ARCHAEOMETRY, Issue 4 2009
C. CARDELL
The pigments, binders and execution techniques used by the Nasrids (1238,1492) to polychrome carpentry in the Hall of the Mexuar Palace at the Alhambra (Granada, Spain) were studied using optical microscopy, scanning electron microscopy with EDX analysis, selective staining techniques and gas chromatography , mass spectrometry. This pioneering investigation presents the first results of a research project devoted to filling gaps in the knowledge of Nasrid art, traditionally approached by stylistic studies. Moreover, it is essential for the polychromy conservation of the studied artworks, and will help to clarify historical and painting uncertainties in the Alhambra monument. The palette consists of a limited range of colours: white (lead-base pigment), red (cinnabar and red lead), blue (lapis lazuli), black (carbon-based) and false gold (golden tin). Tempera grassa was the painting technique identified. Two types of grounds were used: (i) gypsum in calligraphy decoration for the false gold technique, and (ii) synthetic minium in geometric drawings in carpentry. Organic insulating layers of linseed oil were used between paint strata. Artists applied synthetic minium to protect the wood (Juglans regia and conifer) against attack by xylophages. To lighten the surface darkened by this ground layer, powdered tin was added to achieve a metallic lustre. [source]

Direct injection horse-urine analysis for the quantification and confirmation of threshold substances for doping control.

DRUG TESTING AND ANALYSIS, Issue 8 2009

Abstract Levodopa and dopamine have been abused as performance-altering substances in horse racing. Urinary 3-methoxytyramine is used as an indicator of dopaminergic manipulation resulting from dopamine or levodopa administration and is prohibited with a urinary threshold of 4 µg mL,1 (free and conjugated). A simple liquid chromatographic (LC)/mass spectrometric (MS) (LCMS) method was developed and validated for the quantification and identification of 3-methoxytyramine in equine urine. Sample preparation involved enzymatic hydrolysis and protein precipitation. Hydrophilic interaction liquid chromatography (HILIC) was selected as a separation technique that allows effective retention of polar substances like 3-methoxytyramine and efficient separation from matrix compounds. Electrospray ionization (ESI) in positive mode with product ion scan mode was chosen for the detection of the analytes. Quantification of 3-methoxytyramine was performed with fragmentation at low collision energy, resulting in one product ion, while a second run at high collision energy was performed for confirmation (at least three abundant ions). Studies on matrix effects showed ion suppression depending on the horse urine used. To overcome the variability of the results originating from the matrix effects, isotopic labelled internal standard was used and linear regression calibration methodology was applied for the quantitative determination of the analyte. The tested linear range was 1,20 µg mL,1. The relative standard deviations of intra- and inter- assay analysis of 3-methoxytyramine in horse urine were lower than 4.2% and 3.2%, respectively. Overall accuracy (relative percentage error) was less than 6.2%. The method was applied to case samples, demonstrating simplicity, accuracy and selectivity. Copyright © 2009 John Wiley & Sons, Ltd. [source]

Electrocatalytic Oxidation of Sulfur Containing Amino Acids at Renewable Ni-Powder Doped Carbon Ceramic Electrode: Application to Amperometric Detection L -Cystine, L -Cysteine and L -Methionine

ELECTROANALYSIS, Issue 21 2006
Abdollah Salimi
Abstract A sol-gel technique was used here to prepare a renewable carbon ceramic electrode modified with nickel powder. Cyclic voltammograms of the resulting modified electrode show stable and a well defined redox couple due to Ni(II)/Ni(III) system with surface confined characteristics. The modified electrode shows excellent catalytic activity toward L -cystine, L -cysteine and L -methionine oxidation at reduced overpotential in alkaline solutions. In addition the antifouling properties at the modified electrode toward the above analytes and their oxidation products increases the reproducibility of results. L -cystine, L -cysteine and L -methionine were determined chronoamperometricaly at the surface of this modified electrode at pH range 9,13. Under the optimized conditions the calibration curves are linear in the concentration range 1,450,,M, 2,90,,M and 0.2,75,,M for L -cystine, L -methionine and L -cysteine determination, respectively. The detection limit and sensitivity were 0.64,,M, 3.8,nA/ ,M for L -cystine, 2,,M, 5.6,nA/ ,M for L -methionine and 0.2,,M and 8.1,nA/,M for L -cysteine. The advantageous of this modified electrode is high response, good stability and reproducibility, excellent catalytic activity for oxidation inert molecules at reduced overpotential and possibility of regeneration of the electrode surface by potential cycling for 5,minutes. Furthermore, the modified electrode has been prepared without using specific reagents. This sensor can be used as an amperometric detector for disulfides detection in chromatographic or flow systems. [source]

Determination of the chiral and achiral related substances of methotrexate by cyclodextrin-modified micellar electrokinetic chromatography

