Fourier transform infrared imaging analysis in discrimination studies of bladder cancer.

Bladder carcinoma represents more than 4% of all cancer diseases in Austria. The histomorphological evaluation is invasive and remains a subjective and time consuming technique. On account of this it is necessary to find novel non-invasive approaches which support the pathologists for histological recognition to identify malignancy at an early stage. In the present study, Fourier transform infrared (FTIR) microscopic imaging was combined with univariate and multivariate data analysis methods to study bladder carcinoma tissue sections in detail. The possibility to collect IR spectra of bladder carcinoma tissue sections employing an optimized analytical protocol is demonstrated. The correlation between FTIR microscopic imaging and the morphological tissue features obtained by histological staining of the sections demonstrated that many histomorphological tissue patterns can be visualized in the colour images. The routine generation of high quality imaging data is enabled because of the combination of FTIR technology and optimized sample preparation techniques. This opens a new quality of spectroscopic analyses of cancerous tissue, allowing exploration of molecular changes associated with the histopathological morphology.

Chemically modified diamond-like carbon (DLC) for protein enrichment and profiling by MALDI-MS.

The development of new high throughput methods based on different materials with chemical modifications for protein profiling of complex mixtures leads towards biomarkers; used particularly for early diagnosis of a disease. In this work, diamond-like carbon (DLC) is developed and optimized for serum protein profiling by matrix-assisted laser/desorption ionization mass spectrometry (MALDI-MS). This study is carried out in connection with a material-based approach, termed as material-enhanced laser desorption ionization mass spectrometry. DLC is selected as carrier surface which provides large surface to volume ratio and offers high sensitivity. DLC has a dual role of working as MALDI target while acting as an interface for protein profiling by specifically binding peptides and proteins out of serum samples. Serum constituents are bound through immobilized metal ion affinity chromatography (IMAC) functionality, created through glycidyl methacrylate polymerization under ultraviolet light followed by further derivatization with iminodiacetic acid and copper ion loading. Scanning electron microscopy highlights the morphological characteristics of DLC surface. It could be demonstrated that IMAC functionalized DLC coatings represent a powerful material in trapping biomolecules for their further analysis by MALDI-MS resulting in improved sensitivity, specificity and capacity in comparison to other protein-profiling methods.

Fourier transform infrared imaging analysis in discrimination studies of squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) of the oral cavity and oropharynx represents more than 95% of all malignant neoplasms in the oral cavity. Histomorphological evaluation of this cancer type is invasive and remains a time consuming and subjective technique. Therefore, novel approaches for histological recognition are necessary to identify malignancy at an early stage. Fourier transform infrared (FTIR) imaging has become an essential tool for the detection and characterization of the molecular components of biological processes, such as those responsible for the dynamic properties of tumor progression. FTIR imaging is a modern analytical technique enabling molecular imaging of a complex biological sample and is based on the absorption of IR radiation by vibrational transitions in covalent bonds. One major advantage of this technique is the acquisition of local molecular expression profiles, while maintaining the topographic integrity of the tissue and avoiding time-consuming extraction, purification, and separation steps. With this imaging technique, it is possible to obtain unique images of the spatial distribution of proteins, lipids, carbohydrates, cholesterols, nucleic acids, phospholipids, and small molecules with high spatial resolution. Analysis and visualization of FTIR imaging datasets are challenging and the use of chemometric tools is crucial in order to take advantage of the full measurement. Therefore, methodologies for this task based on the novel developed algorithm for multivariate image analysis (MIA) are often necessary. In the present study, FTIR imaging and data analysis methods were combined to optimize the tissue measurement mode after deparaffinization and subsequent data evaluation (univariate analysis and MIAs). We demonstrate that it is possible to collect excellent IR spectra from formalin-fixed paraffin-embedded (FFPE) tissue microarrays (TMAs) of OSCC tissue sections employing an optimised analytical protocol. The correlation of FTIR imaging to the morphological tissue features obtained by histological staining of the sections demonstrated that many histomorphological tissue patterns can be visualized in the colour images. The different algorithms used for MIAs of FTIR imaging data dramatically increased the information content of the IR images from squamous cell tissue sections. These findings indicate that intra-operative and surgical specimens of squamous cell carcinoma tissue can be characterized by FTIR imaging.

