Bioreactor and probe system for magnetic resonance microimaging and spectroscopy of chondrocytes and neocartilage

We have developed a nuclear magnetic resonance (NMR)-compatible hollow fiber chondrocyte bioreactor (HFBR), permitting the noninvasive study of neocartilage under conditions optimized for cell growth and matrix expression. The system was used to investigate the properties of neocartilage which developed from embryonic chick chondrocytes. Histologic studies performed 30 days after inoculation of the HFBR with chondrocytes showed cartilage growth units demarcated by stromal layers surrounding each fiber; the tissue itself was highly cellular with abundant proteoglycan content. Spin-density, spin-lattice, and spin-spin relaxation and magnetization transfer contrast images revealed heterogeneous tissue with NMR properties that correlated well with histologic data. It was found that the apparent free water content of the neocartilage was greater than that seen in mature cartilage, even in regions of relatively low cell density. The bioenergetic profile of the cells in culture was monitored with ³¹P-NMR spectroscopy, and the presence of phosphocreatine was clearly demonstrated. Overall metabolic stability was confirmed between days 10 and 17 after inoculation. A significant decrease in intracellular pH with time was observed during early development of the chondrocyte system. © 1997 John Wiley & Sons, Inc. Int J Imaging Syst Technol, 8, 285–292, 1997     

Analysis of poly(ethyl methacrylate)s by on-line hyphenation of liquid chromatography at the critical adsorption point and nuclear magnetic resonance spectroscopy

Liquid chromatography at the critical adsorption point (LC CAP) with on-line NMR detection (on-line LC CAP NMR) was utilized for analysis of tacticity distribution of stereo-regular poly(ethyl methacrylate)s (PEMAs). The separation of a model PEMA sample composed of four constituents with similar molar masses (Mw = (14-16) x 10(3) g mol-1) differing in their tacticity (rr triad content = 0, 33, 68, and 89%) was achieved by LC CAP with a mixed eluent composed of acetone, acetone-d6, and cyclohexane. The tacticity composition within each peak eluted from the LC CAP column was directly determined by a 750-MHz 1H NMR spectrometer that was used as a real-time detector in the continuous-flow mode. Tacticity distribution in a particular PEMA sample with mm/mr/rr = 2/45/53 and narrow molar mass distribution of Mw/Mn = 1.05 has been revealed by the LC CAP NMR technique.     

Reaction monitoring of aliphatic amines in supercritical carbon dioxide by proton nuclear magnetic resonance spectroscopy and implications for supercritical fluid chromatography

In the recent years, it has repeatedly been stated that amines react with CO2 and can therefore not be chromatographed under supercritical conditions with CO2. The aim of the present work is to elucidate the structural requirements and conditions that can lead to the reaction of an amine analyte with CO2 and, if this occurs, the structure of the formed product. The use of on-line nuclear magnetic resonance (NMR) spectroscopy with a flow probe for supercritical fluid chromatography (SFC) enables the investigation of these unstable analytes in supercritical mediums. Several alkyl-substituted secondary benzylamines and some primary aromatic amines were dissolved in supercritical CO2 and investigated by employing on-line SFC-1H NMR spectroscopy. It was found that the condition of carbamic acid formation depends on the steric properties of the substituents of the amine. A 2-isopropylamino alcohol compound, metoprolol, was also investigated with the setup. No carbamic acid could be detected with the present conditions.     

Analysis of the unresolved organic fraction in atmospheric aerosols with ultrahigh-resolution mass spectrometry and nuclear magnetic resonance spectroscopy: organosulfates as photochemical smog constituents

Complementary molecular and atomic signatures obtained from Fourier transform ion cyclotron resonance (FTICR) mass spectra and NMR spectra provided unequivocal attribution of CHO, CHNO, CHOS, and CHNOS molecular series in secondary organic aerosols (SOA) and high-resolution definition of carbon chemical environments. Sulfate esters were confirmed as major players in SOA formation and as major constituents of its water-soluble fraction (WSOC). Elevated concentrations of SO(2), sulfate, and photochemical activity were shown to increase the proportion of SOA sulfur-containing compounds. Sulfonation of CHO precursors by means of heterogeneous reactions between carbonyl derivatives and sulfuric acid in gas-phase photoreactions was proposed as a likely formation mechanism of CHOS molecules. In addition, photochemistry induced oligomerization processes of CHOS molecules. Methylesters found in methanolic extracts of a SOA subjected to strong photochemical exposure were considered secondary products derived from sulfate esters by methanolysis. The relative abundance of nitrogen-containing compounds (CHNO and CHNOS series) appeared rather dependent on local effects such as biomass burning. Extensive aliphatic branching and disruption of extended NMR spin-systems by carbonyl derivatives and other heteroatoms were the most significant structural motifs in SOA. The presence of heteroatoms in elevated oxidation states suggests a clearly different SOA formation trajectory in comparison with established terrestrial and aqueous natural organic matter.     

