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Reversed-phase High-performance Liquid Chromatography (reversed-phase + high-performance_liquid_chromatography)
Selected AbstractsSimultaneous quantification of eudesmanolides and thymol derivatives from tissues of Inula helenium and I. royleana by reversed-phase high-performance liquid ChromatographyPHYTOCHEMICAL ANALYSIS, Issue 3 2006Anna Stojakowska Abstract A simple and rapid isocratic reversed-phase high-performance liquid chromatographic method for the quantification of alantolactone/isoalantolactone and three thymol derivatives in roots and root cultures of Inula helenium and I. royleana has been developed. The method could be applied to screen raw materials in search for highly productive plants and in vitro cultures. Copyright © 2006 John Wiley & Sons, Ltd. [source] Chiral separation of N -imidazole derivatives, aromatase inhibitors, by cyclodextrin-capillary zone electrophoresis.ELECTROPHORESIS, Issue 16 2004Mechanism of enantioselective recognition Abstract Baseline separation of ten new, substituted [1-(imidazo-1-yl)-1-phenylmethyl)] benzothiazolinone and benzoxazolinone derivatives with one chiral center was achieved using cyclodextrin-capillary zone electrophoresis (CD-CZE). A method for the enantiomeric resolution of these compounds was developed using neutral CDs (native ,-, ,-, ,-CDs or ,-, ,-, ,-hydroxypropyl (HP)-CDs) as chiral selectors. Operational parameters including the nature and concentration of the chiral selectors, pH, ionic strength, organic modifiers, temperature, and applied voltage were investigated. The use of neutral CDs provides enantiomeric resolution by inclusion of compounds in the CD cavity. The HP-,-CD and HP-,-CD were found to be the most effective complexing agents and allowed efficient enantiomeric resolutions. Optimal separation of N -imidazole derivatives was obtained using 50 mM phosphate buffer at pH 2.5 containing either HP-,-CD or HP-,-CD (7.5,12.5 mM) at 25°C, with an applied field of 0.50 kV·cm,1 giving resolution factors Rs superior to 1.70 with migration times of the second enantiomer less than 13 min. The same enantiomer migration order observed for all molecules can be related to a close interaction mechanism with CDs. The influence of structural features of the solutes on Rs and tm was studied. The lipophilic character (log kw) of the solutes and the apparent and averaged association constants of inclusion complexes for four compounds with the six different CDs led us to rationalize the enantioseparation mechanisms. The conclusions were corroborated with reversed-phase high-performance liquid chromatography (HPLC) on chiral stationary phases (CSPs) based on CDs. [source] Narrow-band fractionation of proteins from whole cell lysates using isoelectric membrane focusing and nonporous reversed-phase separationsELECTROPHORESIS, Issue 7-8 2004Yi Zhu Abstract Preparative isoelectric focusing (PIEF) is used to achieve narrow-band fractionation of proteins from whole cell lysates of Escherichia coli (E. coli). Isoelectric membranes create well-defined pH ranges that fractionate proteins by isoelectric point (pI) upon application of an electric potential. A commercial IsoPrime device (Amersham-Pharmacia BioTech) is modified for the PIEF separation to lessen run volumes significantly. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) analysis of chamber contents indicates that excellent pH fractionation is achieved with little overlap between chambers. PIEF pH fractions are further separated using nonporous reversed-phase high-performance liquid chromatography (NPS-RP-HPLC) and HPLC eluent is analyzed on-line by electrospray ionization-time of flight-mass spectrometry (ESI-TOF-MS) for intact protein molecular weight (MW) analysis. The result is a pI versus MW map of bacterial protein content. IEF fractionation down to 0.1 pH units combined with intact protein MW values result in a highly reproducible map that can be used for comparative analysis of different E. coli strains. [source] Proteomics of snake venoms from Elapidae and Viperidae families by multidimensional chromatographic methodsELECTROPHORESIS, Issue 16 2003Jiraporn Nawarak Abstract Snake venoms contain a large number of biologically active substances and the venom components are very useful for pharmaceutical applications. Our goal is to separate and identify components of snake venoms in ten snake species from the Elapidae and Viperidae families using multidimensional chromatographic methods. The multidimensional chromatographic methods include reversed-phase high-performance liquid chromatography (RP-HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), lab-on-a-chip, two-dimensional electrophoresis (2-DE), and mass spectrometry. The venoms of eight snake species demonstrated major differences in hydrophobicity, molecular weight separations, and 2-DE protein distribution patterns. The 2-DE images showed major differences between families, within each family and even between the same species. Venoms of the Elapidae family showed many basic proteins with a wide range of molecular weights, while venoms of the Viperidae family showed wide ranges of pI and molecular weights, especially for Trimeresurus sp. The multidimensional chromatographic methods revealed specific differences in venom proteins intra-species as well as between species and families. We have isolated and identified proteins that may be unique for each species for further studies in the proteome of snake venoms