ELECTROPHORESIS, Issue 16 2004
Roberto Gotti
Abstract A cyclodextrin-modified micellar electrokinetic chromatographic (CD-MEKC) method for the determination of the most important potential impurities of methotrexate (MTX): 2,4-diamino-6-(hydroxymethyl)pteridine, aminopterine hydrate, 4-[N -(2-amino-4-hydroxy-6-pteridinylmethyl)- N -methylamino] benzoic acid, 4-[N -(2,4-diamino-6-pteridinylmethyl)- N -methylamino] benzoic acid, and the distomer D -MTX is presented. The MEKC separation of these compounds was optimized by applying a step-by-step approach. The addition of ,-CD to a conventional MEKC system, based on sodium dodecyl sulfate (SDS) as surfactant, showed to be essential for the enantioresolution of racemic MTX as well as for the separation of the achiral impurities. To achieve high-resolution factor between the peaks adjacent to the main component (L -MTX), as required in the analysis of related impurities, the separation conditions were stressed; in particular, the addition of methanol to the CD-MEKC system resulted in a very effective choice. Under the optimized final conditions (100 mM SDS and 45 mM ,-CD in a mixture of 50 mM borate buffer, pH 9.30-methanol (75:25 v/v)), the method was validated showing a general adequate accuracy (93,106% recovery) in the determination of L -MTX related substances at the impurity level of 0.12% w/w with a relative standard deviation (RSD)% lower than 8% (n = 4). The method was successfully applied to the analysis of pharmaceuticals (tablets and injections) which showed to contain the distomer D -MTX as major impurity and aminopterine hydrate as a further related substance in the commercial tablets. [source]

Comprehensive proteome analysis by chromatographic protein prefractionation

ELECTROPHORESIS, Issue 7-8 2004
Pierre Lescuyer
Abstract Protein copy number is distributed from 7 to 8 orders of magnitude in cells and probably up to 12 orders of magnitude in plasma. Classical silver-stained two-dimensional electrophoresis (2-DE) can only display up to four orders of magnitude. This is a major drawback since it is assumed that most of the regulatory proteins are low-abundance gene products. It is thus clear that the separation of low copy number proteins in amounts sufficient for postseparation analysis is an important issue in proteome studies to complete the comprehensive description of the proteome of any given cell type. The visualization of a polypeptide on a 2-DE gel will depend on the copy number, on the quantity loaded onto the gel and on the method of detection. As the amount of protein that can be loaded onto a gel is limited, one efficient solution is to fractionate the sample prior to 2-DE analysis. Several approaches exist including subcellular fractionation, affinity purification and chromatographic and electrophoretic protein prefractionation. The chromatographic step adds a new dimension in the protein separation using specific protein properties. It allows proteins to be adsorbed to a surface and eluted differentially under certain conditions. This review article presents studies combining chromatography-based methods to 2-DE analysis and draws general conclusions on this strategy. [source]

Signal denoising and baseline correction by discrete wavelet transform for microchip capillary electrophoresis

ELECTROPHORESIS, Issue 18 2003
Bi-Feng Liu
Abstract Signal denoising and baseline correction using discrete wavelet transform (DWT) are described for microchip capillary electrophoresis (MCE). DWT was performed on an electropherogram describing a separation of nine tetramethylrohodamine-5-isothiocyanate labeled amino acids, following MCE with laser-induced fluorescence detection, using Daubechies 5 wavelet at a decomposition level of 6. The denoising efficiency was compared with, and proved to be superior to, other commonly used denoising techniques such as Fourier transform, Savitzky-Golay smoothing and moving average, in terms of noise removal and peak preservation by directly visual inspection. Novel strategies for baseline correction were proposed, with a special interest in baseline drift that frequently occurred in chromatographic and electrophoretic separations. [source]

Production of Taxol fromPhyllosticta spinarum, an endophytic fungus ofCupressus sp.

ENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 4 2008
R. Senthil Kumaran
Abstract Taxol production during the cultivation on a modified liquid and potato dextrose broth medium was indicated for the first time to occur in Phyllosticta spinarum, an endophytic fungus isolated from the needles of Cupressus sp. The presence of taxol in the fungal culture filtrate was confirmed by chromatographic and spectroscopic methods of analysis. The amount of taxol produced by this fungus was quantified by high performance liquid chromatography. The maximum amount of taxol production was obtained in this fungus when grown on M1D medium (235,,g/L) followed by PDB medium (125,,g/L). The results indicate that P.,spinarum is an excellent candidate for taxol production. The production rate was 4.7,×,103 -fold higher than that found in the culture broth of an earlier reported fungus, Taxomyces andreanae. The fungal taxol extracted also showed a strong cytotoxic activity in the in vitro culture of human cancer cells tested in an apoptotic assay. [source]

Ultra-trace analysis of multiple endocrine-disrupting chemicals in municipal and bleached kraft mill effluents using gas chromatography,high-resolution mass spectrometry

ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 2 2008
Michael G. Ikonomou
Abstract A comprehensive gas chromatographic,high-resolution mass spectrometric (GC-HRMS),based method was developed that permitted the simultaneous determination of 30 estrogenic endocrine-disrupting chemicals (EDCs) and related compounds, including surfactants, biogenic and synthetic steroids, fecal sterols, phytoestrogens, and plasticizers, in wastewater. Features of the method include low sample volume (,40 ml), optimized Florisil® cleanup to minimize matrix interferences and optimized analyte derivatization to improve sensitivity via GC-HRMS. Detection limits were in the low- to mid-ng/L range, and recoveries were greater than 60% for most target analytes. This new method allows for high throughput analysis of many organic wastewater contaminants in a complex matrix with relative standard deviation of less than 15% for most measurable compounds. The applicability of the method was demonstrated by examining wastewater samples from different origins. Compounds such as di(2-ethylhex-yl)phthalate, cholesterol, cholestanol, and other cholesterol derivatives were measured in much higher concentrations in untreated sewage and were reduced substantially in concentration by the treatment process. However, steroidal compounds, particularly estrone (E1), 17,-estradiol (E2), and estriol (E3), as well as plant sterols (except stigmastanol), were greater in the treated municipal wastewater versus the untreated effluent. Plant and fungi sterols, stigmastanol and ergosterol, were found largely associated with bleached kraft mill effluent (BKME) as compared to the municipal effluents. [source]

Comparative analysis of triacylglycerols from Olea europaea L. fruits using HPLC and MALDI-TOFMS

EUROPEAN JOURNAL OF LIPID SCIENCE AND TECHNOLOGY, Issue 5 2010
Faouzi Sakouhi
Abstract MALDI-TOFMS and HPLC are two analytical methods that were used to characterize triacylglycerols (TAG) of the Meski, Sayali, and Picholine Tunisian olive varieties. The HPLC chromatograms of the oils showed the presence of 15 TAG species, among which triolein (OOO) was the most abundant (21,48%). In the Sayali cultivar, OOO was the predominant TAG species followed by POO and LOO. However, the minor TAG molecules were represented by LnLO and LnLP. MALDI mass spectra produced sodiated ([M,+,Na]+) and potassiated ([M,+,K]+) TAG molecules; only the major TAG were potassiated [OOO,+,K] ([OOO,+,K]+, [POO,+,K]+, and [LOO,+,K]+). In contrast to the HPLC chromatograms, the MALDI mass spectra showed 13 peaks of TAG. The major peak was detected at m/z,907, which corresponds to OOO with an Na+ adduct. The results from both HPLC and MALDI techniques predict the fatty acid composition and their percentages for each olive variety. Practical applications: TAG are the main components in vegetable oils. These biomolecules determine the physical, chemical, and nutritional properties of the oils. The nutritional benefits of TAG are related to DAG (moderate plasma lipid level) and esterified FA, which are intermediate biosynthetic molecules of TAG. TAG analysis is necessary to discriminate between oils of different origin, since some oils have similar FA profiles. Olive products, oils, and table olives, are the main diet sources of TAG in the Mediterranean countries. In this work, chromatographic and spectrometric methods were used for TAG analysis and characterization of Tunisian olive varieties. [source]

Comparative study on the antimicrobial activities of different sandalwood essential oils of various origin

FLAVOUR AND FRAGRANCE JOURNAL, Issue 3 2006
Leopold Jirovetz
Abstract In total, eight samples of different sandalwoods [Amyris balsamifera L., Santalum album L. and Santalum spicatum (R.Br.) A.DC.] and a mixture of , - and , -santalols, as well as eugenol as reference compound, were tested by an agar dilution and agar diffusion method for their antimicrobial activities against the yeast Candida albicans, the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae. The main compounds of each essential oil were investigated by gas chromatographic,spectroscopic (GC-FID and GC,MS) and ,olfactory methods to obtain information about the inßuence of these volatiles on the observed antimicrobial effects. For the santalol mixture, as well as for one S. album and one S. spicatum sample with moderate concentrations of santalols, antimicrobial activity was found against all the strains used. The A. balsamifera sample, containing only a small quantity of , -santalol and nearly no , -santalol, showed high effects only against Klebsiella pneumoniae, while against the other strains weak or no activity was observed. Therefore, santalols in medium and/or high concentrations in sandalwood oils show a significant inßuence on antimicrobial potential in such natural products. Copyright © 2006 John Wiley & Sons, Ltd. [source]

Chemical composition and olfactory characterization of essential oils of fruits and seeds of African pear (Dacryodes edulis (G. Don) H. J. Lam) from Cameroon

FLAVOUR AND FRAGRANCE JOURNAL, Issue 2 2005
Leopold Jirovetz
Abstract The composition of the essential oil of Dacryodes edulis (G. Don) H. J. Lam (Burseraceae) fruits and seeds from Cameroon were investigated by gas chromatographic,spectroscopic (GC,FID and GC,MS) and olfactory methods to identify those volatiles responsible for the characteristic aroma of this commonly known African pear. Monoterpenes, such as , -pinene (fruits/seeds: 22.3/21.5%), , -pinene (13.7/19.7%), limonene (7.2/27.5%) and , -phellandrene (10.8/12.1%) were found to be main compounds of these essential oils. A correlation of the identi,ed constituents of the two essential oils of African pear from Cameroon with their single odour impressions is also given. Copyright © 2004 John Wiley & Sons, Ltd. [source]