Morphological and tissue characterization of the medicinal fungus Hericium coralloides by a structural and molecular imaging platform.

In this study the potential of new imaging techniques such as Magnetic Resonance Imaging (MRI), Matrix-Assisted Laser Desorption/Ionization (MALDI) profiling mass spectrometry ("MALDI Profiling") and Fourier Transform Infrared (FTIR) spectroscopic imaging was evaluated to study morphological and molecular patterns of the potential medicinal fungus Hericium coralloides. For interpretation, the MALDI profiling, FTIR imaging and MRI results were correlated with histological information gained from Scanning Electron Microscopy (SEM) and Light Microscopy (LM). Additionally we tested several evaluation processes and optimized the methodology for use of complex FTIR images to monitor molecular patterns. It is demonstrated that the combination of these spectroscopic methods enables to gain a more distinct picture concerning morphology and distribution of active ingredients. We were able to obtain high quality FTIR imaging and MALDI-profiling results and to distinguish different tissue types with their chemical ingredients. Beside this, we have created a 3-D reconstruction of a mature Hericium basidioma, based on the MRI dataset: analyses allowed, for the first time, a realistic approximation of the "evolutionary effectiveness" of this bizarrely formed basidioma type, concerning the investment of sterile tissue and its reproductive output (production of basidiospores).

Fourier transform infrared imaging analysis in discrimination studies of St. John's wort (Hypericum perforatum).

In the present study, Fourier transform infrared (FTIR) imaging and data analysis methods were combined to study morphological and molecular patterns of St. John's wort (Hypericum perforatum) in detail. For interpretation, FTIR imaging results were correlated with histological information gained from light microscopy (LM). Additionally, we tested several evaluation processes and optimized the methodology for use of complex FTIR microscopic images to monitor molecular patterns. It is demonstrated that the combination of the used spectroscopic method with LM enables a more distinct picture, concerning morphology and distribution of active ingredients, to be gained. We were able to obtain high-quality FTIR microscopic imaging results and to distinguish different tissue types with their chemical ingredients.

Selected Extracts of Chinese Herbal Medicines: Their Effect on NF-κB, PPARα and PPARγ and the Respective Bioactive Compounds.

Chinese herbal medicinal (CHM) extracts from fourteen plants were investigated in cell-based in vitro assays for their effect on nuclear factor κB (NF-κB), a key regulator of inflammation, as well as on peroxisome proliferator-activated receptors (PPARs) being key regulators of genes involved in lipid and glucose metabolism. 43% of the investigated CHMs showed NF-κB inhibitory and 50% PPARα and PPARγ activating effects. Apolar extracts from cortex and flos of Albizia julibrissin Durazz. and processed rhizomes of Arisaema sp. and Pinellia ternata (Thunb.) Breit. that effectively inhibited TNF-α-induced NF-κB activation and dose-dependently activated PPARα and PPARγ were further investigated. Bioassay-guided fractionation and analysis by GC-MS led to the identification of fatty acids as PPAR agonists, including linoleic and palmitic acid.

Development and application of Fourier-transform infrared chemical imaging of tumour in human tissue.

Fourier-transform infrared (FT-IR) based mapping and imaging is a fast emerging technology which is being increasingly applied to investigate tissues in the high-throughput mode. The high resolution close to the cellular level, the possibility to determine the bio-distribution of molecules of interest (proteins, peptides, lipids, carbohydrates) without any pre-treatment and the offer to yield molecular structure information have brought evidence that this technique allows to gain new insights in cancer pathology. Thus, several individual mainly protein and peptide cancer markers ("biomarkers") can be identified from FT-IR tissue images, enabling accurate discrimination between healthy and tumour areas. Optimal data acquisition (spatial resolution, spectral resolution, signal to noise ratio), classification, and validation are necessary to establish practical protocols that can be translated to the qualitative and quantitative clinical routine analysis. Thereby, the development of modern fast infrared imaging systems has strongly supported its acceptance in clinical histopathology. In this review, the necessity of analysis based on global cancer statistics, instrumental setups and developments, experimental state of the art are summarised and applications to investigate different kinds of cancer (e.g., prostate, breast, cervical, colon, oral cavity) are shown and discussed in detail.

