Applicability of the Kubelka-Munk theory for the evaluation of reflectance spectra demonstrated for haemoglobin-free perfused heart tissue.

Reflectance spectrometry is a useful tool for studying in vivo kinetic changes in the oxygen saturation of haemoglobin and myoglobin as well as the redox state of cytochromes. A method is given which allows the quantification of tissue reflectance spectra using multicomponent analysis. This method utilizes the Kubelka-Munk theory for modelling the measured tissue spectra. To test this approach, reflectance spectra of a haemoglobin-free perfused guinea pig heart were measured by a fast scanning spectrophotometer (100 spectra/s, spectral resolution 1.0 nm) and evaluated using the component absorbance spectra measured separately. A relative mean spectral residual error of 0.15% was achieved by least-squares fitting. Using statistical error propagation, oxygenation of myoglobin is obtained within a relative precision of 1%, and the redox state of cytochromes aa3 and c are determined simultaneously within a margin of 3%; the results for the redox-state of cytochrome b, however, are less precise. Special component error functions are presented to provide a reliability measure for the concentration prediction using this multicomponent assay. The consistency of the theory and the component absorptivity data is tested by regressing the actual concentrations obtained for each of the redox pair components during the various states of tissue oxygenation. A method is described for the recognition and reduction of systematic errors.

Comparison of sun protection factors determined by an in vivo and different in vitro methodologies: a study with 58 different commercially available sunscreen products.

An extensive study on the sun protection factors (SPF) of sun care products was carried out using the COLIPA (The European Cosmetic Toiletry and Perfumery Association) method, which relates to in vivo experiments. Furthermore, in vitro methods were tested with sunscreen formulations that were prepared as films on surface-roughened plates of polymethyl methacrylate (PMMA). One of the in vitro methods, i.e. using the sunscreen tester, has been recently developed, whereas the second has been defined by a pure spectroscopic approach, which is based on spectral transmission measurements of sunscreen films. Altogether 58 different sunscreen formulations, with manufacturer declared SPF values ranging from 4 to 60 and currently available on the European market, were investigated. The quality of correlations with results from the individual products based on the different in vitro methods versus the COLIPA values that were considered as generally accepted standard values was assessed. In this context, also variations because of sample preparation and spectral measurement were discussed. For sunscreen products with in vivo SPF values larger 25, the spectral transmittance within the UVA/UVB range is rapidly decreasing, which is experienced even for products with reduced amounts reaching 0.5 mg cm(-2) and still leading to unsatisfactory correlation of the spectroscopically derived SPF values versus the results from the alternative assays. Opposite to these small amounts, a sunscreen product spread of 2 mg cm(-2) is standard for the in vivo COLIPA method, whereas an area-normalized amount of 1 mg cm(-2) is currently routinely used for the sunscreen tester method. Furthermore, an overview of the individual product characteristics, such as their specific critical wavelengths and their UVA/UVB ratios is provided; both parameters can also be calculated from the spectral absorbances of the standardized sunscreen films.

Penetration studies of vitamin E acetate applied from cosmetic formulations to the stratum corneum of an in vitro model using quantification by tape stripping, UV spectroscopy, and HPLC.

The skin activation and penetration capability of vitamin E acetate as an ingredient in a basic o/w cream (lamellar type), in liposomes (Rovisome) and microparticles (Roviparts), was investigated under in vitro conditions (BUS model) by the adhesive stripping method. The aim of the study was to compare the analytical results obtained by UV spectroscopy (transmission) and the conventional HPLC method. For the quantitative spectrometric assay, a classical least-squares evaluation of the spectra between 265 and 350 nm, based on the constituent spectra, was used. UV spectroscopy is an economic analytical method for evaluating a large population of samples of the horny layer taken by the adhesive tape stripping method, which is an established tool for depth profiling of substances within the stratum corneum. With regard to the irritation test, no cytotoxicity was recorded for all formulations tested. However, the Roviparts and Rovisome cream formulations induced a considerable activation of the epidermal cells that may contribute to the penetration efficiency of Rovisome-formulated vitamin E acetate. The Rovisome-formulated cream delivered a maximum amount of vitamin E acetate into the horny layer compared to the other formulations tested. The difference can be explained by an alteration of the plasticity of the horny layer inducing a strong reservoir capacity and an activation of upper epidermal cells. Moreover, the opening of the potential pathway for a follicular penetration may be part of the increased reservoir capacity.