Classification of cancer types by measuring variants of host response proteins using SELDI serum assays

INTERNATIONAL JOURNAL OF CANCER, Issue 5 2005
Eric T. Fung
Abstract Protein expression profiling has been increasingly used to discover and characterize biomarkers that can be used for diagnostic, prognostic or therapeutic purposes. Most proteomic studies published to date have identified relatively abundant host response proteins as candidate biomarkers, which are often dismissed because of an apparent lack of specificity. We demonstrate that 2 host response proteins previously identified as candidate markers for early stage ovarian cancer, transthyretin and inter-alpha trypsin inhibitor heavy chain 4 (ITIH4), are posttranslationally modified. These modifications include proteolytic truncation, cysteinylation and glutathionylation. Assays using Surface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry (SELDI-TOF-MS) may provide a means to confer specificity to these proteins because of their ability to detect and quantitate multiple posttranslationally modified forms of these proteins in a single assay. Quantitative measurements of these modifications using chromatographic and antibody-based ProteinChip® array assays reveal that these posttranslational modifications occur to different extents in different cancers and that multivariate analysis permits the derivation of algorithms to improve the classification of these cancers. We have termed this process host response protein amplification cascade (HRPAC), since the process of synthesis, posttranslational modification and metabolism of host response proteins amplifies the signal of potentially low-abundant biologically active disease markers such as enzymes. © 2005 Wiley-Liss, Inc. [source]

Determination of average carbon number of petroleum waxes by X-ray diffraction

JOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 5 2008
Sanat Kumar
Eight petroleum waxes, both paraffin as well as microcrystalline, have been analysed by X-ray diffraction. The average carbon number has been estimated by the long-range ordering observed in the diffractograms of these waxes. The average carbon number has also been determined following the standard gas chromatographic (GC) method. The results obtained by X-ray diffractometry compare well with those obtained by the GC method. The former method also permits determination of the average carbon number of high melting point waxes, which is otherwise difficult using GC. [source]

Preparation of normal-phase HPLC stationary phase based on monodisperse hydrophilic polymeric beads and their application

JOURNAL OF APPLIED POLYMER SCIENCE, Issue 4 2007
Bolin Gong
Abstract The monodisperse, 5.0 ,m hydrophilic macroporous poly(glycidymethacrylate- co -ethylenedimethacrylate) beads were first prepared based on monosized linear poly(glycidylmethacrylate) beads as seed by using a single-step swelling and polymerization method. The seed beads prepared by dispersion polymerization exhibited good absorption of the monomer phase. The pore size distribution of the beads was evaluated by mercury instrusion method. The surface area was calculated from the BET isotherms of nitrogen adsorption and desorption. The beads were modified to be a normal-phase liquid chromatographic (NPLC) stationary phase for high performance liquid chromatography (HPLC) in the following steps. First, the beads were completely hydrolyzed. Second, hydrolyzed particles were reacted with epichlorihydrin followed by another hydrolysis of the newly introduced epoxide groups. The retention properties of the NPLC stationary phase were easily modulated by changes in the composition of the mobile phase. The performance of theses beads was demonstrated with the separation of a variety of polar compounds. The satisfactory results were obtained. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007 [source]

High-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry for the determination of flocoumafen and brodifacoum in whole blood

JOURNAL OF APPLIED TOXICOLOGY, Issue 1 2007
Mi-cong Jin
Abstract A high-performance liquid chromatographic,tandem mass spectrometric (HPLC,MS,MS) assay was developed and validated to determine quantitatively flocoumafen and brodifacoum in whole blood using warfarin as an internal standard (IS). Liquid,liquid extraction, using ethyl acetate, was used to isolate flocoumafen, brodifacoum and the IS from the biological matrix. Detection was performed on a mass spectrometer by negative electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode. The calibration curves were linear (r2 > 0.998) in the concentration range of 0.1,100.0 ng ml,1 with a lower limit of quantification of 0.05 ng ml,1 for flocoumafen, and 0.1 ng ml,1 for brodifacoum in whole blood. Intra-day and inter-day relative standard deviations (RSDs) were less than 8.0% and 10.8%, respectively. Recoveries of flocoumafen and brodifacoum ranged from 78.0% to 83.7%. This assay can be used to determine trace flocoumafen and brodifacoum in whole blood to investigate suspected poisoning of human and animals. Copyright © 2006 John Wiley & Sons, Ltd. [source]