and their potentially use in the pharmaceutical applications. [source] Insoluble eggshell matrix proteins , their peptide mapping and partial characterization by capillary electrophoresis and high-performance liquid chromatographyELECTROPHORESIS, Issue 5 2003Ivan Mik Abstract Avian eggshell matrix proteins were studied by two analytical approaches. Peptide mapping was done by trypsin and pepsin followed by collagenase cleavage; analyses were carried out by capillary electrophoresis and reversed-phase high-performance liquid chromatography (HPLC). Comparison of peptide maps obtained by both methods revealed a complex mixture of peptides in the insoluble layers of the eggshell; it was concluded that there are at least three different insoluble protein/peptide layers in the avian eggshell (cuticle, palisade, and mammillary layer). Partial characterization of peptides in each layer was made by HPLC-mass spectrometry analysis. There is an evidence that the eggshell insoluble proteins contain species susceptible to collagenase cleavage, however, the sequences split by this enzyme probably are not those typical for the main triple-helical core of collagenous proteins. It is proposed that the action of collagenase upon eggshell proteins is caused by the side effect of collagenase described previously with synthetic peptides. Some of the proteins present are probably glycosylated. Fatty acid content in the insoluble eggshell layers (after decalcification) was in the range of 2,4% (which reflected both lipid and lipoproteins bound fatty acids). Porphyrin pigments are dominant in the cuticle layer. [source] Vitamins A1 and A2 in hepatic tissue and subcellular fractions in mink feeding on fish-based diets and exposed to Aroclor 1242ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 2 2002Anne Käkelä Abstract Two-month-old female mink were fed diets based on either Baltic herring (Clupea harengus membras) or freshwater smelt (Osmerus eperlanus) for 21 weeks. A portion of the smelt-fed mink were exposed orally to polychlorinated biphenyls (PCBs), Aroclor 1242 (1 mg/d). Retinol (vitamin A1), 3,4-didehydroretinol (vitamin A2), and their different fatty acyl esters were studied in hepatic tissue, microsomes, and cytosol by argentated reversed-phase high-performance liquid chromatography. As a result of Aroclor exposure, concentrations of the fatty acyl esters of vitamins A1 and A2 were about one-tenth and those of unesterified A2 one-fourth those of the control levels. In the fatty acyl esters, percentages of stearates (A1 -18:0 and A2 -18:0) increased at the expense of the other fatty acyl esters. The Aroclor exposure decreased concentrations of alcoholic and esterified forms of the A2 analog more than those of the corresponding A1 analog. In microsomes, Aroclor decreased the alcoholic and esterified vitamin analogs to the same extent (to 9,17%). In the cytosol compared to the control, the concentrations of the vitamin esters fell below 10%, but the alcoholic analogs remained at 30 to 40%. Despite equal dietary supply, in mink fed on Baltic herring, the hepatic levels of vitamin A1 were only about one-third of the values found in the smelt-fed mink. The organochlorines also altered hepatic lipid composition and impaired breeding and kit growth. In the kits of the females fed on Baltic herring, blood hemoglobin was decreased. [source] Wine industry residue as antioxidant in cooked chicken meatINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 5 2010Ligianne Din Shirahigue Summary The effectiveness of grape extracts as food ingredient has been tested in various systems. The objective of this study was to evaluate the efficiency of four concentrations of residues of the wine industry in delaying lipid oxidation in processed chicken meat stored under refrigeration. The development of oxidation during the 14-day storage was evaluated through the thiobarbituric acid reactive substances method (TBAS). The analyses of phenolic compounds and antioxidant activity were performed in grape residue extracts through DPPH (1,1-difenil-2-picrilidrazil) method, lipid peroxidation inhibition and Rancimat. The profile of polyphenols was determined using reversed-phase high-performance liquid chromatography. Isabel grape extract (IGE) and Niagara grape extract (NGE) showed significant content of phenolic compounds. NGE and IGE had high antioxidant activity. The addition of grape extracts significantly increased the oxidative stability of processed and cooked chicken meat during the storage time. The extracts from both grape varieties when applied in concentrations of 40 and 60 mg of GAE, presented results similar to that of Butyl hydroxy toluene (BHT). [source] BIOCHEMICAL CHARACTERIZATION OF BORAGE (BORAGO OFFICINALIS L.) SEEDSJOURNAL OF FOOD BIOCHEMISTRY, Issue 3 2009BAYA MHAMDI ABSTRACT Gas chromatography,mass spectrometry analysis of seed Borago officinalis essential oil (EO) revealed the presence of 16 volatile components. ,-Caryophyllene (26%) and p-cymene-8-ol (19.7%) represented the major components, while nonadecane (0.7%) and hexanol (0.7%) were the minor ones. The EO composition was characterized by higher abundance of oxygenated monoterpenes (27.7%), followed by sesquiterpenes (26%). Fatty acid composition showed the predominance of linoleic (35.4%), oleic (24.2%) and ,-linolenic (20.4%) acids. Polyphenols were analyzed by reversed-phase high-performance liquid chromatography after acid hydrolysis of phenolic acid esters. Six phenolic acids were identified