MS/NMR: a structure-based approach for discovering protein ligands and for drug design by coupling size exclusion chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy

A protocol is described for rapidly screening small organic molecules for their ability to bind a target protein while obtaining structure-related information as part of a structure-based drug discovery and design program. The methodology takes advantage of and combines the inherent strengths of size exclusion gel chromatography, mass spectrometry, and NMR to identify bound complexes in a relatively universal high-throughput screening approach. Size exclusion gel chromatography in the spin column format provides the high-speed separation of a protein-ligand complex from free ligands. The spin column eluent is then analyzed under denaturing conditions by electrospray ionization mass spectrometry (MS) for the presence of small molecular weight compounds formerly bound to the protein. Hits identified by MS are then individually assayed by chemical shift perturbations in a 2D 1H-15N HSQC NMR spectrum to verify specific interactions of the compound with the protein and identification of the binding site on the protein. The utility of the MS/NMR assay is demonstrated with the use of the catalytic fragment of human fibroblast collagenase (MMP-1) as a target protein and the screening of a library consisting of approximately 32 000 compounds for the identification of molecules that exhibit specific binding to the RGS4 protein.     

Hydrogel network formation revised: high-resolution magic angle spinning nuclear magnetic resonance as a powerful tool for measuring absolute hydrogel cross-link efficiencies

In the present work, high-resolution magic angle spinning (hr-MAS) NMR spectroscopy is applied as a straightforward nondestructive technique to quantify unreacted methacrylamide functionalities in cross-linked gelatin hydrogels. By adjusting several cross-linking parameters including the ultraviolet (UV) irradiation time and the photo-initiator concentration, the cross-linking degree can be easily varied. Remarkably, under all experimental conditions typically applied for hydrogel development, no more than 40% of the methacrylamide moieties present have reacted. The hr-MAS based approach to determine the cross-linking efficiency is shown to provide an innovative and more convenient alternative to the well-established classical techniques. In addition, the results obtained are in good correlation with mechanical analysis data.     

Multiresidue method for pesticides in drinking water using a graphitized carbon black cartridge extraction and liquid chromatographic analysis

A general liquid-solid extraction procedure for the isolation of pesticides from groundwater and drinking water for high-performance liquid chromatography (HPLC) is presented. This simple and rapid procedure involved passing a 2-L sample through a 250-mg graphitized carbon black (Carbopack B) cartridge at a flow rate of 150-160 mL/min. By taking advantage of the presence of positively charged active centers on the Carbopack B surface, a stepwise elution system allowed the complete separation of base-neutral pesticides from acidic ones. After partial solvent removal, the components in the two fractions were separated and quantified by gradient elution, reversed-phase HPLC with ultraviolet (UV) detection. The performance of the Carbopack cartridge was compared with that of a 500-mg C-18 bonded silica cartridge. With the Carbopack cartridge, the grand mean measurement accuracy of the 35 pesticides considered was 95%. With the C-18 cartridge, the grand mean measurement accuracy of the analytes was 76%. Compared to the C-18 cartridge, additional advantages of using a Carbopack cartridge are that the extraction procedure is about 7 times shorter, no pH adjustment of the environmental sample is necessary for trapping acidic compounds, and one cartridge instead of two suffices to extract base-neutral and acidic pesticides, making the Carbopack cartridge more adaptable than the C-18 one for field use. The detection limits by this method of all the pesticides considered were between 0.003 and 0.07 micrograms/L.     

Reversed-phase high-performance liquid chromatography combined with on-line UV diode array, FT infrared, and 1H nuclear magnetic resonance spectroscopy and time-of-flight mass spectrometry: application to a mixture of nonsteroidal antiinflammatory drugs

A prototype multiply hyphenated system has been applied to the analysis of a mixture of nonsteroidal antiinflammatory drugs separated by reversed-phase HPLC. Characterization of the model NSAIDs was achieved via a combination of diode array UV, 1H NMR, FT-IR spectroscopy, and time-of-flight mass spectrometry. This combination of spectrometers allowed the collection of UV, 1H NMR, IR, and mass spectra together with atomic composition data enabling almost complete structural characterization to be performed.     