Confocal imaging of chromatographic fouling under flow conditions

JOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 10 2007
Sun Chau Siu
Abstract BACKGROUND: The fouling impact of selected fouling species was assessed by utilising confocal scanning laser microscopy (CSLM) to image a packed chromatographic bed during operation. A custom-made flow cell was packed with Q Sepharose FF and loaded with partially clarified E. coli homogenate. Selective, multicoloured fluorescent dyes were used to label a bovine serum albumin (BSA) test protein (Cy5.5), dsDNA (PicoGreen) and host cell proteins (HCPs) (Cy3). The fouling caused by the various fluorescently labelled components was visualised as a result of the fluorescence emitted by the PicoGreen-labelled dsDNA and the Cy3-labelled protein in the foulant stream, and by testing the adsorptive capacity of a test protein (BSA) onto the resin prior to and post-fouling as well as following the application of a common CIP procedure. RESULTS: Values for the effective diffusivity of BSA (De) were derived from the confocal images and the fouling impact was assessed by comparing De values obtained from different fouling scenarios. Under the most extreme conditions examined, fouling caused a 20% reduction in capacity compared to a fresh bed. BSA diffusivity did not appear to be affected by the fouling conditions studied. Sequential CIP using 15 CVs of 1 mol L,1 NaCl then 15 CVs of 1 mol L,1 NaOH was shown to be effective in removing nucleic acids and HCPs. Subsequent BSA adsorption showed that the CIP regime successfully restored the column capacity to its original value. In contrast, 15 CVs of 1 mol L,1 NaCl were ineffective in removing dsDNA but substantially removed HCPs. CONCLUSION: CSLM was demonstrated to be a useful tool for visualising fouling mechanisms. Comparing the results obtained by this technique using different modes of chromatographic operation provided insights into the fouling characteristics of finite baths versus packed beds. Copyright © 2007 Society of Chemical Industry [source]

A simple and rapid high-performance liquid chromatographic (HPLC) method for 5-fluorouracil (5-FU) assay in plasma and possible detection of patients with impaired dihydropyrimidine dehydrogenase (DPD) activity

JOURNAL OF CLINICAL PHARMACY & THERAPEUTICS, Issue 4 2004
J. Ciccolini PharmD PhD
Summary Background:, Dihydropyrimidine dehydrogenase (DPD) gene polymorphism may lead to severe toxicity with 5-fluorouracil (5-FU), a major anticancer drug extensively used in clinical oncology. Drug monitoring combined with early detection of patients at risk would enable timely dose adaptation so as to maintain drug concentrations within a therapeutic window. However, the best method to identify such patients remains to be determined. Objective:, The aim of this study was to develop a rapid and simple high-performance liquid chromatographic (HPLC) method for estimating uracil/dihydrouracil (U/UH2) ratio in plasma, as an index of DPD status, and for assaying 5-FU as part of drug level monitoring. Method:, Assay of 5-FU, and U/UH2 detection were performed on a HPLC system equipped with UV detector. Analytes were separated at room temperature using a 5 ,m particles, 25 cm RP-18 X-Terra column. The mobile-phase consisted of a KH2PO4 salt solution (0·05 m) + 0·1% triethylamine (TEA) pumped at 0·4 mL/min. Detection of 5-FU and 5-bromouracil were performed at 254 nm; U and UH2 elution was monitored at 210 nm. Results:, The method was sensitive and specific for assaying 5-FU within the 5,500 ng/mL concentration range, which covers exposure levels currently met in clinical practice. The method was simple, and relatively cheap, and rapid, with an analytical run time of about 30 min. Data from a patient with 5-FU toxicity suggest that the method was capable of identifying DPD metabolic phenotype in cancer patients, based on measurement of plasma U/UH2 ratio. Conclusion:, The method described should be suitable both for detecting patients at high risk of 5-FU toxicity, and for drug level monitoring during chemotherapy. [source]

Simple model to predict gel formation in olefin-diene copolymerizations catalyzed by constrained-geometry complexes

AICHE JOURNAL, Issue 5 2010
Job D. Guzmán
Abstract We have developed an analytical model to predict the onset of gel formation in ethylene/1-octene/1,9-decadiene terpolymerizations using constrained-geometry catalysts. The model relies on three kinetic parameters to characterize the catalyst response. Polymer resins have been synthesized in a continuous stirred-tank reactor to determine the model parameters, and to validate the model predictions for polymer properties and for the onset of gel formation and reactor fouling. The experimental results indicate that the free double bonds in 1,9-decadiene are as reactive as those found in 1-octene, and that the reactivity of 1,9-decadiene double bonds decreases after the 1,9-decadiene molecules become part of a polymer chain. The model predictions of polymer properties agree well with chromatographic, density, and mass-balance data. Moreover, the model was successful in preventing unintended reactor fouling during the duration of the experimental campaign. © 2009 American Institute of Chemical Engineers AIChE J, 2010 [source]

