|
| ||
|
|
||
Chromatographic Methods (chromatographic + methods)
Kinds of Chromatographic Methods Selected AbstractsGhanaian Cocoa Bean Fermentation Characterized by Spectroscopic and Chromatographic Methods and ChemometricsJOURNAL OF FOOD SCIENCE, Issue 6 2010Patrick C. Aculey Abstract:, Export of cocoa beans is of great economic importance in Ghana and several other tropical countries. Raw cocoa has an astringent, unpleasant taste, and flavor, and has to be fermented, dried, and roasted to obtain the characteristic cocoa flavor and taste. In an attempt to obtain a deeper understanding of the changes in the cocoa beans during fermentation and investigate the possibility of future development of objective methods for assessing the degree of fermentation, a novel combination of methods including cut test, colorimetry, fluorescence spectroscopy, NIR spectroscopy, and GC-MS evaluated by chemometric methods was used to examine cocoa beans sampled at different durations of fermentation and samples representing fully fermented and dried beans from all cocoa growing regions of Ghana. Using colorimetry it was found that samples moved towards higher,a* and,b* values as fermentation progressed. Furthermore, the degree of fermentation could, in general, be well described by the spectroscopic methods used. In addition, it was possible to link analysis of volatile compounds with predictions of fermentation time. Fermented and dried cocoa beans from the Volta and the Western regions clustered separately in the score plots based on colorimetric, fluorescence, NIR, and GC-MS indicating regional differences in the composition of Ghanaian cocoa beans. The study demonstrates the potential of colorimetry and spectroscopic methods as valuable tools for determining the fermentation degree of cocoa beans. Using GC-MS it was possible to demonstrate the formation of several important aroma compounds such 2-phenylethyl acetate, propionic acid, and acetoin and the breakdown of others like diacetyl during fermentation. Practical Application:, The present study demonstrates the potential of using colorimetry and spectroscopic methods as objective methods for determining cocoa bean quality along the processing chain. Development of objective methods for determining cocoa bean quality will be of great importance for quality insurance within the fields of cocoa processing and raw material control in chocolate producing companies. [source] Chromatographic methods for the separation of biocompatible iron chelators from their synthetic precursors and iron chelatesJOURNAL OF SEPARATION SCIENCE, JSS, Issue 17-18 2004Petra Kova, íková Abstract Chromatographic methods have been developed for the separation of the three novel biocompatible iron chelators pyridoxal isonicotinoyl hydrazone (PIH), salicylaldehyde isonicotinoyl hydrazone (SIH), and pyridoxal 2-chlorobenzoyl hydrazone (o-108) from their synthetic precursors and iron chelates. The chromatographic analyses were achieved using analytical columns packed with 5 ,m Nucleosil 120-5 C18. For the evaluation of all chelators in the presence of the synthetic precursors, EDTA was added to the mobile phase at a concentration of 2 mM. The best separation of PIH and its synthetic precursors was achieved using a mixture of phosphate buffer (0.01 M NaH2PO4, 5 mM 1-heptanesulfonic acid sodium salt; pH 3.0) and methanol (55 : 45, v/v). For separation of SIH and its synthetic precursors, the mobile phase was composed of 0.01 M phosphate buffer (pH 6.0) and methanol (60 : 40, v/v). o-108 was analyzed employing a mixture of 0.01 M phosphate buffer (pH 7.0), methanol, and acetonitrile (60 : 20 : 20, v/v/v). These mobile phases were slightly modified to separate each chelator from its iron chelate. Furthermore, a RP-TLC method has also been developed for fast separation of all compounds. The chromatographic methods described herein could be applied in the evaluation of purity and stability of these drug candidates. [source] Antioxidant diterpenoids from the roots of Salvia barrelieriPHYTOCHEMICAL ANALYSIS, Issue 4 2009Ufuk Kolak Abstract Introduction The phytochemical and biological studies carried out on Salvia species showed that their extracts and constituents have various biological activities. Objective The aim of this study was the isolation of diterpenoids from the roots of Salvia barrelieri Ettling and the determination of the antioxidant activity. Methodology Chromatographic methods were used for fractionation and isolation, respectively. Structure elucidation was established by spectroscopic methods. Five antioxidant assays were performed. Results Three new abietane diterpenoids barreliol, royleanone 12-methyl ether and 7-epi-salviviridinol, and six known diterpenoids, with a known dammarane triterpenoid, pyxinol were isolated. The absolute stereochemistry of pyxinol was confirmed by X-ray analysis. Conclusion Taxodione exhibited the