in seed extract and rosmarinic acid was the predominant one with 1.65 mg/g dry matter weight equivalent to 33% of total phenolic acids. PRACTICAL APPLICATIONS Borage (Borago officinalis L.) is of great interest because of its medicinal and nutritional properties. In fact, thanks to its characteristic composition in fatty acids, particularly high levels of gamma-linolenic acid in its seed oil, borage has gained importance. The potent consumers of this medicinal plant are hypertensive and hypercholesterolemic people. Borage consumption is also recommended for people suffering from rheumatism and eczema. Unfortunately, the knowledge about antioxidative/antiradical properties of borage is very scanty. So, recently, an extensive investigation was focused on the antioxidant properties of borage extracts. These extracts showed excellent antioxidant properties and their effects were attributed to their phenolic constituents. These antioxidants can be concentrated, either as crude extracts or individual phenolic compounds, to be used in highly unsaturated oils such as marine oils. Furthermore, borage consumption has been reported as a possible gastric cancer protective factor. [source] DETERMINATION OF AFLATOXIN CONTAMINATION IN OLIVES BY IMMUNOAFFINITY COLUMN USING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHYJOURNAL OF FOOD QUALITY, Issue 2 2006CAVIT BIRCAN ABSTRACT Eighty-two whole black olive samples gathered from six different olive oil processing facilities were surveyed to determine levels of aflatoxins using immunoaffinity column extraction and reversed-phase high-performance liquid chromatography. Two different analytical procedures adopted for the analysis of aflatoxins were investigated for their suitability by spiking the blank olive samples with five different known levels of aflatoxins to determine which one had higher recovery rates. Although some of the olive samples had been exposed to adverse conditions, such as rain and high temperatures, none were found to contain aflatoxins at the determined detection limit. Although the samples were kept in high relative humidity (75%) and high temperature (30C) for 3 months and were tested at 1-month intervals, no aflatoxins were detected. In addition, the olives were inoculated on a potato dextrose agar medium and incubated for 7 days at 25C to characterize the microflora. Because there is no evidence of aflatoxins in fresh whole olives, the next step of processing the contaminated olives into olive oils and testing them for the aflatoxins was not pursued. [source] Phenolic Acid Content and Composition in Leaves and Roots of Common Commercial Sweetpotato (Ipomea batatas L.) Cultivars in the United StatesJOURNAL OF FOOD SCIENCE, Issue 6 2007V.-D. Truong ABSTRACT:, Phenolic acids in commercially important sweet potato cultivars grown in the United States were analyzed using reversed-phase high-performance liquid chromatography (HPLC). Caffeic acid, chlorogenic acid, 4,5-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, and 3,4-di-O-caffeoylquinic acid were well separated with an isocratic elution in less than 25 min compared to about 120 min for analyzing and re-equilibrating the column with a gradient method. The isocratic elution order of these caffeoylquinic acid derivatives was confirmed by LC-MS/MS. Chlorogenic acid was the highest in root tissues, while 3,5-di-O-caffeoylquinic acid and/or 4,5-di-O-caffeoylquinic acid were predominant in the leaves. Steam cooking resulted in statistically nonsignificant increases in the concentration of total phenolics and all the individual phenolic acids identified. Sweetpotato leaves had the highest phenolic acid content followed by the peel, whole root, and flesh tissues. However, there was no significant difference in the total phenolic content and antioxidant activity between purees made from the whole and peeled sweet potatoes. [source] Isolation and Characterization of Virgin Olive Oil Phenolic Compounds by HPLC/UV and GC-MSJOURNAL OF FOOD SCIENCE, Issue 4 2001M. Tasioula-margari ABSTRACT This research examined the phenolic fraction of extra virgin olive oil samples from Lianolia variety olives grown in the region of Preveza, Greece. Phenolic compounds were extracted from oil samples, separated by reversed-phase high-performance liquid chromatography (HPLC), and characterized by gas chromatography-mass spectrometry (GC-MS). Both simple and complex phenols were detected with the latter being the most abundant. 3,4-Dihydroxyphenyl ethanol (hydroxytyrosol) and p-hydroxyphenylethanol (tyrosol) predominated among the simple phenols. Complex phenolic compounds were further separated by preparative HPLC and analyzed by GC-MS before and after hydrolysis. The presence of hydroxytyrosol and tyrosol derivatives was confirmed. Both derivatives were always present in greater quantities and made up an average exceeding 70% in all samples analyzed. [source] A sequence optimization strategy for chromatographic separation in reversed-phase high-performance liquid chromatographyAICHE JOURNAL, Issue 2 2010Xueling Du Abstract A sequence optimization strategy combining an artificial neural network (ANN) and a chromatographic response function (CRF) for chromatographic separation in reversed-phase high-performance liquid chromatography has been proposed. Experiments were appropriately designed to obtain unbiased data concerning the effects of varying the mobile phase composition, flow-rate, and temperature. The ANN was then used to simultaneously