Non-invasive monitoring of metabolites using near infrared spectroscopy: state of the art.

Non-invasive instrumentation for metabolite monitoring can be based on near-infrared spectroscopy. The main research emphasis is on the development of glucose assays, which could be used for patient self-monitoring. Some histological and physiological variability during in-vivo measurements with integral tissue probing is discussed. The state of non-invasive monitoring is presented and compared with other competing methodologies. Different techniques have been proposed so far, which include transmission and diffuse reflectance measurements. Other methods employ photoacoustic laser spectroscopy or the measurement of changes in radiation scattering due to variations in glucose concentration. Currently available technology requires further improvements, in particular for the normal and hypoglycemic ranges, if it is to be used for devices for self- and bedside-monitoring or during surgery. The necessary selectivity for reliable glucose concentration prediction may only be gained by multivariate data analysis exploiting information from broad spectral ranges because of overlapping spectral features from several other biocomponents in the body tissue under investigation.

Human oral mucosa studies with varying blood glucose concentration by non-invasive ATR-FT-IR-spectroscopy.

During the last few years infrared spectrometry has been investigated as a non-invasive clinical tool for improved understanding of in-vivo processes. Oral mucosa has been suggested as an especially suited subject for drug delivery and in vivo monitoring of endogenous body metabolites due to histological and physicochemical reasons. The attenuated total reflection (ATR) technique was used to characterize the outmost epidermal layer of human oral mucosa by means of Fourier-transform infrared spectroscopy. The penetration depth of the probing radiation in the mid-infrared fingerprint region, using a ZnSe-crystal for the horizontal ATR accessory, is in the order of a few micrometers so that microlayer information can be obtained by such a technique. Spectra of outer human lip and saliva components are presented for comparison. For several test persons, lip spectra were recorded during oral glucose tolerance tests. The individually varying blood glucose concentration was followed by means of frequent blood testing. Variability of the outmost microlayer has been studied using factor analysis of the ATR inner-lip spectra. There is no clear evidence that blood glucose concentration can be followed by ATR-spectroscopy of oral mucosa. Non-invasive spectroscopic methods exploiting trace signals require special attention paid to the variability due to person-to-person differences and changes in physiological conditions.

Optical diffuse reflectance accessory for measurements of skin tissue by near-infrared spectroscopy.

An optimized accessory for measuring the diffuse reflectance spectra of human skin tissue in the near-infrared spectral range is presented. The device includes an on-axis ellipsoidal collecting mirror with efficient illumination optics for small sampling areas of bulky body specimens. The optical design is supported by the results of a Monte Carlo simulation study of the reflectance characteristics of skin tissue. Because the results evolved from efforts to measure blood glucose noninvasively, the main emphasis is placed on the long-wavelength near-infrared range where sufficient penetration depth for radiation into tissue is still available. The accessory is applied for in vivo diffuse reflectance measurements.

Near-infrared reflectance spectroscopy for noninvasive monitoring of metabolites.

An important class of substances in clinical chemistry are metabolites in body fluids, which are accessible by near-infrared spectroscopy without sample treatment using reagentless, fast and readily automated in vitro assays. Furthermore, noninvasive sensing systems are under development for the determination of blood glucose, especially for diabetic patients or for monitoring in intensive care and surgery. Near-infrared diffuse reflectance spectrometry of skin was employed allowing a certain tissue volume to be integrally probed. For calibration, the partial least-squares (PLS) algorithm was used either based on wide spectral intervals or using special spectral variable selection. Capillary blood glucose reference concentrations were obtained by finger pricking and an automated laboratory method (hexokinase/G6P-DH). Clear evidence is provided for the physical effect, as manifested by the spectral glucose absorptivities, underlying the individual single-person calibration models, which still require improvements in the methodology in the normo- and hypoglycemic concentration range. In extending the potential of noninvasive blood assays by infrared spectroscopy, a novel technique is presented for probing the intravascular fluid space by using fast spectral near-infrared measurements of skin tissue. The pulsatile blood spectrum can be derived from reflectance spectra of oral mucosa by Fourier analysis (near-infrared plethysmography). Future applications and prospects for noninvasive blood assays are discussed.