highest antioxidant activity among the tested compounds. Copyright © 2009 John Wiley & Sons, Ltd. [source] Methods for detecting and identifying retinoids in tissueDEVELOPMENTAL NEUROBIOLOGY, Issue 7 2006Thomas E. Gundersen Abstract Methods for retinoid analysis in tissue include direct spectrophotometry or fluorometry and retinoid responsive reporter constructs in the form of cell reporter assays or transgenic reporter animals, but chromatographic methods dominate and posses several superior features in quantitative analysis. The multitude of extraction protocols used can coarsely be divided into manual liquid-liquid extraction protocols and semi- or fully automated solid phase extraction-based protocols. Liquid chromatographic separation in reversed phase dominates although normal phase is also used. Detection is mainly performed with UV detectors although electrochemical and fluorescence detection is also used. Mass spectrometry in combination with LC is more often used in retinoid analysis and is likely to dominate in the future. © 2006 Wiley Periodicals, Inc. J Neurobiol 66: 631,644, 2006 [source] Murder by poisoning: successful analytical investigations of spectacular cases in AustriaDRUG TESTING AND ANALYSIS, Issue 4 2009Walter Vycudilik Abstract To prove murder by poisoning requires the application of analytical toxicology to detect the fatal substance and clear up the cause of death. Improvements in the development of mass spectrometry in combination with high-resolution chromatographic methods are steadily enhancing detection and identification power but making use of these advances relies on proper sample preparation as well as on knowledge about the chemical nature of the substances and their bio-transformation products. This review gives examples of case reports with successful analytical investigations of murder by poisoning in spectacular Austrian cases involving low molecular weight. Copyright © 2009 John Wiley & Sons, Ltd. [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] Synthesis of Carboxyl-Tethered Symmetric Conjugated Polyenes as Fluorescent Transmembrane Probes of Lipid BilayersEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 7 2003Ernesto Quesada Abstract The synthesis of a new series of fluorescent transmembrane probes in which two hydrophilic methyl ester or carboxyl groups are connected by a polymethylene chain, with four, five or six conjugated double bonds in a central position, is reported. The length of the linear structures was designed to match the width of typical lipid bilayers. These bolaamphiphilic compounds result, with overall yields higher than 80%, from an easy PdII -catalyzed double cross-coupling between terminal acetylene esters and conjugated 1,,-dihalopolyenes, followed by selective triple bond partial reduction with activated zinc, and iodine isomerization to the all-(E) isomer. An alternative approach, based on a Stille double cross-coupling between the appropriate all-(E)-,-halopolyenes and (E)-bis(tributylstannyl)ethene, yielded mixtures that could not be resolved by standard chromatographic methods due to the presence of other simultaneous coupling reactions, which are also discussed in detail. Nevertheless, the Stille method can be of utility for the obtention of carbonyl-polyene conjugated analogs. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003) [source] Synthesis of 2-amino-4h -thiazolo[5,4- b]indole and characterization of its colored conversion products with protein tyrosine phosphatase inhibitory activityJOURNAL OF HETEROCYCLIC CHEMISTRY, Issue 3 2001Jens Breinholt In DMSO-solution 2-amino-4H -thiazolo[5,4- b]indole is converted into a complex mixture of colored products. The three major conversion end-products, of which two are inhibitors of protein tyrosine phos-phatases (PTPs), were isolated by chromatographic methods and their structures characterized by spectro-scopic analysis, including NMR and MS combined with computer assisted structure elucidation, and, finally, confirmed by independent chemical synthesis. Synthesis of 2-amino-4H -thiazolo[5,4- b]indole as well as its N -acetyl derivatives prepared from either oxindole or 2-bromo-1-(2-nitro-phenyl)ethanone is described. [source] Immunological Detection of in Vitro Formed Phosphatidylethanol,An Alcohol Biomarker,With Monoclonal AntibodiesALCOHOLISM, Issue 6 2008Antti E. Nissinen Background:, Phosphatidylethanol (PEth) is a promising new marker for detecting long-term alcohol abuse with excellent sensitivity and specificity. Current methods are based on the high performance liquid chromatography,mass spectrometric method and therefore require high levels of expertise and expensive instrumentation. This study was designed to generate PEth-specific monoclonal antibodies for PEth immunoassay development. Methods:, C57/BL6 mice were immunized with PEth in 3 different carriers, mouse serum albumin, mouse high-density