predict the resolution and analysis time, which are the two most important features of chromatographic separation. Subsequently, a CRF consisting of resolution and analysis time was used to predict the optimum operating conditions for different specialized purposes. The experimental chromatograms were consistent with those predicted for given conditions, which verified the applicability of the method. Furthermore, the proposed optimization strategy was applied to literature data and very good agreement was obtained. The results show that a strategy of sequential combination of ANN and CRF can provide a more flexible and efficient optimization method for chromatographic separation. © 2009 American Institute of Chemical Engineers AIChE J, 2010 [source] Purification and initial characterization of a novel protein with factor Xa activity from Lonomia obliqua caterpillar spiculesJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 3 2005S. Lilla Abstract A novel protein with factor Xa-like activity was isolated from Lonomia obliqua caterpillar spicules by gel filtration chromatography and reversed-phase high-performance liquid chromatography. The protein had a mass of 20745.7 Da, as determined by mass spectrometry, and contained four Cys residues. Enzymatic hydrolysis followed by de novo sequencing by tandem mass spectrometry was used to determine the primary structure of the protein and the cysteine residues linked by disulfide bridges. The positions of 24 sequenced tryptic peptides, including the N-terminal, were deduced by comparison with a homologous protein from the superfamily Bombycoidea. Approximately 90% of the primary structure of the active protein was determined. Copyright © 2005 John Wiley & Sons, Ltd. [source] Effect of instrument tuning on the detectabilityof biopolymers in electrospray ionization mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 1 2003Herbert Oberacher Abstract Electrospray ionization mass spectrometry of multiply charged biopolymer ions of different molecular size revealed a strong influence of tuning parameters on their detectability in quadrupole ion trap and triple quadrupole mass spectrometers. Hence, after optimizing the ion optical parameters with the signal of the 4, charge state of (dT)24 (low charge state tuning), a tenfold increase in the signal-to-noise ratio for a mixture of oligodeoxythymidylic acids (n = 12,18) was obtained compared with the results achieved with tune parameters optimized with a synthetic 80-mer oligodeoxynucleotide. By contrast, a detection limit in the upper femtomole region could only be reached for a 104-mer oligodeoxynucleotide utilizing the 24, charge state of the 80-mer (high charge state tuning). The same effect was observed for proteins investigated in the positive ion mode using low and high charge states of cytochrome c and carbonic anhydrase, respectively, for instrument tuning. By comparing the settings for low and high charge state tuning, it became obvious that the most significant difference was observed in the potential applied to the heated metal capillary used to transfer ions from the atmospheric pressure to the vacuum region of the ion source. Taking advantage of the optimized tuning procedure, the molecular mass of a 61 base pair product of polymerase chain reaction was accurately determined by electrospray ionization mass spectrometry on-line interfaced to ion-pair reversed-phase high-performance liquid chromatography. Copyright © 2003 John Wiley & Sons, Ltd. [source] Negative and positive ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and positive ion nano-electrospray ionization quadrupole ion trap mass spectrometry of peptidoglycan fragments isolated from various Bacillus speciesJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 2 2001Gerold Bacher Abstract A general approach for the detailed characterization of sodium borohydride-reduced peptidoglycan fragments (syn. muropeptides), produced by muramidase digestion of the purified sacculus isolated from Bacillus subtilis (vegetative cell form of the wild type and a dacA mutant) and Bacillus megaterium (endospore form), is outlined based on UV matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and nano-electrospray ionization (nESI) quadrupole ion trap (QIT) mass spectrometry (MS). After enzymatic digestion and reduction of the resulting muropeptides, the complex glycopeptide mixture was separated and fractionated by reversed-phase high-performance liquid chromatography. Prior to mass spectrometric analysis, the muropeptide samples were subjected to a desalting step and an aliquot was taken for amino acid analysis. Initial molecular mass determination of these peptidoglycan fragments (ranging from monomeric to tetrameric muropeptides) was performed by positive and negative ion MALDI-MS using the thin-layer technique with the matrix ,-cyano-4-hydroxycinnamic acid. The results demonstrated that for the fast molecular mass determination of large sample numbers in the 0.8,10 pmol range and with a mass accuracy of ±0.07%, negative ion MALDI-MS in the linear TOF mode is the method of choice. After this kind of muropeptide screening often a detailed primary structural analysis is required owing to ambiguous data. Structural data could be obtained from peptidoglycan monomers by post-source decay (PSD) fragment ion analysis, but not from dimers or higher oligomers and not with the necessary sensitivity. Multistage collision-induced dissociation (CID) experiments performed on an nESI-QIT instrument