Application of infrared spectroscopy to monitoring gas insulated high-voltage equipment: electrode material-dependent SF(6) decomposition.

Sulfur hexafluoride is a chemically inert gas which is used in gas insulated substations (GIS) and other high-voltage equipment, leading to a significant enhancement of apparatus lifetime and reductions in installation size and maintenance requirements compared to conventional air insulated substations. However, component failures due to aging of the gas through electrical discharges may occur, and on-site monitoring for risk assessment is needed. Infrared spectroscopy was used for the analysis of gaseous by-products generated from electrical discharges in sulfur hexafluoride gas. An infrared monitoring system was developed using a micro-cell coupled to an FTIR spectrometer by silver halide fibers. Partial least-squares calibration was applied by using a limited number of optimally selected spectral variables. Emphasis was placed on the determination of main decomposition products, such as SOF(2), SOF(4), and SO(2)F(2). Besides the different electrical conditions, the material of the plane counter electrode of the discharge chamber was also varied between silver, aluminum, copper, tungsten, or tungsten/copper alloy. For the spark experiments the point electrode was the same material as chosen for the plane electrode, whereas for partial discharges a stainless steel needle was employed. Complementary investigations on the chemical composition within the solid counter electrode material by secondary neutral mass spectrometry (SNMS) were also carried out. Under sparking conditions, the electrode material plays an important role in the decomposition rates of the gas-phase, but no relevant material dependence could be observed under partial discharge conditions.

Novel infrared optical probes for process monitoring and analysis based on next-generation silver halide fibers.

Mid-infrared spectroscopy has proved to be a powerful method for the study of various samples and chemical media as found in different industrial processes. In general, the analysis of such samples takes advantage of the fact that multiple analytes can be quantified simultaneously and rapidly without the need for additional reagents. When compared to near-infrared spectroscopy, for which quartz fiber probes can be successfully applied, the application of previously used mid-infrared fiber materials was restricted due to deficiencies with regard to their optical transmission and mechanical properties. Progress in the quality of infrared transparent silver halide fibers and their extrusion with different cross-sections enabled us to construct several flexible fiber-optic probes of different geometries which are particularly suitable and inert for process monitoring. Transmission and attenuated total reflection measurement techniques have mainly been employed for the analysis of liquid and gaseous media. One larger field, for which results are reported, is chemical reactor monitoring. Other applications are concerned with bio-reactor monitoring, or quasi-continuous measurements for the food industry. Infrared spectroscopic cosmetic assays for determining the chemical composition of skin-care formulations are a further promising field of application, for which an example is given.

Reflectance spectroscopy can quantify cutaneous haemoglobin oxygenation by oxygen uptake from the atmosphere after epidermal barrier disruption.

BACKGROUND: The supply of oxygen to the viable skin tissue within the upper layers is not only secured by the cutaneous blood vascular system, but to a significant part also by oxygen diffusion from the atmosphere through the horny layer. The aim of this study was to examine whether changes in haemoglobin oxygenation can be observed within the isolated perfused bovine udder skin used as a skin model by removing the upper horny layer by adhesive tape stripping. METHODS: Diffuse reflectance spectroscopy in the visible spectral range was used for non-invasive characterisation of haemoglobin oxygenation in skin under in vitro conditions. Mid-infrared attenuated total reflectance spectroscopy was employed for analysing the surface layer of the stratum corneum with respect to keratin, water and lipid components. Skin barrier disruption was achieved by repeated stripping of superficial corneocyte layers by adhesive tape. RESULTS AND CONCLUSION: Significant changes in skin haemoglobin oxygenation were observed for skin areas with reduced lipid concentration and a reduced stratum corneum layer, as determined from the quantitative evaluation of the diffuse reflectance skin spectra. The result can be interpreted as an increase of oxygen diffusion after the removal of the upper horny layer.

Multivariate determination of glucose in whole blood by attenuated total reflection infrared spectroscopy.