lipoproteins, and human low-density lipoprotein (LDL). Mouse splenocytes were fused with a mouse myeloma cell line using the hybridoma technique. Mouse IgM-producing cell lines were selected by limiting dilutions. Binding characteristics of the anti-PEth antibodies were studied using luminometric immunoassays and sequence analysis of the variable region mRNA sequences of the antibodies. Produced antibodies were purified by chromatographic methods. PEth was detected with these antibodies in fluorescence immunoassay and flow cytometric analysis. Results:, We generated monoclonal cell lines (2B1 and 2E9) that produce IgM antibodies binding specifically to PEth but not to structurally or chemically similar phospholipids, such as phosphatidylcholine, phosphatidic acid, and cardiolipin. We show here that these anti-PEth antibodies can be used to detect PEth in a fluorescent PEth assay and FACS analysis of human red blood cell samples spiked with PEth. Conclusions:, The present study shows that PEth-specific monoclonal antibodies can be generated using traditional hybridoma technique. Immunogenicity of PEth was enhanced using human LDL as an immunization carrier. The generated monoclonal anti-PEth antibodies, 2B1 and 2E9 bind to PEth in fluid phase and in biological membranes. [source] HPLC enantioseparation of ,2 -homoamino acids using crown ether-based chiral stationary phaseJOURNAL OF SEPARATION SCIENCE, JSS, Issue 7 2009Róbert Berkecz Abstract RP high-performance liquid chromatographic methods were developed for the enantioseparation of eleven unusual ,2 -homoamino acids. The underivatized analytes were separated on a chiral stationary phase containing (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid as chiral selector. The effects of organic (alcoholic) and acidic modifiers, the mobile phase composition and temperature on the separation were investigated. The structures of the substituents in the ,-position of the analytes substantially influenced the retention and resolution. The elution sequence was determined in some cases: the S enantiomers eluted before the R enantiomers. [source] Panose, a new prebiotic candidateLETTERS IN APPLIED MICROBIOLOGY, Issue 6 2009H. Mäkeläinen Abstract Aims:, To investigate the prebiotic potential of two novel candidates, sophorose and panose, with in vitro methods. Methods and Results:, The growth of single microbial strains was first assessed for both substrates in pure cultures, and panose was further analysed in the simulated colon model with mixed human faecal culture. Quantitative PCR and flow cytometry were used to determine the microbial group and strain densities after the simulated colonic fermentation of panose, and chromatographic methods were utilized to analyse metabolite concentrations. In pure cultures, sophorose and panose were both fermented only by few beneficial strains, and in the colon simulator, panose gave a significant increase in the numbers of Bifidobacterium and Bifidobacterium lactis, concomitantly decreasing Bacteroides group. Butyrate and acetate production was significantly increased together with decreased markers of protein fermentation as a result of panose fermentation. Conclusions:, Panose had bifidogenic activities in vitro, and these potential beneficial effects should be further assessed in vitro and in vivo. Significance and Impact of the Study:, The current study has provided the first data on pure panose fermentation by the endogenous microbiota and extends our knowledge of the selective fermentation of oligosaccharides by the intestinal microbes. [source] Melt Spinning of Bacterial Aliphatic Polyester Using Reactive Extrusion for Improvement of CrystallizationMACROMOLECULAR BIOSCIENCE, Issue 6 2007Roland Vogel Abstract This paper reports on an attempt to use reactive extrusion with peroxide as a comfortable pathway for improvement of the crystallization of poly(3-hydroxybutyrate) in a melt spinning process. At first, rheological and thermal properties of the modified melts are determined in order to assess the effect of nucleation. Then spinning tests are carried out. Molecular weights and molecular weight distributions of the spun fibers are determined by chromatographic methods. Average crystallite size is measured by wide angle X-ray scattering. Thermal and textile properties of the spun PHB fibers are also determined. An estimation of the improvement of the crystallization in the spinline and of the inhibition of the secondary crystallization in the fibers from the use of the described way of reactive extrusion is given. [source] Two-Dimensional Chromatography of Complex Polymers, 7 , Detailed Study of Polystyrene- block -Polyisoprene Diblock Copolymers Prepared by Sequential Anionic Polymerization and Coupling ChemistryMACROMOLECULAR CHEMISTRY AND PHYSICS, Issue 19 2008Valentina Mass Abstract Two-dimensional chromatographic methods were developed using LC-CC in the first and SEC in the second dimension. These methods were applied for the investigation of PS- b -PI diblock copolymers synthesized by different approaches: sequential living anionic polymerization and coupling of living precursor blocks. The first dimension separates according to the individual block length of PS or PI blocks, whereas the second dimension separates with respect to the total molar masses of components. 