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Reversed-phase HPLC (reversed-phase + hplc)
Terms modified by Reversed-phase HPLC Selected AbstractsTrue and Apparent Temperature Dependence of Protein Adsorption Equilibrium in Reversed-Phase HPLCBIOTECHNOLOGY PROGRESS, Issue 6 2002Szabelski The adsorption behavior of bovine insulin on a C8 -bonded silica stationary phase was investigated at different column pressures and temperatures in isocratic reversed-phase HPLC. Changes in the molar volume of insulin (, Vm) upon adsorption were derived from the pressure dependence of the isothermal retention factor ( k,). The values of , Vm were found to be practically independent of the temperature between 25 and 50 °C at ,96 mL/mol and to increase with increasing temperature, up to ,108 mL/mol reached at 50 °C. This trend was confirmed by two separate series of measurements of the thermal dependence of ln( k,). In the first series the average column pressure was kept constant. The second series involved measurements of ln( k,) under constant mobile-phase flow rate, the average column pressure varying with the temperature. In both cases, a parabolic shape relationship was observed between ln( k,) and the temperature, but the values obtained for ln k, were higher in the first than in the second case. The relative difference in ln( k,), caused by the change in pressure drop induced by the temperature, is equivalent to a systematic error in the estimate of the Gibbs free energy of 12%. Thus, a substantial error is made in the estimates of the enthalpy and entropy of adsorption when neglecting the pressure effects associated with the change in the molar volume of insulin. This work proves that the average column pressure must be kept constant during thermodynamic measurements of protein adsorption constants, especially in RPLC and HIC. Our results show also that there is a critical temperature, Tc , 53 °C, at which ln( k,) is maximum and the insulin adsorption process changes from an exothermic to an endothermic one. This temperature determines also the transition point in the molecular mechanism of insulin adsorption that involves successive unfolding of the protein chain. [source] Electrospray ionization ion trap mass spectrometry for structural characterization of oligosaccharides derivatized with 2-aminobenzamideRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 9 2005Willy Morelle The use of electrospray ionization (ESI) quadrupole ion trap mass spectrometry and reversed-phase high-performance liquid chromatography (HPLC) for the characterization of 2-aminobenzamide (2AB)-labeled oligosaccharides and N-linked protein oligosaccharide mixtures is described. The major signals were obtained under these conditions from the [M+Na]+ ions for all 2AB-derivatized oligosaccharides. Under collision-induced dissociation, sodiated molecular species generated in the ESI mode yield simple and predictable mass spectra. Tandem mass spectrometry (MS/MS) experiments with orders higher than two offer a number of ways to enhance MS/MS spectra and to derive information not present in MS and MS2 spectra. Information on composition, sequence, branching and, to some extent, interglycosidic linkages can be deduced from fragments resulting from the cleavage of glycosidic bonds and from weak cross-ring cleavage products. Reversed-phase HPLC and derivatization by reductive amination using 2-aminobenzamide were finally applied to characterize a glycan pool enzymatically released from glycoproteins. Copyright © 2005 John Wiley & Sons, Ltd. [source] Guanosine diphosphate-4-keto-6-deoxy- d -mannose reductase in the pathway for the synthesis of GDP-6-deoxy- d -talose in Actinobacillus actinomycetemcomitansFEBS JOURNAL, Issue 23 2002Nao Suzuki The serotype a-specific polysaccharide antigen of Actinobacillus actinomycetemcomitans is an unusual sugar, 6-deoxy- d -talose. Guanosine diphosphate (GDP)-6-deoxy- d -talose is the activated sugar nucleotide form of 6-deoxy- d -talose, which has been identified as a constituent of only a few microbial polysaccharides. In this paper, we identify two genes encoding GDP-6-deoxy- d -talose synthetic enzymes, GDP-,- d -mannose 4,6-dehydratase and GDP-4-keto-6-deoxy- d -mannose reductase, in the gene cluster required for the biosynthesis of serotype a-specific polysaccharide antigen from A. actinomycetemcomitans SUNYaB 75. Both gene products were produced and purified from Escherichia coli transformed with plasmids containing these genes. Their enzymatic reactants were analysed by reversed-phase HPLC (RP-HPLC). The sugar nucleotide produced from GDP-,- d -mannose by these enzymes was purified by RP-HPLC and identified by electrospray ionization-MS, 1H nuclear magnetic resonance, and GC/MS. The results indicated that GDP-6-deoxy- d -talose is produced from GDP-,- d -mannose. This paper is the first report on the GDP-6-deoxy- d -talose biosynthetic pathway and the role of GDP-4-keto-6-deoxy- d -mannose reductase in the synthesis of GDP-6-deoxy- d -talose. [source] Antioxidant polyphenols from the mycelial culture of the medicinal fungi Inonotus xeranticus and Phellinus linteusJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2008J.