Near-infrared spectroscopic study on guest-host interactions among G0-G7 amine-terminated poly(amidoamine) dendrimers and porous silica materials for simultaneously determining the molecular weight and particle diameter by multivariate calibration techniques.

The guest-host interactions of poly(amidoamine) (PAMAM) dendrimers and porous silica surfaces were investigated by near-infrared (NIR) diffuse reflection spectroscopy. G0-G7 of amine-terminated PAMAM (PAMAM-NH2) dendrimers were analyzed comprising early, mid, and late generations. For early stages, the adsorption process of the partly protonated dendrimers to the negatively charged silica surface strongly depends on the size/shape characteristics of the guest (PAMAM-NH2 dendrimers) and host (porous silica) materials. G0-G4 (15-45 A) show smaller particle sizes than the pore diameter of the silica (60 A) and thus have access to the interior surface of the host material. For mid and later stages (G5-G7; 54-81 A) only low amounts of the dendrimers adsorb to the silica surface due to the inaccessibility to the interior surface. The loading capacity of the silica material with adsorbed PAMAM-NH(2) was evaluated by means of capillary zone electrophoresis (CZE), whereas deviations from the theoretical to the effective particle size and molecular weight (MW) was determined by gas-phase electrophoretic mobility molecular analysis (GEMMA) and matrix-assisted laser desorption/ionization linear time-of-flight mass spectrometry (MALDI-lin TOF-MS). Deviations from the theoretical to the actual values showed a maximum of 13.8% and 28.0% for the particle size and MW, respectively. The NIR absorption spectra show a distinct band at 4932 cm(-1) (nu(sym) (NH) + amide II) due to the adsorbed dendrimers. It was found that the absorbance tends to increase with decreasing generation number. On this basis multivariate calibration was performed with the theoretical data and the data obtained by GEMMA and MALDI-lin TOF-MS. All in all, the calculated partial least-squares regression (PLSR) model containing the GEMMA/MALDI-lin TOF-MS reference values showed better results than the models exclusively calculated from the theoretical values. This indicates that the theoretical values do not imply the structural imperfections arising during the synthesis that may be present in the PAMAM-NH2 dendrimers.

Simultaneous determination of the micro-, meso-, and macropore size fractions of porous polymers by a combined use of Fourier transform near-infrared diffuse reflection spectroscopy and multivariate techniques.

Fourier transform near-infrared (FT-NIR) diffuse reflection spectroscopy was used in combination with principal component analysis and partial least-squares regression to simultaneously determine the physical and the chemical parameters of a porous poly(p-methylstyrene-co-1,2-bis(p-vinylphenyl)ethane) (MS/BVPE) monolithic polymer. Chemical variations during the synthesis of the polymer material can alter the pore volume and pore area distributions within the polymer scaffold. Furthermore, mid-infrared and near-infrared (NIR) spectroscopic chemical imaging was implemented as a tool to assess the uniformity of the samples. The presented study summarizes the comparative results derived from the spectral FT-NIR data combined with chemometric techniques. The relevance of the interrelation of physical and chemical parameters is highlighted whereas the amount of MS/BVPE (%, v/v) and the quantity (%) of micropores (diameter, d < 6 nm), mesopores (6 nm < d < 50 nm), and macropores (50 nm < d < 200 nm) could be determined with one measurement. For comparison of the quantitative data, the standard error of prediction (SEP) was used. The SEP for determining the MS/BVPE amount in the samples showed 0.35%, for pore volume quantiles 1.42-8.44%, and for pore area quantiles 0.38-1.45%, respectively. The implication of these results is that FT-NIR spectroscopy is a suitable technique for the screening of samples with varying physicochemical properties and to quantitatively determine the parameters simultaneously within a few seconds.

Nano-structured support materials, their characterisation and serum protein profiling through MALDI/TOF-MS.