2D-LC analysis provides information on the purity of the reaction products, the presence of by-products, the chemical compositions and the molar masses of all product components. The accuracy and selectivity of 2D-LC is discussed. [source] Chromatographic analysis of simple phenols in some species from the genus SalixPHYTOCHEMICAL ANALYSIS, Issue 5 2010Loretta Pob, ocka-Olech Abstract Introduction , Salicis Cortex, made from willow bark is a herbal remedy, which is standardised based on the content of salicin, a compound with analgesic and antiphlogistic properties. However, clinical trials suggest that other compounds also present in Salicis Cortex can contribute to the pharmacological effects. Objective , To characterise the composition of phenolic acids in the barks of different species and clones from the genus Salix by use of chromatographic methods,HPTLC and HPLC. Methodology , The phenolic acid composition was analysed by MGD (multiple gradient development),HPTLC technique. The separation was performed on HPTLC Diol plates with gradient elution using a mixture of chloroform:hexane:ethyl acetate with increasing concentration of ethyl acetate from 10 to 25%. Derivatisation with thymol reagent was employed for the first time for specific detection of phenolic acids containing methoxyl groups. Results , The presence of all phenolic acids previously reported in the genus Salix was confirmed, namely p -hydroxybenzoic, vanillic, cinnamic, p -coumaric, ferulic and caffeic acids. Furthermore, pyrocatechol as a constituent of willow bark was revealed. The highest concentration of this compound was observed in the S. purpurea bark (2.25,mg/g). Conclusion , The presence of a relatively high content of pyrocatechol in Salix species may raise doubts about the safe application of this herbal medicine. Copyright © 2010 John Wiley & Sons, Ltd. [source] Analysis of metabolic variation and galanthamine content in Narcissus bulbs by 1H NMR,PHYTOCHEMICAL ANALYSIS, Issue 1 2010Andrea Lubbe Abstract Introduction , Galanthamine is a benzazepine alkaloid used as a drug to relieve symptoms of Alzheimer's disease. For pharmaceutical use this natural product has been extracted from the plant Leucojum aestivum (Amaryllidaceae) or produced synthetically. Limited supply of the natural source and high cost of synthetic production has led to a search for alternative sources of galanthamine. The bulbs of Narcissus pseudonarcissus (Amaryllidaceae) have been identified as a potential source of raw material for galanthamine extraction. Since inconsistent chemical composition can be an issue with medicinal plant material, it is of interest to know whether large variations occur between Narcissus bulbs grown in different geographical locations. Objective , To evaluate whether large differences exist in the overall metabolic profiles of Narcissus bulbs grown in the two most important cultivation regions. Methodology , 1H NMR and principal component analysis were used for an unbiased comparison of the bulb samples. Results , Overall metabolite profiles were quite similar, but galanthamine levels could slightly discriminate samples by geographical region. 1H NMR was used for quantitation of galanthamine, and was found to be comparable to quantitation by HPLC. Compared with conventional chromatographic methods, sample preparation for 1H NMR analysis is simple and rapid, and only a small amount of plant material is required. Conclusions , Since useful qualitative and quantitative information about the metabolic state of Narcissus bulbs can be obtained by 1H NMR, this method is useful for agricultural applications, and for quality control of raw material used in the pharmaceutical industry. Copyright © 2009 John Wiley & Sons, Ltd. [source] Near Infrared Spectroscopy as a Tool for the Determination of Eumelanin in Human HairPIGMENT CELL & MELANOMA RESEARCH, Issue 4 2004Marina Zoccola Eumelanins are brown-black pigments present in the hair and in the epidermis which are acknowledged as protection factors against cell damage caused by ultraviolet radiation. The quantity of eumelanin present in hair has recently been put forward as a means of identifying subjects with a higher risk of skin tumours. For epidemiological studies, chromatographic methods of determining pyrrole-2,3,5-tricarboxylic acid (PTCA; the principal marker of eumelanin) are long, laborious and unsuitable for screening large populations. We suggest near infrared (NIR) spectroscopy as an alternative method of