Gas phase isomeric differentiation of oleanolic and ursolic acids associated with heptakis-(2,6-di- O -methyl)-,-cyclodextrin by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 4 2010
Zhan Yu
Abstract Oleanolic acid (OA) and ursolic acid (UA) are isomeric triterpenoid compounds with similar pharmaceutical properties. Usually, modern chromatographic and electrophoretic methods are widely utilized to differentiate these two compounds. Compared with mass spectrometric (MS) methods, these modern separation methods are both time- and sample-consuming. Herein, we present a new method for structural differentiation of OA and UA by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) with the association of heptakis-(2,6-di- O -methyl)-,-cyclodextrin (DM-,-CD). Exact MS and tandem MS (MS/MS) data showed that there is no perceptible difference between OA and UA, as well as their ,-cyclodextrin and ,-cyclodextrin complexes. However, there is a remarkable difference in MS/MS spectra of DM-,-CD complexes of OA and UA. The peak corresponding to the neutral loss of a formic acid and a water molecule could only be observed in the MS/MS spectrum of the complex of DM-,-CD : OA. Molecular modeling calculations were also employed to further investigate the structural differences of DM-,-CD : OA and DM-,-CD : UA complexes. Therefore, by employing DM-,-CD as a reference reagent, OA and UA could be differentiated with purely MS method. Copyright © 2010 John Wiley & Sons, Ltd. [source]

Development and validation of a liquid chromatographic/tandem mass spectrometric assay for the quantitation of nucleoside HIV reverse transcriptase inhibitors in biological matrices

JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 1 2005
Séverine Compain
Abstract Besides liquid chromatographic (LC)/UV methods adapted to therapeutic drug monitoring, there is still a need for more powerful techniques that can be used for pharmacological research and clinical purposes. We developed an LC method coupled with tandem mass spectrometry (MS/MS) to separate, detect and quantify with high sensitivity the nucleoside analogues used in multitherapies (zidovudine, stavudine, zalcitabine, didanosine, lamivudine and abacavir) in plasma and in the intracellular medium. We worked on two essential issues: (i) the need to use two ionization modes in order to achieve the best sensitivity, which leads to the optimization of the chromatographic separation of drugs detected in the positive ionization mode and drugs detected in the negative ionization mode, and (ii) the need to optimize the extraction step in order to enhance sample recovery. The peripheral blood mononuclear cells were lysed in Tris buffer,MeOH. A clean-up procedure was performed by solid-phase extraction only for plasma samples. The LC separation was carried out on a Zorbax Stable Bond C18 column followed by MS/MS analysis after electrospray ionization in either the negative or positive mode. The positive ionization mode was applied at the beginning of the run to detect zalcitabine and lamivudine, then the ionization mode was changed to negative for the detection of didanosine, stavudine, internal standard and zidovudine. The calibration range for all the analytes was 0.5,200 ng ml,1. The recoveries were between 64 and 90%, with coefficients of variation (CVs) lower than 15%. The inaccuracy (bias) was ±15% with CVs always lower than 12%. The analytes were stable at room temperature and in the extraction solvent for at least 24 h, after storage at ,80 °C for 3 months, after three freeze,thaw cycles and in the injection solvent after 48 h at 4 °C. Together with the measurement of intracellular triphosphorylated metabolites thanks to the powerful plasma and intracellular assay method for intact drugs, it is possible to describe the behaviour of nucleoside analogues against HIV through plasma pharmacokinetics, cell membrane diffusion including drug transport involvement, and also the intracellular metabolism. Copyright © 2005 John Wiley & Sons, Ltd. [source]

Advances in membrane receptor screening and analysis

JOURNAL OF MOLECULAR RECOGNITION, Issue 4 2004
Matthew A. Cooper
Abstract During the last decade there has been significant progress in the development of analytical techniques for the screening of ligand binding to membranes and membrane receptors. This review focuses on developments using label-free assays that facilitate ligand,membrane,receptor screening without the need for chemical-, biological- or radiological-labelled reagents. These assays include acoustic, optical surface plasmon resonance biosensing, sedimentation (analytical ultracentrifugation), chromatographic assays, isothermal titration calorimetry and differential scanning calorimetry. The merits and applications of cell-based screening systems and of different model membrane systems, including planar supported lipid layers, bead-supported membranes and lipid micro-arrays, are discussed. Recent advances involving more established techniques including intrinsic fluorescence, FRET spectroscopy, scintillation proximity assays and automated patch clamping are presented along with applications to peripheral membrane proteins, ion channels and G protein-coupled receptors. Novel high-throughput assays for determination of drug- and protein-partitioning in membranes are also highlighted. To aid the experimenter, a brief synopsis of the techniques commonly employed to purify and reconstitute membranes and membrane receptors is included. Copyright © 2004 John Wiley & Sons, Ltd [source]

Analysis of the reverse flow chromatographic reactor

AICHE JOURNAL, Issue 9 2004
Hugo S. Caram
A reverse flow chromatographic reactor with side feed (RFCR) can be used to improve the conversion and selectivity for irreversible and reversible reaction systems beyond the values obtained from conventional steady-state reactors in the case where the reactants are more strongly adsorbed than the products. It is also simpler than conventional chromatographic, moving, and simulated moving beds and other systems that combine reaction and adsorption separation. The results of the study show the robustness and stability of the system, as well as its capacity to significantly improve conversion and selectivity for several reaction systems, including consecutive reactions. Simulation results of this mathematical model for the hydrogenation of 1,3,5-trymethylbenzene (MES) compare well with previous studies, using countercurrent moving-bed reactors and simulated countercurrent bed reactors. © 2004 American Institute of Chemical Engineers AIChE J, 50: 2266,2275, 2004 [source]