In the bioanalytical era, novel nano-materials for the selective extraction, pre-concentration and purification of biomolecules prior to analysis are vital. Their application as affinity binding in this regard is needed to be authentic. We report here the comparative application of derivatised materials and surfaces on the basis of nano-crystalline diamond, carbon nanotubes and fullerenes for the analysis of marker peptides and proteins by material enhanced laser desorption ionisation mass spectrometry MELDI-MS. In this particular work, the emphasis is placed on the derivatization, termed as immobilised metal affinity chromatography (IMAC), with three different support materials, to show the effectiveness of MELDI technique. For the physicochemical characterisation of the phases, near infrared reflectance spectroscopy (NIRS) is used, which is a well-established method within the analytical chemistry, covering a wide range of applications. NIRS enables differentiation between silica materials and different fullerenes derivatives, in a 3-dimensional factor-plot, depending on their derivatizations and physical characteristics. The method offers a physicochemical quantitative description in the nano-scale level of particle size, specific surface area, pore diameter, pore porosity, pore volume and total porosity with high linearity and improved precision. The measurement takes only a few seconds while high sample throughput is guaranteed.

Characterisation of different types of hay by solid-phase micro-extraction-gas chromatography mass spectrometry and multivariate data analysis.

The volatile organic compounds of different ground hay samples from Austria, Italy and Switzerland were collected at 50 degrees C on a Supelco Carbowax Divinylbenzene headspace solid-phase microextraction fibre, separated by capillary gas chromatography on an HP5-ms column running a temperature programme and using helium as carrier gas, detected with a mass sensitive detector and studied with principal component analyses after autoscaling selected variables. The analytes, mainly mono- and sesquiterpenes, were able to cluster differences resulting from the site of production. Coumarin can be used to differentiate hay grown north and south of the main chain of the Alps. Acetic acid is appropriate for distinguishing between hay from Kastelruth and Passeier Valley, two South Tyrolean regions. The average linalool content in aftermath is higher than in hay.

Quantitative and qualitative investigation of the main flavonoids in heartsease (Viola tricolor L.).

Liquid chromatography coupled to electrospray ionization tandem mass spectrometry (MSn) is used for the analysis of flavonoids in heartsease (Viola tricolor L.). Our data suggested that the two main flavonoid components were violanthin (6-C-glucosyl-8-C-rhamnosyl apigenin) and rutin (3-O-rutinosyl quercetin). The identification of rutin was confirmed by comparing its retention time, UV spectrum, molecular mass, and fragmentation pattern with the reference standard. In this paper, we also report on the quantitative analysis of rutin by high-performance liquid chromatography. According to our results, heartsease herb contained 420+/-1.17 microg/g rutin.

Role of carbon nano-materials in the analysis of biological materials by laser desorption/ionization-mass spectrometry.

At present, carbon nano-materials are being utilized in various procedures, especially in laser desorption/ionization-mass spectrometry (LDI-MS) for analyzing a range of analytes, which include peptides, proteins, metabolites, and polymers. Matrix-oriented LDI-MS techniques are very well established, with weak organic acids as energy-absorbing substances. Carbon materials, such as nano-tubes and fullerenes are being successfully applied in the small-mass range, where routine matrices have strong background signals. In addition, the role of carbon nano-materials is very well established in the fractionation and purification fields. Modified diamond powder and surfaces are utilized in binding peptides and proteins from complex biological fluids and analyzed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). Polylysine-coated diamond is used for solid-phase extraction to pre-concentrate DNA oligonucleotides. Graphite is useful for desalting, pre-concentration, and as energy-absorbing material (matrix) in desorption/ionization. Carbon nano-tubes in their different derivatized forms are used as matrix materials for the analysis of a range of analytes, such as carbohydrates, amino acids, peptides, proteins, and some environmental samples by LDI-MS. Fullerenes are modified in different ways to bind serum entities analyzed through MALDI/TOF-MS and are subsequently utilized in their identifications. In addition, the fullerenes are a promising matrix in LDI-MS, but improvements are needed.

Silica particles encapsulated poly(styrene-divinylbenzene) monolithic stationary phases for micro-high performance liquid chromatography.