analysing eumelanin in hair samples. PCTA was determined on 93 samples of hair by means of oxidizing with hydrogen peroxide in a basic environment followed by chromatographic separation. The same 93 samples were then subjected to NIR spectrophotometric analysis. The spectra were obtained in reflectance mode on hair samples which had not undergone any preliminary treatment, but had simply been pressed and placed on the measuring window of the spectrophotometer. The PTCA values obtained by means of HPLC were correlated with the near infrared spectrum of the respective samples. A correlation between the PTCA values obtained by means of HPLC and the PTCA values obtained from an analysis of the spectra was obtained using the principal component regression (PCR) algorithm. The correlation obtained has a coefficient of regression (R2) of 0.89 and a standard error of prediction (SEP) of 13.8 for a mean value of 108.6 ng PTCA/mg hair. Some considerations about the accuracy of the obtained correlation and the main sources of error are made and some validation results are shown. [source] Development of a liquid chromatography/electrospray selected reaction monitoring method for the determination of organoarsenic species in marine and freshwater samplesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 19 2006Richard Schaeffer Cation- and anion-exchange high-performance liquid chromatography/electrospray selected reaction monitoring (HPLC/ES-SRM) methods were developed for the determination of 15 organoarsenic compounds in marine and freshwater samples. The results demonstrate that the developed HPLC/ES-SRM methods are powerful approaches for the identification of organoarsenic species in crude sample extracts. The detection limits, linearity as well as reproducibility for most of the species are comparable or even better than those measured by the HPLC/inductively coupled plasma mass spectrometry (ICPMS) technique. The qualitative analysis of the extracts shows that the developed methods allow for the identification of arsenicals which were not detectable by ICPMS. It was also demonstrated that the signal suppression caused by matrix effects means a significant limitation in the quantification of arsenicals by ES-SRM detection. This drawback is manifested especially in the case of the slightly retained species. The three sample-cleanup chromatographic methods including off-line size-exclusion, on-line reversed-phase and on-line oppositely charged ion-exchange approaches proved to be ineffective for separation of the signal-suppressive matrix from the analytes. The standard addition calibration seems to be a suitable solution for such problems. Copyright © 2006 John Wiley & Sons, Ltd. [source] Observation of different ceramide species from crude cellular extracts by normal-phase high-performance liquid chromatography coupled to atmospheric pressure chemical ionization mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 11 2003Benjamin J. Pettus Normal-phase high-performance liquid chromatography (NP-HPLC) coupled to atmospheric pressure chemical ionization mass spectrometry (APCI-MS) allows qualitative analysis of endogenous ceramide and dihydroceramide species from crude lipid extracts utilizing chromatographic methods readily adaptable from commonly used thin layer chromatography (TLC) conditions. Qualitative information for the species comes from observation of differences in chromatographic and mass spectrometric behavior between species. Application to the analysis of ceramide and dihydroceramide from various cell lines is demonstrated. The results show the species profile in each cell line to be unique despite growth under identical conditions. The results from APCI-MS analysis corroborate and enhance information acquired from use of the diacylglycerol kinase assay for total ceramide measurement. This technique readily allows the previously difficult distinction between ceramide and dihydroceramide species. Copyright © 2003 John Wiley & Sons, Ltd. [source] Liquid chromatography/tandem mass spectrometric quantification with metabolite screening as a strategy to enhance the early drug discovery processRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 12 2002Philip R. Tiller Throughput for early discovery drug metabolism studies can be increased with the concomitant acquisition of metabolite screening information and quantitative analysis using ultra-fast gradient chromatographic methods. Typical ultra-fast high-performance liquid chromatography (HPLC) parameters used during early discovery pharmacokinetic (PK) studies, for example, employ full-linear gradients over 1,2,min at very high flow rates (1.5,2,mL/min) on very short HPLC columns (2,×,20,mm). These conditions increase sample throughput by reducing analytical run time without sacrificing chromatographic integrity and may be used to analyze samples generated from a variety of in vitro and