-Y. Jung Abstract Aims:, The medicinal fungi Inonotus xeranticus and Phellinus linteus in the family Hymenochaetaceae have been used as traditional medicines for the treatment of various diseases. However, the compound responsible for the antioxidant activity is still unknown. Therefore, this study was conducted to characterize the antioxidant substances present in cultured broths made from these fungi. Methods and Results:, Antioxidant fractions of the cultured broths obtained from I. xeranticus and P. linteus were analysed using reversed-phase HPLC, which revealed several peaks that exhibited a potent free radical scavenging activity. To identify these antioxidant peaks, an I. xeranticus strain was mass-cultured, and the cultured broth was separated using antioxidant activity-guided fractionation. Four major active substances were purified and identified as hispidin and its dimers, 3,14,-bihispidinyl, hypholomine B, and 1,1-distyrylpyrylethan based on spectroscopic analyses. All compounds exhibited a significant scavenging activity against these radical species in a concentration-dependent manner. Conclusions:, Antioxidant substances found in the cultured broths of the medicinal fungi I. xeranticus and P. linteus were identified as hispidin and its dimers, 3,14,-bihispidinyl, hypholomine B, and 1,1-distyrylpyrylethan. Significance and Impact of the Study:, Polyphenol antioxidants were isolated from the cultured broth of the medicinal fungi I. xeranticus and P. linteus and identified based on extensive spectroscopic analyses. These compounds exhibited a strong antioxidant activity. [source] Photodegradation study of a new activator of the cystic fibrosis chloride channel, the 6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-07)JOURNAL OF PHARMACEUTICAL SCIENCES, Issue 2 2002Jean-Christophe Olivier Abstract The photodegradation of 6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-07), a new activator of the transmembrane conductance regulator chloride channel, was studied in aqueous solutions exposed to artificial daylight (2300 Lux intensity). Various conditions of pH, concentration, and temperature were used. MPB-07 concentration was determined at regular time intervals by reversed-phase HPLC. MPB-07 stability was also studied at pH 7.4 in the dark. Results showed that in all the conditions tested MPB-07 underwent rapid photodegradation, apparently following first-order kinetics. Rate constants were dependent on the initial MPB-07 concentration, temperature, and pH. At pH 7.4, and for concentrations from 1 to 125 ,M, half-lives ranged from 0.681,±,0.047 to 4.54,±,0.28 h. The Arrhenius plot was linear and activation energy was calculated to be 20.7 kJ,·,mol,1. Analysis by chemical ionization-mass spectrometry showed that the chlorine atom of the MPB-07 molecule might be replaced by an OH group during the photodegradation process. In the dark, MPB-07 in solutions at pH 7.4 was found to be stable over a 6-week period. In conclusion, MPB-07 is a highly photolabile molecule that should be carefully protected from light when used. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:324,330, 2002 [source] Comparative analysis of the chemical profile of wild and cultivated populations of Corydalis saxicola by high-performance liquid chromatographyPHYTOCHEMICAL ANALYSIS, Issue 5 2007Hui-liang Li Abstract Studies on the simultaneous determination and chemical fingerprinting of alkaloids in Corydalis saxicola Bunting. (Yanhuanglian) were performed for authentication purposes. Ninety samples prepared from different parts of C. saxicola, including whole plants, roots, stems, leaves and flowers, from wild and cultivated populations, were submitted to quantitative determination and fingerprint analysis. Five major alkaloids, namely, tetradehydroscoulerine, dehydroapocavidine, dehydroisoapocavidine, coptisine and dehydrocavidine, were quantitatively analysed by reversed-phase