Microwave-assisted TFA cleavage of peptides from Merrifield resin

JOURNAL OF PEPTIDE SCIENCE, Issue 1 2010
Alicja Kluczyk
Abstract Microwave-assisted (MW) reactions are of special interest to the chemical community due to faster reaction times, cleaner reactions and higher product yields. The adaptation of MW to solid phase peptide synthesis resulted in spectacular syntheses of difficult peptides. In the case of Merrifield support, used frequently in synthesis of special peptides, the conditions used in product cleavage are not compatible with off-resin monitoring of the reaction progress. The application of MW irradiation in product removal from Merrifield resin using trifluoroacetic acid (TFA) was investigated using model tetrapeptides and the effects were compared with standard trifluoromethanesulphonic acid (TFMSA) cleavage using elemental analysis as well as chromatographic (HPLC) and spectroscopic (IR) methods. The deprotection of benzyloxycarbonyl and benzyl groups in synthetic bioactive peptides was analyzed using LC-MS and MS/MS experiments. In a 5 min microwave-assisted TFA reaction at low temperature, the majority of product is released from the resin, making the analytical scale MW-assisted procedure a method of choice in monitoring the reactions carried out on Merrifield resin due to the short reaction time and compatibility with HPLC and ESI-MS conditions. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd. [source]

Synthesis and characterization of water-soluble barbiturate- and thiobarbiturate-functionalized polystyrene

JOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 6 2002
S. Raja
Abstract The synthesis and characterization of barbiturate- and thiobarbiturate-functionalized polystyrene from polystyrene homopolymer by polymer-modification reactions is discussed. Polystyrene homopolymer quantitatively functionalized at the para postion with diethyl oxomalonate functionality was subjected to a condensation reaction with urea and thiourea in the presence of sodium methoxide in methanol. This reaction proceeded essentially to quantitative conversion to the barbiturate- (BAPS) and thiobarbiturate-functionalized polystyrenes (TBAPS) as estimated by 1H NMR, UV, and IR spectroscopies. Thus, several copolymers of styrene with barbiturate- and thiobarbiturate-functionalized styrene were synthesized. The detailed characterizations of quantitatively functionalized polystyrene using gel permeation chromatographic, IR, UV, and 1H NMR spectroscopic techniques as well as thermogravimetric analysis are discussed. An application of the newly synthesized polymer in removing Cu(II) ions from aqueous solution is demonstrated. This is the first report on the synthesis of BAPS and TBAPS by the polymer-modification route or otherwise. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 731,737, 2002; DOI 10.1002/pola.10154 [source]

Instrumental planar chromatographic method for determination of carbamazepine in human serum

JOURNAL OF SEPARATION SCIENCE, JSS, Issue 9 2009
Sigrid Mennickent
Abstract An instrumental planar chromatographic (HPTLC) method for quantification of carbamazepine in human serum was developed using liquid-liquid extraction with dichloromethane, fluorescence activation with perchloric acid 60%/ethanol/water (1:1:1, v/v) and fluorescence detection. Planar chromatographic separation was performed on precoated silica gel F254 HPTLC plates using a mixture of ethyl acetate/toluene/methanol/acetic acid glacial (5:4:0.5:0.5, v/v) as mobile phase. Densitometric detection was done at 366 nm. The method was validated for linearity, precision and accuracy. Linear calibration curves in the range of 3 and 20 ng/,L showed correlation coefficient of 0.998. The intra-assay and inter-assay precision, expressed as the RSD, were in the range of 0.41,1.24% (n = 3) and 2.17,3.17% (n = 9), respectively. The LOD was 0.19 ng, and the LOQ was 0.57 ng. Accuracy, calculated as percentage recovery, was between 98.98 and 101.96%, with a RSD not higher than 1.52%. The method was selective for the active principle tested. In conclusion, the method is useful for quantitative determination of carbamazepine in human serum. [source]

New ,-amino phenylalanine tetrazole ligand for immobilized metal affinity chromatography of proteins

JOURNAL OF SEPARATION SCIENCE, JSS, Issue 16-17 2008
Genhu Lei
Abstract A new chelating compound has been developed for use in the immobilized metal affinity chromatographic (IMAC) separation of proteins. The bidentate ligand, ,-amino phenylalanine tetrazole, 4, was synthesized via a five-step synthesis from N -fluorenylmethoxycarbonyl phenylalanine and then immobilized onto silica through the epoxide coupling procedure. The binding behavior of the resulting IMAC sorbent, following chelation with Zn2+ to a density of 183 ,mol Zn2+ ions/g silica, was characterized by the retention of proteins in the pH range of 5.0,8.0, and by the adsorption behavior of lysozyme with frontal chromatography at pH 6.0 and 8.0. The prepared column showed the separation ability to four test proteins and the retention time of these proteins increased with an increase in pH. From the derived isotherms, the adsorption capacity, qm, for the binding of lysozyme to immobilized Zn2+ -,-amino phenylalanine tetrazole,silica was found to be 1.21 ,mol/g at pH 6.0 and 1.20 ,mol/g sorbent at pH 8.0, respectively, whilst the dissociation constants KD at these pH values were 5.22×10,6 and 3.49×10,6 M, respectively, indicating that the lysozyme was retained more stable under alkaline conditions, although the binding capacity in terms of micromole protein per gram sorbent remained essentially unchanged. [source]