were found to be the superior method for structural characterization of not only monomeric but also of dimeric and trimeric muropeptides. Up to MS4 experiments were sometimes necessary to obtain unambiguous structural information. Three examples are presented: (a) CID MSn (n = 2,4) of a peptidoglycan monomer (disaccharide-tripeptide) isolated from B. subtilis (wild type, vegetative cell form), (b) CID MSn (n = 2,4) of a peptidoglycan dimer (bis-disaccharide-tetrapentapeptide) obtained from a B. subtilis mutant (vegetative cell form) and (c) CID MS2 of a peptidoglycan trimer (a linear hexasaccharide with two peptide side chains) isolated from the spore cortex of B. megaterium. All MSn experiments were performed on singly charged precursor ions and the MS2 spectra were dominated by fragments derived from interglycosidic bond cleavages. MS3 and MS4 spectra exhibited mainly peptide moiety fragment ions. In case of the bis-disaccharide-tetrapentapeptide, the peptide branching point could be determined based on MS3 and MS4 spectra. The results demonstrate the utility of nESI-QIT-MS towards the facile determination of the glycan sequence, the peptide linkage and the peptide sequence and branching of purified muropeptides (monomeric up to trimeric forms). The wealth of structural information generated by nESI-QIT-MSn is unsurpassed by any other individual technique. Copyright © 2001 John Wiley & Sons, Ltd. [source] Pharmacokinetics and tissue distribution of idarubicin-loaded solid lipid nanoparticles after duodenal administration to ratsJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 5 2002Gian Paolo Zara Abstract Idarubicin-loaded solid lipid nanoparticles (IDA-SLN) and idarubicin in solution were prepared and the two formulations were administered to rats, either by the duodenal route or intravenously (iv). The aim of this research was to study whether the bioavailability of idarubicin can be improved by administering IDA-SLN duodenally to rats. Idarubicin and its main metabolite idarubicinol were determined in plasma and tissues by reversed-phase high-performance liquid chromatography. The pharmacokinetic parameters of idarubicin found after duodenal administration of the two formulations were different: area under the curve of concentration versus time (AUC) and elimination half-life were ,21 times and 30 times, respectively, higher after IDA-SLN administration than after the solution administration. Tissue distribution also differed: idarubicin and idarubicinol concentrations were lower in heart, lung, spleen, and kidneys after IDA-SLN administration than after solution administration. The drug and its metabolite were detected in the brain only after IDA-SLN administration, indicating that SLN were able to pass the blood,brain barrier. After iv IDA-SLN administration, the AUC of idarubicin was lower than after duodenal administration of the same formulation. Duodenal administration of IDA-SLN modifies the pharmacokinetics and tissue distribution of idarubicin. The IDA-SLN act as a prolonged release system for the drug. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:1324,1333, 2002 [source] Resolution of triacylglycerol positional isomers by reversed-phase high-performance liquid chromatographyJOURNAL OF SEPARATION SCIENCE, JSS, Issue 12 2004Svetlana Momchilova Abstract The ability of reversed-phase high-performance liquid chromatography (RP-HPLC) to separate some positionally isomeric disaturated and monounsaturated triacylglycerols (TAGs) as intact species is demonstrated for the first time. Mobile phases of acetonitrile modified with methanol, ethanol, 2-propanol, 1-propanol, 1-butanol, acetone, or dichloromethane were tested for the separation of POP-PPO, PLP-PPL, PEP-PPE, and PDP-PPD (P , palmitic, O , oleic, L , linoleic, E , eicosapentaenoic, D , docosahexaenoic acid residue) on a single RP-HPLC column. The resolution improved with increasing number of double bonds in the acyl residues. While POP and PPO were only partially resolved, PDP and PPD were fully separated with all tested mobile phases, except those containing methanol. Also separated were the four TAGs having the same equivalent carbon number (ECN = 42), PEP, PPE, PDP, and PPD, on a single RP-HPLC column with mobile phase acetonitrile,2-propanol (70:30, v/v) at 0.8 mL/min. In all cases the isomer with the unsaturated acyl residue in either 1- or 3-position was retained more strongly than the respective 2-isomer. [source] Comparison of two sample clean-up methodologies for the determination of polycyclic aromatic hydrocarbons in edible oilsJOURNAL OF SEPARATION SCIENCE, JSS, Issue 17 2003Alejandro Barranco Abstract An off-line high-performance normal-phase liquid chromatography procedure with a silica column followed by reversed-phase high-performance liquid chromatography (HPLC) with fluorescence detection for the determination of polycyclic aromatic hydrocarbons (PAHs) in edible oils is reported. The method was validated using certified reference materials and compared with a standardized method widely used in the food industry, consisting in low pressure column chromatography with alumina as stationary phase followed by reversed phase HPLC determination. The limits of detection were lower than 1 ng/g and good selectivity was achieved for both methods. There were no significant differences in accuracies and precisions obtained for each approach. The advantages and disadvantages of the two methods are discussed. [source] Separation and determination of small