HPLC with acceptable recoveries (>98.2%). Chemical fingerprinting of C. saxicola was established and involved 11 markers. The results indicated that there were no obvious differences between the chemical profiles of wild and of cultivated C. saxicola populations, and that the mean alkaloid contents of the five marker compounds in cultivated populations were significantly higher than those of the wild plants. The highest content of total alkaloids (up to 28.8 mg/g) was found in roots of C. saxicola. The total alkaloids of the leaves were approximately 50% of those of roots, suggesting that the leaves may be employed as an alternative source of alkaloids. Chemical fingerprints and quantitative HPLC analysis will have a positive impact on the conservation and cultivation of this medicinal plant. Copyright © 2007 John Wiley & Sons, Ltd. [source] Determination of diarylheptanoids from Alpinia officinarum (lesser galangal) by HPLC with photodiode array and electrochemical detection,PHYTOCHEMICAL ANALYSIS, Issue 4 2005Zhihua Liu Abstract Normal-phase column chromatography followed by semi-preparative reversed-phase HPLC has been used to isolate, from the rhizomes of Alpinia officinarum, five diarylheptanoids identified as 5-hydroxy-7-(4,-hydroxy-3,-methoxyphenyl)-1-phenyl-3-heptanone, 5-methoxy-7-(4,-hydroxy-3,-methoxyphenyl)-1-phenyl-3-heptanone, 7-(4,-hydroxyphenyl)-1-phenylhept-4-en-3-one, 7-(4,-hydroxy-3,-methoxyphenyl)-1-phenyl-hept-4-en-3-one, 1,7-diphenylhept-4-en-3-one. The levels of these five diarylheptanoids in root material were determined quantitatively by HPLC with UV detection and the assay methods so developed were simple, rapid and accurate. Four of the diarylheptanoids could also be detected by HPLC with electrochemical detection (ECD) in the oxidative mode, and ECD was found to have a higher sensitivity than photodiode array detection. Copyright © 2005 John Wiley & Sons, Ltd. [source] Analytical characterisation of crude extracts from an african Ancistrocladus species using high-performance liquid chromatography and capillary electrophoresis coupled to ion trap mass spectrometryPHYTOCHEMICAL ANALYSIS, Issue 1 2004Matthias Unger Abstract The analysis by HPLC, CE and CE-MS/MS of root bark extracts of a, so far undescribed, Central-African Ancistrocladus species (family Ancistrocladaceae) is described. Owing to the complexity of the extract, the application of reversed-phase HPLC resulted in a partially incomplete separation of the naphthylisoquinoline alkaloids, whilst CE using a non-aqueous buffer proved to be a very valuable complementary method for a ,rst characterisation of the crude extract. By performing additional CE-MS/MS experiments, in combination with parallel isolation studies and structural elucidation using conventional methods, six alkaloidal substances present in the plant could be identi,ed. Copyright © 2004 John Wiley & Sons, Ltd. [source] Quanti,cation of sideroxylonals in Eucalyptus foliage by high-performance liquid chromatographyPHYTOCHEMICAL ANALYSIS, Issue 6 2003Ian R. Wallis Abstract This paper describes the extraction and quanti,cation of sideroxylonals, a group of formylated phloroglucinol compounds found in the foliage of some eucalypt species. Samples of dry, ground foliage were Soxhlet-extracted with light petroleum spirit:acetone (4:1) and the resultant extract analysed (in the presence of internal standard) by reversed-phase HPLC without further puri,cation. The yield of sideroxylonals was exponential with time and showed an in,ection at ca. 4 h of extraction. It is recommended that samples be extracted for 6 h, giving a 92% recovery of the sideroxylonals. The title compounds deteriorate under various conditions, e.g. 10% are lost when foliage is oven-dried at 40C compared to freeze-drying. Storing samples in mobile phase led to a slow deterioration of sideroxylonals with a 7% loss after 4 days, while 22% of these compounds were lost from dry, ground eucalypt leaf stored at room temperature for 20 months. Copyright © 2003 John Wiley & Sons, Ltd. [source] Evaluation of detection methods for the reversed-phase HPLC determination of 3,,4,,5,-trimethoxyflavone in different phytopharmaceutical products and in human serumPHYTOCHEMICAL ANALYSIS, Issue 2 2001Christian W. Huck Abstract Quantitative determination of the major compound, 3,,4,,5,-trimethoxyflavone (1), in plant extracts, in tablets of Flos and of Radix Primulae veris and in human serum has been accomplished using reversed-phase HPLC with UV, fluorescence and mass spectrometric (MS) detection. Compared to UV