In the paper we demonstrate a new approach for the preparation and application of continuous silica bed columns that involve encapsulation (entrapment) of functionalized silica microparticles, which can be used as packing material in micro high performance liquid chromatography (micro-HPLC) and capillary electrochromatography (CEC). Like traditional packed columns, these capillaries possess characterized silica particles that offer high phase ratio and narrow pore size distribution leading to high retention and separation efficiency, respectively. More importantly, immobilization of the microparticles stabilizes the separation bed and eliminates the need for retaining frits. The developed capillary columns were fabricated in exactly the same way as a packed capillary column (slurry packing) but with an additional entrapment step. This immobilization of the packed bed was achieved by in situ polymerization of styrene and divinylbenzene in presence of decanol as a porogen and azobisisobutyronitrile as thermal initiator. Silica particles with different particle sizes and pore sizes ranging from 60 to 4000 A were studied. In addition different modified silica was used, including C-18 reversed phase, anion exchange and chiral stationary phases. Efficient separation of polyphenolic compounds, peptides, proteins and even DNA mutation were achieved using the developed technique depending on the properties of the silica particles used (particles pore size). For example, using 3 microm ProntoSIL C-18 particles with 300 A pore size, separation efficiencies in the range of 120,000-200,000 plates/m were obtained for protein separation, in a 6 cm x 200 microm i.d. capillary column. Using encapsulated silica C-18 with 1000 A pore size, separation of DNA homo and hetero duplexes were achieved under denaturing HPLC conditions for mutation detection. In addition, nucleotides were separated using anion exchange material encapsulated with poly(styrene-divinylbenzene) (PS/DVB), which indicated that the chromatographic properties of the silica packing material were still active after polymerization. The prepared capillary columns were found to be stable and could easily be operated continuously up to a pressure of 350 bar without column damage and capillary can be cut to any desired length.

Characterisation and evaluation of metal-loaded iminodiacetic acid-silica of different porosity for the selective enrichment of phosphopeptides.

Silica particles of different porosity were functionalised with iminodiacetic acid (IDA) and loaded with Fe(III) to yield immobilised metal affinity chromatography stationary phases (Fe(III)-IDA-silica) for phosphopeptide enrichment. The elution step of bound phosphopeptides was optimised with a 32P radioactive labelled peptide by a comprehensive study. Several elution systems, including phosphate buffers of different pH and concentration and ethylenediaminetetraacetic acid solutions were employed. Furthermore the effect of support porosity on elution behaviour was investigated. Under best conditions recoveries higher than 90% were achieved. A solid-phase extraction (SPE) protocol was developed for fractionation of phosphorylated and non-phosphorylated peptides and desalting of the fractions which is essential for subsequent mass spectrometric analysis by the combination of Fe(III)-IDA-silica and C18-silica particles. The pH of the loading buffer was found to be a critical parameter for the efficiency of the SPE protocol. As tryptic digests of alpha-lactalbumin, lysozyme and ribonuclease A mixed with three synthetic phosphopeptides were fractionated, pH 2.5 provided minimal proportion of unspecific bound peptides when comparing the fractions after mu-LC-electrospray ionization MS separation. The effect of a sample derivatisation reaction (methylation) on the efficiency of phosphopeptide enrichment was further investigated. Blocking carboxylate groups by methyl ester formation totally prevented unspecific interaction with the immobilised Fe(III) ions, but generated partially methylated phosphopeptides that increased the complexity of the phosphorylated fraction.

High-performance liquid chromatography for routine analysis of hepatitis C virus cDNA/PCR products.

Ion-pair reversed-phase high-performance liquid chromatography on alkylated nonporous polystyrene-divinylbenzene particles with a mean diameter of 2.1 microns was used to analyze PCR products according to their chain length within a few minutes. The simple and reliable procedure allows the simultaneous separation and isolation of DNA fragments differing in chain length by 1%-5% up to a size of 500 base pairs with recovery rates exceeding 97%. A greater than 70-fold increase in sensitivity could be achieved through the use of a fluorescein-labeled primer, which allowed the determination of a 127-bp hepatitis C virus cDNA/PCR product with a lower mass detection limit of 2 fmol. Calibration curves showed excellent linearity over a range of at least 4 magnitudes. Finally, the stationary phase allowed the routine analysis of hundreds of PCR products with high reproducibility of both retention times and peak areas.

Recent developments in polymer-based sorbents for solid-phase extraction.

A review with 136 references on the principles and recent developments in the solid-phase extraction based on polymer sorbents is presented. New polymer-based materials, chromatographic modes, experimental configurations are described and their advantages for a rapid sample preparation of certain classes of compounds with different functional groups are discussed and compared to silica-based sorbents.

