIMS), X-ray photoelectron spectroscopy (XPS) and glow discharge optical emission spectroscopy (GDOES). In addition, we predict future trends in these techniques regarding improvements in their depth resolutions. Subnanometric resolution can now be achieved in RBS using magnetic spectrometry systems. In SIMS, the use of rotating sample holders and oxygen flooding during analysis as well as the optimization of floating low-energy ion guns to lower the impact energy of the primary ions improves the depth resolution of the technique. Angle-resolved XPS provides a very powerful and nondestructive technique for obtaining depth profiling and chemical information within the range of a few monolayers. Finally, the application of mathematical tools (deconvolution algorithms and a depth-profiling model), pulsed sources and surface plasma cleaning procedures is expected to greatly improve GDOES depth resolution.     

Confocal micro-X-ray fluorescence analysis as a new tool for the non-destructive study of the elemental distributions in pharmaceutical tablets

Chemical imaging studies of pharmaceutical tablets are currently an important emerging field in the pharmaceutical industry. Finding the distribution of the different compounds inside the tablet is an important issue for production quality control but also for counterfeit detection.Most of the currently used techniques are limited to the study of the surface of the compacts, whereas the study of the bulk requires a time-consuming sample preparation. In this paper, we present the use of 3D micro-X-ray fluorescence analysis (3D μXRF) for the non-destructive study of pharmaceutical tablets.Based on two different examples, it was shown that it was possible to measure the distribution of several inorganic elements (Zn, Fe, Ti, Mn, Cu) from the surface to a depth of several hundred microns under the surface. The X-ray absorption, depending on both matrix composition and energy, is one of the most critical factors of this analytical method while performing depth profiling or mapping. Therefore, an original method to correct the absorption, in order to accurately measure the true elemental distribution, was proposed.Moreover, by using the presence of titanium dioxide in a pharmaceutical coating, we proved that this technique is also suited to the non-destructive measurement of coating thickness.     

Vibrational Spectroscopy Study of Selenate and Sulfate Adsorption Mechanisms on Fe and Al (Hydr)oxide Surfaces

The coordination and speciation of selenate (SeO(4)) and sulfate (SO(4)) on goethite and Al oxide were studied using Raman and ATR-FTIR spectroscopy. Raman spectra were collected from pastes of suspensions containing 4 mM SeO(4) or SO(4). For SO(4), complementary data were collected by ATR-FTIR spectroscopy in goethite systems with 1 mM SO(4) and in Al oxide systems with 4 mM SO(4). The combined data set of Raman and ATR-FTIR spectra indicate that both inner- and outer-sphere surface complexes of SeO(4) and SO(4) occur on these metal (hydr)oxide surfaces. These spectral data show that SeO(4) and SO(4) have a similar complexation behavior on the same adsorbent. On goethite, these form predominantly monodentate inner-sphere surface complexes at pH <6, while at pH >6 these anions exist predominantly as outer-sphere surface complexes. On Al oxide, in contrast, these anions exist predominantly as outer-sphere surface complexes, but a small fraction is also present as an inner-sphere complex at pH <6. A comparison of the spectral intensities of these anions on goethite and Al oxide shows that complexation of these anions with Al oxide is weaker than with Fe oxide. Copyright 2000 Academic Press.