amounts of sulfur in technical thiophanate-methyl by reversed-phase high-performance liquid chromatographyJOURNAL OF SEPARATION SCIENCE, JSS, Issue 9-10 2003R. Nageswara Rao Abstract A simple and rapid reversed phase high-performance liquid chromatographic method for separation and determination of elemental sulfur in technical thiophanate-methyl using a reversed-phase C18 column and methanol-water-tetrahydrofuran (90 : 8 : 2 v/v/v) as eluent with UV detection at 254 nm has been developed and validated with respect to accuracy, precision, specificity, linearity range, and limits of detection and quantification. The method was found to be suitable not only for detection but also determination of elemental sulfur at levels of 5×10,9 g. [source] Effect of thermal processing on genistein, daidzein and glycitein content in soymilkJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 7 2006Huihua Huang Abstract Soymilk was subjected to various heat treatments at 95, 121 and 140 °C for various lengths of time. The contents of the aglycones of isoflavone (daidzein, glycitein and genistein) of the soymilk were determined using C18 reversed-phase high-performance liquid chromatography. Genistein showed greater stability to heat treatment than daidzein and glycitein. Both the daidzein and glycitein contents decreased rapidly during the early stage of heating, but on continued heating the rates of decrease were much slower. Heating may cause an increase or decrease in the genistein content of soymilk depending on the temperature and time used. Upon heating at 95 and 121 °C, there was an increase in the genistein content in the early stage of heating, possibly due the conversion of genistin to genistein. Heating at 140 °C for more than 15 s and prolonged heating at 95 and 121 °C, however, caused a slow decline in the genistein content. Copyright © 2006 Society of Chemical Industry [source] Biogenic amine production by lactic acid bacteria isolated from ciderLETTERS IN APPLIED MICROBIOLOGY, Issue 5 2007G. Garai Abstract Aims:, To study the occurrence of histidine, tyrosine and ornithine decarboxylase activity in lactic acid bacteria (LAB) isolated from natural ciders and to examine their potential to produce detrimental levels of biogenic amines. Methods and Results:, The presence of biogenic amines in a decarboxylase synthetic broth and in cider was determined by reversed-phase high-performance liquid chromatography (RP-HPLC). Among the 54 LAB strains tested, six (five lactobacilli and one oenococci) were biogenic amine producers in both media. Histamine and tyramine were the amines formed by the LAB strains investigated. Lactobacillus diolivorans were the most intensive histamine producers. This species together with Lactobacillus collinoides and Oenococcus oeni also seemed to produce tyramine. No ability to form histamine, tyramine or putrescine by Pediococus parvulus was observed, although it is a known biogenic amine producer in wines and beers. Conclusions:, This study demonstrated that LAB microbiota growing in ciders had the ability to produce biogenic amines, particularly histamine and tyramine, and suggests that this capability might be strain-dependent rather than being related to a particular bacterial species. Significance and Impact of the Study:, Production of biogenic amines by food micro-organisms has continued to be the focus of intensive study because of their potential toxicity. The main goal was to identify the microbial species capable of producing these compounds in order to control their presence and metabolic activity in foods. [source] Reversed-phase HPLC-ESI/MS analysis of birch leaf proanthocyanidins after their acidic degradation in the presence of nucleophilesPHYTOCHEMICAL ANALYSIS, Issue 5 2007Maarit Karonen Abstract Mountain birch leaves contain large amounts of structurally variable polymeric proanthocyanidins. Their isolation procedure was enhanced by the addition of liquid,liquid extractions prior to column chromatography over Sephadex LH-20. Isolated polymeric proanthocyanidins were depolymerised by acid-catalysis in the presence of benzyl mercaptan or phloroglucinol in order to study their composition. The resulting degradation products, flavan-3-ols and flavan-3-ol adducts, were analysed with reversed-phase high-performance liquid chromatography using UV photodiode array detection for quantification and electrospray ionisation mass spectrometry for identification. The results showed that polymeric proanthocyanidins contained (epi)gallocatechins and (epi)catechins as the extension units and, mainly, (+)-catechin as the terminal unit. The mean degree of polymerisation was found to be 26 based on thiolysis and 31 based on phloroglucinol degradation. Copyright © 2007 John Wiley & Sons, Ltd. [source] Solid-phase extraction and reversed-phase high-performance liquid chromatography of the five major alkaloids in Narcissus confususPHYTOCHEMICAL ANALYSIS, Issue 6 2002Susana López Abstract A novel, fast and precise method, combining solid-phase extraction and reversed-phase high-performance liquid chromatography is described for the quantitative determination of five alkaloids (galanthamine, N -formylnorgalanthamine, haemanthamine, homolycorine and tazettine/pretazettine) from bulbs of wild Narcissus confusus, a high galanthamine-containing plant species growing in the Iberian Peninsula. Copyright © 2002 John Wiley & Sons, Ltd. [source] Phytochelatin synthesis plays a similar role in shoots of the cadmium hyperaccumulator