detection, fluorescence detection showed greater selectivity, was 10-fold more sensitive and allowed the determination of 1 in human serum after sample pre-treatment by solid-phase extraction. MS detection of 1 using electrospray ionisation (ESI) interface could be improved by substituting trifluoroacetic acid with the more polar and less conductive additive acetic acid, giving rise to a 230-fold improvement in analyte detectability at the cost of an increase of only 45% in the peak width of the eluting peak at its half height. Further optimisation of the acetic acid concentration showed the highest signal intensity at 1.25% for HPLC-atmospheric pressure chemical ionisation (APCI)-MS and at 0.75% for HPLC-ESI-MS. The optimised MS method proved to be extremely selective, 50 times more sensitive than UV detection and 5 times more sensitive than fluorescence detection. Furthermore, fragment-ion spectra produced by collision induced dissociation-MS have been used as "fingerprints" for identifying compounds in the highly complex mixtures examine. Copyright © 2001 John Wiley & Sons, Ltd. [source] Facile chemical synthesis and equilibrium unfolding properties of CopGPROTEIN SCIENCE, Issue 7 2004Thomas E. Wales Abstract The 45-amino acid polypeptide chain of the homodimeric transcriptional repressor, CopG, was chemically synthesized by stepwise solid phase peptide synthesis (SPPS) using a protocol based on Boc-chemistry. The product obtained from the synthesis was readily purified by reversed-phase HPLC to give a good overall yield (21% by weight). Moreover, the synthetic CopG constructs prepared in this work folded into three-dimensional structures similar to the wild-type protein prepared using conventional recombinant methods as judged by far UV-CD spectroscopy. A fluorescent CopG analog, (Y39W)CopG, was also designed and chemically synthesized to facilitate biophysical studies of CopG's protein folding and assembly reaction. The guanidinium chloride-induced equilibrium unfolding properties of the wild-type CopG and (Y39W)CopG constructs in this work were characterized and used to develop a model for CopG's equilibrium unfolding reaction. Our results indicate that CopG's folding and assembly reaction is well modeled by a two-state process involving folded dimer and unfolded monomer. Using this model, ,Gf and m -values of ,13.42 ± 0.04 kcal/mole dimer and 1.92 ± 0.01 kcal/(mole M) were calculated for CopG. [source] Development of a multi-residue method for the determination of 18 carbamates in tobacco by high-performance liquid chromatography/positive electrospray ionisation tandem mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 4 2006B. Mayer-Helm A multi-residue method for the determination of carbamates in tobacco was developed by high-performance liquid chromatography (HPLC) triple quadrupole mass spectrometry (MS). A rapid sample preparation consisted of an extraction step with methanol, centrifugation and 1:1 dilution with aqueous 10,mM ammonium acetate. After filtration these extracts were directly analysed by reversed-phase HPLC coupled to positive electrospray ionisation tandem mass spectrometry operated in the multiple reaction monitoring mode. Capillary voltage and dwell times were optimised to reduce matrix effects and to increase sensitivity. The method was validated for the determination of 18 carbamates in three main types of raw tobacco and three tobacco products. The interday accuracy ranged between 80 and 110% with a relative standard deviation (RSD) of <30%. The limits of quantification (LOQs) ranged between 0.01 and 0.04,ppm for almost all carbamates, except aldicarb sulfone, carbofuran, and pebulate, with LOQs between 0.10 and 0.20,ppm. These LOQs were clearly below the guidance residue levels defined by the Agrochemical Advisory Committee of CORESTA, an association of organisations having scientific research relative to tobacco. Copyright © 2006 John Wiley & Sons, Ltd. [source] Analysis of tannins in seeds and skins of Shiraz grapes throughout berry developmentAUSTRALIAN JOURNAL OF GRAPE AND WINE RESEARCH, Issue 1 2003MARK O. DOWNEY Abstract The flavan-3-ol and proanthocyanidin composition of both seeds and skin of Vitis vinifera L. cv. Shiraz grapes was determined by reversed-phase HPLC after acetone extraction and acid-catalysis in the presence of excess phloroglucinol. Samples were taken at weekly intervals from fruit-set until commercial harvest. The main period of proanthocyanidin accumulation in grape seeds occurred immediately after fruit-set with maximum levels observed