Stability-indicating assay of sodium cromoglicate in ophthalmic solution using mixed-mode hydrophilic interaction chromatography

JOURNAL OF SEPARATION SCIENCE, JSS, Issue 9 2008
Mohammed Shahid Ali
Abstract A hydrophilic interaction chromatographic (HILIC) procedure for the quantification of Sodium Cromoglicate (SCG) in ophthalmic solution is developed. Mobile phase consists of ACN and buffer, 86:14 v/v. Atlantis HILIC,Si column, 25 cm×4.6 mm, is used as stationary phase. Detection is carried out using a variable wavelength UV-Vis detector at 326 nm. Linearity range and percent recoveries for SCG were 50,400 ,g/mL and 100.44%, respectively. The SCG HILIC-UV assay was validated according to the International Conference on Harmonization guidelines. The method separates two impurities and degradation products resulting from stress environment. Influence of organic solvent, ionic strength and mobile phase pH on the retention of SCG is studied. The paper provides optimization of polar anionic solute (SCG) on unmodified silica by HILIC. Proposed method can be used as a stability-indicating assay for SGC and can be proved to be beneficial in ESI-MS for enhanced sensitivity. [source]

Gradient HPLC of antibiotics in urine, ground water, chicken muscle, hospital wastewater, and pharmaceutical samples using C-18 and RP-amide columns

JOURNAL OF SEPARATION SCIENCE, JSS, Issue 2 2008
Ashwini Kumar
Abstract A simple and highly sensitive high pressure liquid chromatographic (HPLC-UV) method has been developed for the determination of ofloxacin, lomefloxacin, cinoxacin, and nalidixic acid, in mobile phase citrate buffer (0.001 M) of pH 4.5 prepared in water (X), methanol (Y), and ACN (Z) using gradient at a flow rate of 1.0 mL/min by direct UV absorbance detection at , = 280 nm. Separation of analytes was studied on the C-18 and RP-amide columns and best results were observed on the RP-amide column with LODs (3.3×S/m) 0.89, 0.55, 0.67, and 1.41 ng/mL for ofloxacin, lomefloxacin, cinoxacin, and nalidixic acid, respectively, and better RSD than the C-18 column. The recovery of Fluoroquinolones (FQs) in urine, ground water, hospital wastewater, and chicken muscle using this method is more than 90%. The method was successfully applied to the analysis of ofloxacin, lomefloxacin, cinoxacin, and nalidixic acid in urine, ground water, pharmaceutical dosage forms, hospital wastewater, and chicken muscle. [source]

Fingerprint chromatogram analysis of Pseudostellaria heterophylla (Miq.) Pax root by high performance liquid chromatography

JOURNAL OF SEPARATION SCIENCE, JSS, Issue 14 2006
Chao Han
Abstract A simple and reliable high performance liquid chromatographic (HPLC) method has been developed and validated for the fingerprinting of extracts from the root of Pseudostellaria heterophylla (Miq.) Pax. HPLC with gradient elution was performed on an authentic reference standard of powdered P. heterophylla (Miq.) Pax root and 11 plant samples of the root were collected from different geographic locations. The HPLC chromatograms have been standardized through the selection and identification of reference peaks and the normalization of retention times and peak intensities of all the common peaks. The standardized HPLC fingerprints show high stability and reproducibility, and thus can be used effectively for the screening analysis or quality assessment of the root or its derived products. Similarity index calculations based on cosine angle values or correlation methods have been performed on the HPLC fingerprints. As a group, the fingerprints of the P. heterophylla (Miq.) Pax samples studied are highly correlated with closely similar fingerprints. Within the group, the samples can be further divided into subgroups based on hierarchical clustering analysis (HCA). Sample grouping based on HCA coincides nicely with those based on the geographical origins of the samples. The HPLC fingerprinting techniques thus have high potential in authentication or source-tracing types of applications. [source]

Rapid and sensitive determination of morphine in street opium samples by thermal desorption gas chromatography using a microfurnace pyrolyzer

JOURNAL OF SEPARATION SCIENCE, JSS, Issue 12 2004
Minemasa Hida
Abstract Thermal desorption of the alkaloids in opium samples at 300°C using a vertical microfurnace pyrolyzer was followed by their on-line gas chromatographic (GC) analysis on a large-bore glass capillary column. This method permitted rapid and sensitive determination of the content of the main alkaloid, morphine, in the small (ca. 100 ,g) opium samples with a relative standard deviation within 4% for 5 runs. The observed morphine contents of about 12 to 15 w/w% in the given opium samples were in fairly good agreement with those estimated by a conventional GC-MS method. [source]