Determination of airborne, volatile amines from polyurethane foams by sorption onto a high-capacity cation-exchange resin based on poly(succinic acid).

A high-capacity carboxylic acid-functionalized resin prepared by ring-opening metathesis polymerization based on cross-linked endo,endo-poly(norborn-2-ene-5,6-dicarboxylic acid) was used for the sampling of volatile, airborne amines from polyurethane (PU) foams. Six tertiary amines which represent commonly used promotors for the formation of PUs from diisocyanates and polyols, namely pentamethyldiethylenetriamine, diazabicyclooctane, N-methylmorpholine, N-ethylmorphine, 1,4-dimethylpiperazine and N,N-dimethylethanolamine, were sorbed onto the new resin. The sorption behavior of the new material was investigated in terms of loading capacities, the influence of concentration, flow-rate as well as of the amount of resin. Breakthrough curves were recorded from each single component as well as of mixtures thereof. Finally, the resin was used for the sampling of amines evaporating from PU foams applied in buildings. Further information about time dependent concentration profiles were obtained using a combination of GC-MS and Fourier transform IR spectroscopy.

Fast analysis of flavonoids in plant extracts by liquid chromatography-ultraviolet absorbance detection on poly(carboxylic acid)-coated silica and electrospray ionization tandem mass spectrometric detection.

A highly hydrophilic poly(7-oxonorbornene-5,6-dicarboxylic acid-block-norbornene) [=poly-(ONDCA-b-NBE)]-coated silica was investigated for the liquid chromatographic (LC) determination of flavonoids in plant extracts of complex biological origin using UV absorbance and mass spectrometric (MS) detection. Compared to the most commonly used octadecyl derivatized silica this sorbent allowed fast separations even at extreme pH values. Furthermore, UV absorbance and MS detection were evaluated. As we found, UV detection at 254 nm allows the determination of flavonoids down to the ng range with a linearity of R2>0.9906. For the more selective characterization the validated LC system was coupled to a quadrupole ion trap mass spectrometer via an electrospray ionization (ESI) interface. MS detection showed high linearity (R2>0.9904) for all investigated flavonoids. Due to the relatively high flow-rate of 1 ml/min the limits of detection were found in the lower-microg range. Collision induced dissociation was applied to obtain characteristic fragmentation fingerprints. Finally, the validated LC-ESI-MS-MS method demonstrated that this poly-(ONDCA-b-NBE) stationary phase allows fast characterization and quantitation in onion, elderflower blossom, lime blossom, St. John's Wort and red wine.

Isolation and characterization of methoxylated flavones in the flowers of Primula veris by liquid chromatography and mass spectrometry.

Characterization of six flavones, which were named substances G1, G2, G3, G4, G5 and G6 according to their R(F) values in normal-phase thin-layer chromatography, is reported. The pure flavones were purified after maceration with methanol by normal-phase solid-phase extraction, normal-phase medium-pressure liquid chromatography, normal-phase preparative thin-layer chromatography and preparative reversed-phase high-performance liquid chromatography (RP-HPLC). The collected fractions of several isolation steps were analyzed by normal-phase (NP) and RP-HPLC. Detection and identification of the substances G was accomplished by UV detection at 213-216 nm, diode array UV detection, or fluorescence detection (lambda(ex)=330 nm; lambda(cm)=440 nm). The molecular mass, the elementary composition, and the structure of the six components was determined by electron-impact high-resolution mass spectrometry (EI-HRMS). Substance G4 was identified as 3',4',5'-trimethoxyflavone. The substances G1-G6 were shown to be mono-, di- tri- and pentamethoxyflavones. HPLC-electrospray ionization tandem mass spectrometry (ESI-MS-MS) of the flavones was carried out employing a 150x2 mm I.D. column packed with a 3 microm/100 A octadecylsilica stationary phase and a mobile phase comprising 1.0% acetic acid in water-acetonitrile (50:50). Comparative RP-HPLC-ESI-MS of the raw methanol extract and the isolated substances G1-G6 proved that the isolated compounds were pure and were not artifacts. Finally, RP-HPLC-ESI-MS-MS was used to identify substances G1-G6 in phytopharmaceutical drugs.