around veraison. Over two seasons there was variation in both the timing and content of proanthocyanidins in seeds. In skin, proanthocyanidin accumulation occurred from fruit set until 1,2 weeks after veraison. Proanthocyanidin subunit composition was different in seeds and skin and changed during berry development but the mean degree of polymerisation of the tannin polymers in skins was higher than in the seeds at all stages of berry development. Proanthocyanidin levels in both seeds and skin decreased between veraison and harvest. Additional proanthocyanidin subunits were released when the residues remaining after acetone extraction were subjected to direct acid-catalysis in the presence of phloroglucinol. In the seeds, these accounted for much of the post-veraison decrease, but not in grape skin. At harvest, 75% of extractable berry proanthocyanidin was in the seeds. Accumulation of proanthocyanidins in the seeds appears to be independent of that in the skins, but in both tissues synthesis occurs early in berry development and maximum levels are reached around veraison. [source] Long-term streptozotocin-induced diabetes alters prostanoid production in rat aorta and mesenteric bedAUTONOMIC & AUTACOID PHARMACOLOGY, Issue 4 2006H. A. Peredo Summary 1 Vascular disease is a major cause of mortality and morbidity in chronic diabetes mellitus. 2 Prostanoids, metabolites of arachidonic acid, include vasoactive substances produced and released from the vascular wall. Alterations in prostanoid production have been reported in the vasculature of diabetic humans and experimental animals. 3 The aim of the present work was to study the influence of three different periods of long-term streptozotocin-induced diabetes, 30, 120 and 180 days in the production of prostanoids in the thoracic aorta and in the mesenteric vascular bed of the rat. The prostanoids released to the incubation medium by the tissues were extracted and measured by reversed-phase HPLC. 4 In the diabetic groups, body weight was reduced and glycaemia was increased when compared with the corresponding non-diabetic controls. 5 In the aorta, 30 days of diabetes did not modify the prostanoid release pattern, meanwhile 120 and 180 days of incubation decreased prostacyclin (PGI2) production. In the mesenteric bed, at 30 days the release of the vasodilators PGI2 and prostaglandin (PGE2) and the vasoconstrictor thromboxane (TXA2) was reduced. At 120 days the vasodilators were reduced and at 180 days such reduction was joined by an increase of the release of vasoconstrictor metabolites. 6 Thirty days of diabetes did not modify the PGI2/TXA2 ratio in the aorta or mesenteric bed. On the other hand, 120 and 180 days of diabetes reduced significantly the ratio when compared with the corresponding controls. 7 In conclusion, the mesenteric bed, a resistance vascular bed, seems to be more sensitive than the aorta, a conductance vessel, to the effects of diabetes on prostanoid production. The observed effects contribute to a displacement of the balance of prostanoid release in favour of the vasoconstrictor metabolites, a phenomenon that could be related to the vascular complications of diabetes mellitus. [source] Determination of carboplatin in canine plasma by high-performance liquid chromatographyBIOMEDICAL CHROMATOGRAPHY, Issue 8 2010Nicolas Villarino Abstract Carboplatin is an antineoplastic drug administered to treat different tumoral conditions in canine oncology. The objective of this study was to validate a high-performance chromatographic (HPLC) method which could be applied in canine pharmacokinetic studies. Following ultrafiltration using a Centrifree device, standards, quality controls and plasma samples were separated by isocratic reversed-phase HPLC on an Inertsil ODS-2 (250 × 4.6,mm i.d.) analytical column and quantified using UV detection at 220,nm. The mobile phase was potassium phosphate (pH 4.5), with a flow-rate of 1.0,mL/min. The procedure produced a linear curve (r2 > 0.999) over the concentration range 1,200,,g/mL. The lower limit of quantification was 1,,g/mL. The intra-assay and inter-assay precision was ,90%. The overall recovery was ,90%. The method was illustrated with a preliminary pharmacokinetic analysis on nine dogs treated with carboplatin at our hospital. Carboplatin disposition followed a monocompartmental structure in dogs and was characterized by a short half-life (50,min). Copyright © 2009 John Wiley & Sons, Ltd. [source] Development of a fluorimetric detection method for cinnabarinic acid using ortho -tolyl hydrazine as the