Sedum alfredii as in non-resistant plantsPLANT CELL & ENVIRONMENT, Issue 8 2010ZHONG-CHUN ZHANG ABSTRACT Phytochelatin (PC) synthesis is considered necessary for Cd tolerance in non-resistant plants, but roles for PCs in hyper-accumulating species are currently unknown. In the present study, the relationship between PC synthesis and Cd accumulation was investigated in the Cd hyperaccumulator Sedum alfredii Hance. PCs were most abundant in leaves followed by stems, but hardly detected by the reversed-phase high-performance liquid chromatography (HPLC) in roots. Both PC synthesis and Cd accumulation were time-dependent and a linear correlation between the two was established with about 1:15 PCs : Cd stoichiometry in leaves. PCs were found in the elution fractions, which were responsible for Cd peaks in the anion exchange chromatograph assay. About 5% of the total Cd was detected in these elution fractions as PCs were found. Most Cd was observed in the cell wall and intercellular space of leaf vascular cells. These results suggest that PCs do not detoxify Cd in roots of S. alfredii. However, like in non-resistant plants, PCs might act as the major intracellular Cd detoxification mechanism in shoots of S. alfredii. [source] Recombinant decorsin: Dynamics of the RGD recognition sitePROTEIN SCIENCE, Issue 8 2000Andrzej M. Krezel Abstract Decorsin is an antagonist of integrin ,IIb,3 and a potent platelet aggregation inhibitor. A synthetic gene encoding decorsin, originally isolated from the leech Macrobdella decora, was designed, constructed, and expressed in Escherichia coli. The synthetic gene was fused to the stII signal sequence and expressed under the transcriptional control of the E. coli alkaline phosphatase promoter. The protein was purified by size-exclusion filtration of the periplasmic contents followed by reversed-phase high-performance liquid chromatography. Purified recombinant decorsin was found to be indistinguishable from leech-derived decorsin based on amino acid composition, mass spectral analysis, and biological activity assays. Complete sequential assignments of 1H and proton bound 13C resonances were established. Stereospecific assignments of 21 of 25 nondegenerate ,-methylene groups were determined. The RGD adhesion site recognized by integrin receptors was found at the apex of a most exposed hairpin loop. The dynamic behavior of decorsin was analyzed using several independent NMR parameters. Although the loop containing the RGD sequence is the most flexible one in decorsin, the conformation of the RGD site itself is more restricted than in other proteins with similar activities. [source] The use of acetone as a substitute for acetonitrile in analysis of peptides by liquid chromatography/electrospray ionization mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 1 2010Theodore R. Keppel The recent worldwide shortage of acetonitrile has prompted interest in alternative solvents for liquid chromatography/mass spectrometry (LC/MS). In this work, acetone was substituted for acetonitrile in the separation of a peptide mixture by reversed-phase high-performance liquid chromatography (RP-HPLC) and in the positive electrospray ionization mass spectrometry (ESI-MS) of individual peptides. On both C12 and C18 stationary phases, the substitution of acetone for acetonitrile as the organic component of the mobile phase did not alter the gradient elution order of a five-peptide retention standard, but did increase peak width, shorten retention times, and increase peak tailing. Positive ESI mass spectra were obtained for angiotensin I, bradykinin, [Leu5]-enkephalin, and somatostatin 14 dissolved in both acetonitrile/water/formic acid (25%/75%/0.1%) and acetone/water/formic acid (25%/75%/0.1%). Under optimized ESI-MS conditions, the mass spectral response of [Leu5]-enkephalin was increased two-fold when the solvent contained acetone. The substitution of acetone for acetonitrile resulted in only slight changes in the responses of the remaining peptides. A higher capillary voltage was required for optimum response when acetone was used. Compared with acetonitrile/water/formic acid (50/50/0.1%), more interfering species below m/z,=,140 were found in the ESI-MS spectra of acetone/water/formic acid (50/50/0.1%). Copyright © 2009 John Wiley & Sons, Ltd. [source] Rapid structural determination of alkaloids in a crude extract of Stemona saxorum by high-performance liquid chromatography/electrospray ionization coupled with tandem mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 23 2009Shu-Ying Peng The electrospray ionization (ESI) mass spectrometric behavior of five Stemona alkaloids, stemokerrin, oxystemokerrin, oxystemokerrilactone, oxystemokerrin N -oxide and stemokerrin N -oxide, was studied using an ESI tandem mass technique (MSn). These compounds, isolated from Stemonasaxorum endemic in Vietnam, represent a class of alkaloids containing a pyrido[1,2-a]azepine A,B-ring core with a 1-hydroxypropyl side chain attached to C-4. Their fragmentation pathways were elucidated by ESI-MSn results and the elemental composition of the major product ions was confirmed by accurate mass measurement. In order to rationalize some fragmentation pathways, the relative Gibbs free energies of some product ions were estimated using the B3LYP/6-31+G(d) method. Based on the ESI-MSn results of five reference compounds, a reversed-phase high-performance liquid chromatography with tandem mass spectrometry (RP-HPLC/MSn) method was developed for the characterization