in vivo studies. This approach allows acquisition of more information about a lead candidate while maintaining rapid analytical turn-around time. Some examples of this approach are discussed in further detail. Copyright © 2002 John Wiley & Sons, Ltd. [source] Structural studies of MIP synthaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2000Adam J. Stein The conversion of glucose 6-phosphate to 1- l - myo -inositol 1-phosphate (MIP) by 1- l - myo -inositol 1-phosphate synthase (MIP synthase) is the first committed and rate-limiting step in the de novo biosynthesis of inositol in all eukaryotes. The importance of inositol-containing molecules both as membrane components and as critical second messenger signal-transduction species make the function and regulation of this enzyme important for a host of biologically important cellular functions including proliferation, neurostimulation, secretion and contraction. MIP synthase has been overexpressed in Esherichia coli and purified to homogeneity by chromatographic methods. Two crystal forms of MIP synthase were obtained by the hanging-drop vapor-diffusion method. Native data sets for both crystal forms were collected in-house on a Rigaku R-AXIS IIC imaging-plate detector. Crystal form I belongs to space group C2, with unit-cell parameters a = 153.0, b = 96.6, c = 122.6,Å, , = 126.4°, and diffracts to 2.5,Å resolution. Crystal form II belongs to space group P21, with unit-cell parameters a = 94.5, b = 186.2, c = 86.5,Å, , = 110.5°, and diffracts to 2.9,Å resolution. [source] 5-in-1 biodiesel: an approach to combining five biodiesel gas chromatographic methods on a single instrumentBIOFUELS, BIOPRODUCTS AND BIOREFINING, Issue 3 2009James D. McCurry James D. McCurry and Wesley Norman report on a new technology developed at Agilent Technologies. A small external, isothermal, capillary-column oven is used to isolate a polar wax column from a high-temperature column on a single gas chromatograph (GC). This allows five different biodiesel methods to reside on the same GC which would otherwise require two separate instruments. The isothermal biodiesel methods are used to test the external column oven performance and demonstrate the ability to combine these methods on a single GC. © 2009 Society of Chemical Industry and John Wiley & Sons, Ltd [source] Chromatography of amino acids and short peptides.BIOMEDICAL CHROMATOGRAPHY, Issue 8 2007New advances Abstract The newest results in the application of various chromatographic methods (gas,liquid chromatography, liquid chromatographic techniques, electrically driven systems) for the separation and quantitative determination of amino acids and short peptides in pure state and in complicated matrices are compiled. The results are concisely described and critically evaluated. The future trends of the chromatographic analysis of amino acids and short peptides are briefly discussed. Copyright © 2007 John Wiley & Sons, Ltd. [source] Investigation of lipophilicity of anticancer-active thioquinoline derivativesBIOMEDICAL CHROMATOGRAPHY, Issue 2 2007Marek Bajda Abstract The lipophilicity of a series of anticancer propargylthioquinoline derivatives has been investigated using chromatographic and computational methods. The parameters of relative lipophilicity (RMO and logk0) of the tested compounds were determined experimentally both by reversed-phase thin layer (RP-TLC), and high-performance liquid chromatographic methods (RP-HPLC, LiChrospher RP18 column), with mixtures of acetonitrile and water as mobile phases. Their phospholipophilicity (logk0IAM) was determined using immobilized artificial membrane HPLC (IAM. PC DD2 Regis column). Mobile phase acetonitrile concentrations were in the ranges 50,90% (RP-TLC), 55,90% (RP-HPLC) and 35,60% (IAM-HPLC). The RM, logk and logkIAM values of the compounds investigated were linearly dependent on acetonitrile concentration. The analysis led to the calculation of RMO, logk0 and logk0IAM parameter values for each of the tested compounds. Their partition coefficients (logP) were also calculated with the Pallas and CAChe programs. The obtained results indicated that, among experimental methods, both RP-TLC and RP-HPLC gave similar results, and these methods enable the determination of lipophilicity of derivatives of thioquinolines. Using the IAM-HPLC technique a simple method of estimation of phospholipoplilicity was described. The CAChe program might better predict calculated lipophilicity logP values, and therefore is a useful tool for the early stage of design of new propargyl thioquinolines. Copyright © 2006 John Wiley & Sons, Ltd. [source] Comparison between micellar liquid chromatography and capillary zone electrophoresis for the determination of hydrophobic basic drugs in pharmaceutical preparationsBIOMEDICAL CHROMATOGRAPHY, Issue 1 2007S. Torres-Cartas Abstract The determination of highly hydrophobic basic compounds by means