derivatization reagentBIOMEDICAL CHROMATOGRAPHY, Issue 3 2010Hideaki Iizuka Abstract A fluorimetric detection method for one of the tryptophan metabolites, cinnabarinic acid (CA), which has recently been reported to have the ability to induce apoptosis in thymocytes, was developed using o -tolyl hydrazine (TH) as the derivatization reagent. The carbonyl group at position 3 in CA was tagged with the hydrazino moiety of TH at 100°C for 30 min, and the generated derivative, CA tagged with TH, fluoresced at 412 nm with a 316 nm excitation wavelength. The CA tagged with TH was separated on a reversed-phase HPLC and detected fluorometrically. The relative standard deviation was in the range of 1.1,8.9% (n = 3), and the detection limit was approximately 12?fmol (signal-to-noise ratio, 3). The proposed HPLC method can be useful for the sensitive detection of CA. Copyright © 2009 John Wiley & Son, Ltd. [source] Liquid chromatographic method for the determination of sirolimus in blood using electrochemical detectionBIOMEDICAL CHROMATOGRAPHY, Issue 3 2009Nobuo Mochizuki Abstract Therapeutic drug monitoring of sirolimus (rapamycin) is important for immunosuppressive therapy in solid organ transplantation. We have developed a simple and reliable method for determining blood concentrations of sirolimus using reversed-phase HPLC with electrochemical detection (ECD). The E2 potential was set at +900 mV. The potential of guard cell was set at +950 mV and that of the E1 cell at +400 mV. The method was linear for a concentration range of 1,50 ng/mL when 0.5 mL blood was used. The correlation coefficients of all standard curves were greater than or equal to 0.999. The limit of detection was 0.5 ng/mL. The inter-assay precision ranged from 3.22 to 7.48%, and the coefficient of variation (CV) for a quality control sample at 10 ng/mL was 7.48% with a bias of 8.4% from the target value. The intra-assay precision ranged from 0.72 to 3.71%, and the CV for a quality control sample at 10 ng/mL was 0.72% with a bias of 6.8% from the target value. In a solid organ transplant recipient, trough concentrations of sirolimus were well within the analytic range of the HPLC/ECD procedure. The method described here is suitable for management of sirolimus therapy in solid organ transplantation. Copyright © 2008 John Wiley & Sons, Ltd. [source] Liquid chromatographic fluorescence determination of amino acids in plasma and urine after derivatization with phanquinoneBIOMEDICAL CHROMATOGRAPHY, Issue 2 2008Rita Gatti Abstract Phanquinone (4,7-phenanthroline-5,6-dione) has been investigated as a pre-column derivatization fluorogenic reagent for liquid chromatographic determination of primary amino acids in biological samples. The derivatization reaction was carried out at 68°C both in the presence of aqueous phosphate buffer (pH 8) for 30 min and without buffer for 60 min to allow the determination of basic amino acids (Orn, Lys, Arg). The resulting derivatives were separated under reversed-phase HPLC and detected at ,em = 460 nm with ,ex = 400 nm. The proposed method was validated and applied to the determination of a variety of amino acids directly in urine and after deproteinization with 5-sulfosalicylic acid in plasma samples. The detection and quantitation limits were found in the range 10,450 and 35,1400 fmol, respectively. Copyright © 2007 John Wiley & Sons, Ltd. [source] A sensitive and selective determination method of histamine by HPLC with intramolecular excimer-forming derivatization and ,uorescence detectionBIOMEDICAL CHROMATOGRAPHY, Issue 8 2003Takashi Yoshitake Abstract A highly sensitive, selective and simple method is described for the determination of histamine by high-performance liquid chromatography (HPLC) with ,uorescence detection. The method is based on an intramolecular excimer-forming ,uorescence derivatization of histamine with 4-(1-pyrene)butyric acid N -hydroxysuccinimide ester (PSE), followed by reversed-phase HPLC. Histamine, having two amino moieties in a molecule, was converted to the dipyrene-labeled derivative by reaction with PSE. The derivative afforded intramolecular excimer ,uorescence (450,540 nm), which can clearly be discriminated from the monomer ,uorescence (370,420 nm) emitted from PSE. Typically, a 10 µL sample solution was mixed with 100 µL of derivatization reagent solution, which was a mixture of 0.5 mm PSE in acetonitrile and 0.5 mm potassium carbonate in water (8:2, v/v). The derivatization was carried out at 100°C for 90 min. The PSE derivative of histamine could be separated by reversed-phase ODS