Combined use of nuclear magnetic resonance,electron microscopy,and diffraction techniques as a probe of the uniqueness of zeolite structures: zeolites KZ-2, Theta-1, ZSM-22, and NU-10

Scanning electron microscopy, powder X-ray diffraction, and ²⁹Si MAS n.m.r. spectroscopy have been used as complementary techniques to probe the uniqueness of the crystal structures of a series of closely related zeolites. Based on the above criteria, KZ-2, Theta-1, ZSM-22, and NU-10 in their completely siliceous forms were found to have identical crystal structures although differing in their external morphologies.     

Surface-enhanced Raman scattering spectroscopy as a sensitive and selective technique for the detection of folic acid in water and human serum

Surface-enhanced Raman scattering (SERS) is shown to give linear and sensitive concentration-dependent detection of folic acid using silver nanoparticles created via ethylene-diaminetetraacetic acid (EDTA) reduction. Optical detection by SERS overcomes the primary limitation of photodissociation encountered during the application of other shorter wavelength ultraviolet (UV)/near-UV techniques such as fluorescence based microscopy. The SERS approach in water-based samples was demonstrated and optimized using several longer wavelengths of excitation (514.5, 632.8, and 785 nm). Excitation in the green (514.5 nm) was found to achieve the best balance between photodissociation and SERS efficiency. Linear concentration dependence was observed in the range of 0.018 to 1 microM. The importance of folic acid in a clinical setting and the potential applications of this technique in a biological environment are highlighted. We demonstrate the potential to transfer this technique to real biological samples by the detection of folic acid in human serum samples by SERS.     

On-line solid-phase extraction with surfactant accelerated on-column derivatization and micellar liquid chromatographic separation as a tool for the determination of biogenic amines in various food substrates

A sensitive method was developed for the determination of biogenic amines at very low levels by combining solid phase extraction (SPE) and derivatization on-line with HPLC. The on-line derivatization and SPE were performed simultaneously on a commercially available ODS guard column, which was installed instead of the filling loop on the HPLC apparatus. Resolution of the peaks and quantification was further enhanced with micellar liquid chromatography and sensitization of the benzene ring absorption at 254 nm. Detection limits of the benzoyl derivatives of biogenic amines were in the vicinity of 0.1 microg L(-)(1), which is even lower than those obtained by fluorescence detection and is unparallel to any other UV approach. The correlation coefficients of determinations were 0.9850-0.9998. The method was applied to the determination of Biogenic amines, that is, putrescine, cadaverine, agmatine, tyramine, tryptamine, phenylethylamine, spermine, spermidine and histamine in fish, chicken, and wine samples. Recovery of the proposed method ranged from 94 to 106%.     

Pressurized liquid extraction with water as a tool for chemical and toxicological screening of soil samples at army live-fire training ranges

Significant discrepancies in the results of risk assessments based on chemical and toxicity analyses of soils may arise through differences in the efficiency of the extraction or leaching methods used. A rapid technique that may be used in the screening phase of live-fire training ranges and suitable for extracting explosive residues is pressurized liquid extraction (PLE) with water. Therefore, PLE and the commonly used batch leaching method EN-124 57-2 were compared for their utility to extract specific residues from soil samples collected from the Canadian Forces Base (CFB) Petawawa, Ontario. After extraction the cytotoxicity of the samples were assessed in the L-929 growth inhibition assay. The PLE method yielded extracts suitable for direct use in the toxicity assay within 20 min as compared to 24h for the batch leaching method. Analysis of the extracts showed that the PLE water extracts tended to give higher recoveries of explosive residues and the resulting exposure concentrations were confirmed by higher cytotoxicities. Furthermore, gas chromatography-mass spectrometry analyses showed that the samples contained significant amounts of several munition-related stabilizers and plasticizers of toxicological significance in addition to the analysed explosive residues. In conclusion, PLE using water is a promising extraction technique for both chemical and toxicological screening of soil samples from areas that may be contaminated with explosive residues.     