A spectral analysis of whole EDTA blood was undertaken by using attenuated total reflection and Fourier-transform infrared spectroscopy. The concentration of blood glucose was measured by an enzymatic method using glucose dehydrogenase and ranged between 40 and 290 mg/dL with an average concentration of 90.4 mg/dL. Multivariate calibration with the partial least-squares (PLS) algorithm was performed on spectral data between 1500 and 750 cm-1 showing a varying background from different unidentified interfering compounds. Cross validation was carried out for optimizing the PLS model. PRESS was 19.8 mg/dL, which was calculated on the basis of 127 standards, whereas the estimated standard deviation for the calibration fit was computed to be 11.9 mg/dL. Infrared spectroscopy can be used for monitoring glucose levels within the normal physiological range in a complex matrix like whole blood as an alternative to electrochemical sensors.

Multivariate calibration for assays in clinical chemistry using attenuated total reflection infrared spectra of human blood plasma.

A spectral analysis of human blood plasma was undertaken by use of a Fourier-transform infrared spectrometer with a circular attenuated total reflection cell. The concentrations of total protein, glucose, triglycerides, total cholesterol, urea, and uric acid were measured by chemical or enzymatic methods. For these constituents the partial least-squares (PLS) algorithm was used for a multivariate calibration including the infrared fingerprint region of the plasma spectra. Best results were achieved for total protein with an average prediction error (PRESS based on cross validation) of 2.1 g/L; other PRESS results were for glucose 22 mg/dL, triglycerides 33 mg/dL, cholesterol 31 mg/dL, urea 4.4 mg/dL, and uric acid 1.6 mg/dL.

New tool for epidermal and cosmetic formulation studies by attenuated total-reflection spectroscopy using a flexible mid-infrared fiber probe.

The potential of mid-infrared attenuated total-reflection spectroscopy for dermatology studies has been increased by the development of a flexible fiber-optic probe from silver halide material. Such a tool significantly eases epidermal surface characterization. Spectra of human forearm skin and of bovine udder skin (BUS-model), which is used as a substitute for in-vivo tests on man, were compared, and as an example low concentrations of vitamin E acetate (alpha-tocopherol acetate) in the stratum corneum were measured after topical skin application. Depth profiling was enabled by repeated steps of tape stripping and subsequent skin measurement. The infrared methodology is rapid and can be applied to small areas of skin. It is also sufficiently sensitive for analysis of a large variety of cosmetic formulations and dermatopharmaca. Discrimination of vehicle formulations is possible because of the large information content of mid-infrared spectra.

Noninvasive blood glucose sensors based on near-infrared spectroscopy.

Research into noninvasive devices for self-monitoring of blood glucose is mainly based on near-infrared spectroscopy. Such a device is particularly desirable in the intensive therapy of patients with diabetes mellitus to achieve optimal metabolic control through frequent glucose testing. The state of noninvasive assay technology is presented. Using diffuse reflectance spectra of mucous lip tissue has advantages and drawbacks compared with tissue transmittance experiments. Different approaches have been proposed in the patent literature; however, current technology requires further significant improvements, particularly within the lower normal and hypoglycemic glucose concentration ranges.

Monitoreo no invasivo de metabolitos mediante el uso de espectroscopia casi infrarroja: grado de avance / Non-invasive monitoring of metabolites using near infrared spectroscopy: state of the art

El monitoreo no invasivo de metabolitos se puede efectuar mediante espectroscopía infrarroja. El principal énfasis de la investigación radica en el desarrollo de ensayos de glucosa, que podrían usarse para que el paciente se automonitoree. Se discute sobre la existencia de variabilidad histológica y fisiológica durante las mediciones in vivo con exámenes de tejido integral. Se presenta la situación del monitoreo no invasivo, comparándolo con otras metodologías competitivas. Se han propuesto diferentes técnicas, incluyendo las mediciones de transmisión y de reflectancia difusa. Otros métodos utilizan la espectroscopía láser fotoacústica o la medición de cambios en la dispersión de la radiación debido a variaciones en la concentración de glucosa. La tecnología disponible actualmente requiere mayores desarrollos, particularmente para los rangos de glucosa normal e hipoglucémico, si va a ser utilizada en dispositivos para automonitoreo o monitoreo del paciente cuando está internado, o durante la cirugía. La selectividad necesaria para predecir una concentración de glucosa confiable sólo se obtiene mediante análisis de datos multivariado, utilizando la información obtenida de rangos amplios del espectro al superponer las características del espectro de otros varios biocomponentes en el tejido corporal que se está investigando