of Stemona alkaloids with a pyrido[1,2-a]azepine A,B-ring core from the extract of S. saxorum. A total of 41 components were rapidly identified or tentatively characterized, of which 12 compounds were identified as Stemona alkaloids with a pyrido[1,2-a]azepine A,B-ring core, including four new compounds. This method is convenient and sensitive, especially for minor components in complex natural product extracts. Copyright © 2009 John Wiley & Sons, Ltd. [source] Sequence and phosphorylation level determination of two donkey , -caseins by mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 13 2009Vincenzo Cunsolo Two coeluting components, with experimentally measured Mr values of 25529 and 24606 Da, were identified by reversed-phase high-performance liquid chromatography (RP-HPLC) and mass spectrometric analysis in the dephosphorylated casein fraction of a milk sample collected from an individual donkey belonging to the Ragusano breed of the east of Sicily. By coupling enzymatic digestions, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and RP-HPLC/nano-electrospray ionization tandem mass spectrometry (nESI-MS/MS) analysis, the two proteins were identified as donkey , -CNs and their sequences characterized completely, using the two known , -CNs from mare as references. The two donkey , -CNs, showing a mass difference of 923 Da, differ by the presence of the domain E27SITHINK34 in the full-length component (Mr 25529 Da). In comparison with the mare's , -CNs used as reference, they present nine amino acid substitutions: L,S37, R,H52, S,N81, P,V84, L,V91, R,Q203, P,L/I206, L,F210 and A,P219. Together, these substitutions account for the increase of 18 Da in the Mr of the donkey , -CNs with respect to the counterparts from the mare. The molecular mass determination by ESI-MS for the phosphorylated proteins showed that the full-length component was composed of highly multi-phosphorylated isoforms with five to seven phosphate groups. By analogy with the homologous mare's , -CNs, the full-length (226 amino acids) , -CN was termed variant A, whereas the shorter (218 amino acids) , -CN was termed variant A,5. Copyright © 2009 John Wiley & Sons, Ltd. [source] Analysis of electrochemical degradation products of sulphonated azo dyes using high-performance liquid chromatography/tandem mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 19 2006Dana Van, rková Electrochemical treatment of wastewaters containing azo dyes in the textile industry is a promising approach for their degradation. The monitoring of the course of the decomposition of azo dyes in wastewaters is essential due to the environmental impact of their degradation products. In this work, aqueous solutions of a simple azo dye with a low molecular weight (C.I. Acid Yellow 9) and more complex commercial dye (C.I. Reactive Black 5) were electrochemically treated in a laboratory-scale electrolytic cell in sodium chloride or ammonium acetate as supporting electrolytes. Ion-pairing reversed-phase high-performance liquid chromatography coupled with negative-ion electrospray ionization mass spectrometry is applied for the identification of electrochemical degradation products. In addition to simple inorganic salts, the formation of aromatic degradation products obtained due to the cleavage of azo bonds and further degradation reactions is shown, as well as chlorination where sodium chloride is the supporting electrolyte. Degradation mechanisms are suggested for the treatment with sodium chloride as the supporting electrolyte. Copyright © 2006 John Wiley & Sons, Ltd. [source] Analysis of the composition of immunoconjugates using size-exclusion chromatography coupled to mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 13 2005Alexandru C. Lazar Recombinant monoclonal antibody drug products play an increasingly important role in the treatment of various diseases. Antibodies are large, multi-chain proteins and antibody preparations often contain several molecular variants, which renders them heterogeneous. The heterogeneity is further increased in immunoconjugates prepared by covalently linking several drug molecules per antibody molecule. As part of the product characterization, the molecular weights of the antibodies or their drug conjugates need to be measured. Electrospray ionization mass spectrometry (ESI-MS) is well suited for the analysis of recombinant antibodies and immunoconjugates. Sample preparation is an important element of ESI-MS analysis, in particular samples need to be freed of interfering charged species, such as salts and buffer components. In this paper, Amicon centrifugal filters, reversed-phase high-performance liquid chromatography (HPLC), and size-exclusion HPLC were evaluated for sample desalting. Size-exclusion HPLC, using aqueous acetonitrile as the mobile phase, directly coupled to ESI-MS provided the best performance and was optimized for the study of immunoconjugates. The results showed that antibodies carrying covalently linked maytansinoid molecules generated charge envelope profiles that differ from those of the non-conjugated antibody. For the determination of the distribution of the various conjugate species in an immunoconjugate sample prepared by randomly linking in the average 3.6 drug molecules per antibody molecule, the experimental conditions needed to be carefully selected to allow acquisition of the whole spectrum containing the charge envelopes of all species. Copyright © 2005 John Wiley & Sons, Ltd. [source] | |||||||||||||||||||||||||