Spray-drying as a method for microparticulate controlled release systems preparation: advantages and limits. I. Water-soluble drugs

Spray-drying was used for the preparation of paracetamol/eudragit RS or RL or ethylcellulose microspheres to verify the possibility of their use in controlled-release solid-dosage forms formulation and try to determine advantages and limits of the technique of such use. Microspheres were first characterized by scanning electron microscopy, differential scanning calorimetry, x-ray diffractometry, and in vitro dissolution studies and then used for the preparation of tablets. During this step, the compressibility of the spray-dried powders was also evaluated. In vitro dissolution studies were performed also on the tablets and their release control was accessed. Although powders were unable to slow down drug release, tablets obtained from microsphere compression showed a good capability of controlling paracetamol release when eudragit RS or ethylcellulose was used, even at low polymer amounts.     

Sputtering yields for C60 and Au3 bombardment of water ice as a function of incident kinetic energy

The total sputtering yields for water ice due to kiloelectronvolt cluster bombardment have been measured and compared to the predictions made by the mesoscale energy deposition footprint (MEDF) model. For C60 bombardment, the experimental yield varies almost linearly from 820 water molecule equivalents at an incident kinetic energy of 10 keV to 10 100 water molecule equivalents at a kinetic energy of 120 keV. For Au3 bombardment, the experimental yield varies almost linearly from 630 water molecule equivalents at an incident energy of 10 keV and rises to 1200 water molecule equivalents at 25 keV. The MEDF model is used to calculate relative yield trends with respect to incident energy using short-time molecular dynamics simulations. The results of these calculations indicate that the model can effectively predict the yield trends observed for these two clusters in experiments, although there is a consistent overestimate of the predicted induced C60 yield. It is hypothesized that this overestimate can be explained by the absence of reactions and ionization processes in the current simulations. Despite this omission, experimental yield trends can be accurately predicted using relatively small amounts of computer time. The success of the model in predicting the yield of water from ice films using a variety of energies and projectiles suggests this approach may greatly aid in the optimization of experimental configurations.     

Synthesis and characterization of Piperidine-4-carboxylic acid functionalized Fe3O4 nanoparticles as a magnetic catalyst for Knoevenagel reaction

Piperidine-4-carboxylic acid (PPCA) functionalized Fe₃O₄ nanoparticles as a novel organic–inorganic hybrid heterogeneous catalyst was fabricated and characterized by XRD, FT-IR, TGA, TEM and VSM techniques. Composition was determined as Fe₃O₄, while particles were observed to have spherical morphology. Size estimations using X-ray line profile fitting (10 nm), TEM (11 nm) and magnetization fitting (9 nm) agree well, revealing nearly single crystalline character of Fe₃O₄ nanoparticles. Magnetization measurements reveal that PPCA functionalized Fe₃O₄ NPs have superparamagnetic features, namely immeasurable coercivity and absence of saturation. Small coercivity is established at low temperatures. The catalytic activity of Fe₃O₄–PPCA was probed through one-pot synthesis of nitro alkenes through Knoevenagel reaction in CH₂Cl₂ at room temperature. The heterogeneous catalyst showed very high conversion rates (97%) and could be recovered easily and reused many times without significant loss of its catalytic activity.     

Laser-excited atomic fluorescence in a flame as a high-sensitivity detector for organomanganese and organotin compounds following separation by high-performance liquid chromatography.

The coupling of a high-performance liquid chromatograph with a sensitive and selective laser-excited atomic fluorescence spectrometry (LEAFS) detector is described. In connection with this, a study of the signal and noise characteristics of instrumentation for dispersive, nondispersive, and front surface LEAFS is reported together with a comparison of the sensitivity and selectivity achieved with high-performance liquid chromatography (HPLC)-flame LEAFS, HPLC-ultraviolet (UV), and HPLC-continuum source excited flame atomic fluorescence spectrometry (AFC) instrumentation. The HPLC-flame LEAFS instrumentation was applied to an investigation of the Mn species responsible for (methylcyclopentadienyl)manganese tricarbonyl (MMT) toxicity in rats. The detection limits for various organomanganese species by HPLC-flame LEAFS ranged from 8 to 22 pg of manganese. Recovery of these compounds from rat urine varied between 80% and 100%, with a reproducibility of between 4% and 8% relative standard deviation. Preliminary data for the HPLC-flame LEAFS determination of toxic alkyltin compounds are reported.     

Nanoalumina-supported Mn2O3 as efficient adsorbent for removal of fluoride and arsenic from water: a study from lab to field

Journal of Materials Science - Water pollution with heavy metals has been a problem for the environment and human health. The cohabitation of As(III) and fluoride in groundwater has gotten...