of conventional reversed-phase liquid chromatographic methods has several drawbacks. Owing to the characteristics of micellar liquid chromatography (MLC) and capillary electrophoresis (CE), these techniques could be advantageous alternatives to reversed-phase chromatographic methods for the determination of these kinds of compounds. The objective of this study was to develop and compare MLC and CE methods for the determination of antipsychotic basic drugs (amitryptiline, haloperidol, perphenazine and thioridazine) in pharmaceutical preparations. The chromatographic determination of the analytes was performed on a Kromasil C18 analytical column; the mobile phase was 0.04 m cetyltrimethylammonium bromide (CTAB), at pH 3, containing 5% 1-butanol, at a flow rate of 1 mL/min. The CE separation was performed in a fused-silica capillary with a 50 mm tris-(hydroxymethyl)-aminomethane buffer, pH 7, at an applied voltage of 20 kV, using barbital as internal stardard. The proposed methods are suitable for a reliable quantitation of these compounds in the commercial tablets and drops in terms of accuracy and precision and require a very simple pre-treatment of the samples. By comparing the performance characteristics and experimental details of the MLC and CE methods we conclude that CE seems to be slightly better than MLC in the determination of highly hydrophobic compounds in pharmaceuticals in terms of resolution and economy, taking into account that the limits of detection are not a handicap in pharmaceutical samples. Copyright © 2006 John Wiley & Sons, Ltd. [source] Evaluation of 3-hydroxy-3-methylglutaryl-COA-reductase, cholesterol-7, -hydroxylase and acyl-COA:cholesterol acyltransferase activities: alternative chromatographic methods to separate metabolitesBIOMEDICAL CHROMATOGRAPHY, Issue 9 2004Alain Montoudis Abstract Alternative HPLC and solid-phase extraction column methods were developed to separate metabolites of enzymes involved in cholesterol metabolism in rabbit liver microsomes: hydroxyl-methylglutaryl-CoA reductase, cholesterol-7, -hydroxylase and acyl-CoA:cholesterol acyltransferase. A comparison method of thin-layer chromatography and solid-phase extraction column were assayed to separate substrate and metabolite of hydroxy-methylglutaryl-CoA reductase, whereas for cholesterol-7, -hydroxylase and acyl-CoA:cholesterol acyltransferase, this comparison was done between thin layer chromatography and HPLC. The results obtained by the new analytical chromatographic methods are not signi,cantly different than those observed in literature. Moreover a larger percentage recovery was obtained for analysed metabolites. Our results demonstrate the reliability of these alternative chromatographic techniques and showed that they are valuable tools to precisely and rapidly measure the activity of those enzymes. Copyright © 2004 John Wiley & Sons, Ltd. [source] Chromatographic determination of herbicide residues in various matricesBIOMEDICAL CHROMATOGRAPHY, Issue 6 2004Tibor Cserháti Abstract The newest results in the use of various extraction techniques and chromatographic methods such as gas,liquid and high-performance liquid chromatography used for the assessment of herbicide residues in various matrices have been compiled and critically evaluated. Practical employments in water and soil research, environmental protection, clinical and food chemistry are presented. Copyright © 2004 John Wiley & Sons, Ltd. [source] Determination of terbinafine hydrochloride in cat hair by two chromatographic methodsBIOMEDICAL CHROMATOGRAPHY, Issue 8 2001Jernej Kuz Terbinafine hydrochloride (terbHCl) concentration on the site of infection with Microsporum canis is a very important indicator of drug effectiveness. Several chromatographic methods exist that can be used for the determination of terbHCl concentration in biological samples. A high performance liquid chromatographic (HPLC) method and a gas chromatographic (GC) method have been compared and critically evaluated for the determination of a terbHCl levels in cat hair. The sensitivity and the linearity of the previously developed HPLC method were 0.25,ng/mL and 0.25,3000,ng/mL, respectively. The limit of quantification (LOQ) was 0.01,µg/g of terbHCl in cat hair, and reproducibility of 96.6% and recovery of 93.8% were achieved using appropriate sample pre-treatment and optimal chromatographic conditions. The sensitivity of the GC method, 25,ng/mL (LOQ 625 ppb), was much lower than that of the HPLC method. The GC method still enables determination of terbHCl in a range of concentrations in cat hair. The reproducibility of terbHCl for the cat hair samples was 95.3% and the recovery was only 70.0%. Both methods can be used for the evaluation of drug effectiveness in cats and both of them require only basic chromatographic equipment that can be found in most analytical laboratories. Copyright © 2001 John Wiley & Sons, Ltd. [source] Crystallization and preliminary X-ray analysis of three dUTPases from Gram-positive bacteriaACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2009Gui-Lan Li All organisms examined to date possess a dUTPase that performs the important function of efficiently hydrolyzing dUTP to dUMP in order to prevent the incorporation of dUTP into DNA. Three putative dUTPases from Gram-positive bacteria have been studied in this work. Two dUTPase-encoding genes, yncF and yosS, have been identified in Bacillus subtilis. The gene dut, encoding dUTPase from the dental pathogen Streptococcus mutans, was amplified from S. mutans genomic DNA. The three genes were cloned into expression vectors and overexpressed at high levels in Escherichia coli. Each protein was purified in two steps using chromatographic methods. Crystals of the YosS and YncF proteins and of S. mutans dUTPase were obtained using the vapour-diffusion method. X-ray diffraction data sets were collected from crystals of selenomethionine-labelled YosS and S. mutans dUTPase to resolutions of 2.3 and 1.7,Å, respectively. The crystal of native YncF diffracted to 2.7,Å resolution. [source] Ultra scale-down of protein refold screening in microwells: Challenges, solutions and applicationBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2009Gareth J. Mannall Abstract Steps for the refolding of proteins from solubilized inclusion bodies or misfolded product often represent bottlenecks in process development, where optimal conditions are typically derived empirically. To expedite refolding optimization, microwell screening may be used to test multiple conditions in parallel. Fast, accurate, and reproducible assays are required for such screening processes, and the results derived must be representative of the process at full scale. This article demonstrates the use of these microscale techniques to evaluate the effects of a number of additives on the refolding of IGF-1 from denatured inclusion bodies, using an established HPLC assay for this protein. Prior to this, microwell refolding was calibrated for scale-up using hen egg-white lysozyme (HEWL) as an initial model protein, allowing us to implement and compare several assays for protein refolding, including turbidity, enzyme activity, and chromatographic methods, and assess their use for microwell-based experimentation. The impact of various microplate types upon protein binding and loss is also assessed. Solution mixing is a key factor in protein refolding, therefore we have characterized the effects of different methods of mixing in microwells in terms of their impact on protein refolding. Our results confirm the applicability and scalability of microwell screening for the development of protein refolding processes, and its potential for application to new inclusion body-derived protein products. Biotechnol. Bioeng. 2009;103: 329,340. © 2008 Wiley Periodicals, Inc. [source] Preclinical Manufacture of Anti-HER2 Liposome-Inserting, scFv-PEG-Lipid Conjugate.BIOTECHNOLOGY PROGRESS, Issue 1 2005Analytical methods optimized for micellar F5cys-MP-PEG(2000)-DPSE protein-lipopolymer conjugate are presented. The apparent micelle molecular weight, determined by size exclusion chromatography, ranged from 330 to 960 kDa. The F5cys antibody and conjugate melting points, determined by differential scanning calorimetry, were near 82 °C. Traditional methods for characterizing monodisperse protein species were inapplicable to conjugate analysis. The isoelectric point of F5cys (9.2) and the conjugate (8.9) were determined by capillary isoelectric focusing (cIEF) after addition of the zwitterionic detergent CHAPS to the buffer. Conjugate incubation with phospholipase B selectively removed DSPE lipid groups and dispersed the conjugate prior to separation by chromatographic methods. Alternatively, adding 2-propanol (29.4 vol %) and n -butanol (4.5 vol %) to buffers for salt-gradient cation exchange chromatography provided gentler, nonenzymatic dispersion, resulting in well-resolved peaks. This method was used to assess stability, identify contaminants, establish lot-to-lot comparability, and determine the average chromatographic purity (93%) for conjugate lots, described previously. The F5cys amino acid content was confirmed after conjugation. The expected conjugate avidity for immobilized HER-2/neu was measured by bimolecular interaction analysis (BIAcore). Mock therapeutic assemblies were made by conjugate insertion into preformed doxorubicin-encapsulating liposomes for antibody-directed uptake of doxorubicin by HER2-overexpressing cancer cells in vitro. Together these developed assays established that the manufacturing method as described in the first part of this study consistently produced F5cys-MP-PEG(2000)-DSPE having sufficient purity, stability, and functionality for use in preclinical toxicology investigations. [source] | |||||||||||||||||||||||||||||||