column with isocratic elution using acetonitrile:water (82:18, v/v) containing 0.03% triethylamine. The detection limit (singnal-to-noise ratio = 3) of histamine was 0.5 fmol for a 30 µL injection. The method was successfully applied to the determination of histamine in human urine, and had enough selectivity and sensitivity for urinary histamine quanti,cation. Copyright © 2003 John Wiley & Sons, Ltd. [source] Analysis of lipoprotein lipase activity using high-performance liquid chromatographyBIOMEDICAL CHROMATOGRAPHY, Issue 8 2002Yukinori Eguchi Lipoprotein lipase (LPL) is a key enzyme which regulates the plasma triglyceride concentration by hydrolyzing triglycerides in chylomicrons and very-low-density lipoprotein (VLDL). The activity of LPL was conventionally analyzed using radio-labeled residues or direct sandwich-ELISA. An assay for lipoprotein lipase activity which used a nonradioactive substrate, tri-olein, is described. In this method, LPL activity was detected fluorometrically by reacting 9-anthryldiazomethane (ADAM) with the oleic acid generated from tri-olein by enzyme activity and separated by reversed-phase HPLC. This method has been optimized and the optimum enzyme incubation time and reaction time of the generated oleic acid with ADAM were both at 20,min. The method correlated well with the conventional method. Copyright © 2002 John Wiley & Sons, Ltd. [source] Simultaneous determination of ondansetron and tropisetron in human plasma using HPLC with UV detectionBIOMEDICAL CHROMATOGRAPHY, Issue 3 2002Steffen Bauer A rapid and sensitve HPLC method for the simultaneous quantitation of ondansetron and tropisetron, two serotonin (5-HT) receptor antagonists frequently used in treatment and prevention of nausea and emesis, is described. The procedure involves liqid,liquid extraction of human plasma with dichloromethane coupled with reversed-phase HPLC and UV detection. The lower limits of quantification (LOQ) were 0.62,ng/mL for ondansetron and 1.25,ng/mL or tropisetron. Intra- and inter-assay coefficients of variation ranged from 1.5 to 7.5% and 5.3 to 13.7%, respectively. The sensitivity and precision were sufficient for determination of plasma concentrations after therapeutic administration of both drugs and the method can be used for the estimation of pharmacokinetic parameters. Copyright © 2002 John Wiley & Sons, Ltd. [source] True and Apparent Temperature Dependence of Protein Adsorption Equilibrium in Reversed-Phase HPLCBIOTECHNOLOGY PROGRESS, Issue 6 2002Szabelski The adsorption behavior of bovine insulin on a C8 -bonded silica stationary phase was investigated at different column pressures and temperatures in isocratic reversed-phase HPLC. Changes in the molar volume of insulin (, Vm) upon adsorption were derived from the pressure dependence of the isothermal retention factor ( k,). The values of , Vm were found to be practically independent of the temperature between 25 and 50 °C at ,96 mL/mol and to increase with increasing temperature, up to ,108 mL/mol reached at 50 °C. This trend was confirmed by two separate series of measurements of the thermal dependence of ln( k,). In the first series the average column pressure was kept constant. The second series involved measurements of ln( k,) under constant mobile-phase flow rate, the average column pressure varying with the temperature. In both cases, a parabolic shape relationship was observed between ln( k,) and the temperature, but the values obtained for ln k, were higher in the first than in the second case. The relative difference in ln( k,), caused by the change in pressure drop induced by the temperature, is equivalent to a systematic error in the estimate of the Gibbs free energy of 12%. Thus, a substantial error is made in the estimates of the enthalpy and entropy of adsorption when neglecting the pressure effects associated with the change in the molar volume of insulin. This work proves that the average column pressure must be kept constant during thermodynamic measurements of protein adsorption constants, especially in RPLC and HIC. Our results show also that there is a critical temperature, Tc , 53 °C, at which ln( k,) is maximum and the insulin adsorption process changes from an exothermic to an endothermic one. This temperature determines also the transition point in the molecular mechanism of insulin adsorption that involves successive unfolding of the protein chain. [source] Hydrolytic Reactions of Thymidine 5,- O -Phenyl- N -Alkylphosphoramidates, Models of Nucleoside 5,-Monophosphate ProdrugsCHEMISTRY - A EUROPEAN JOURNAL, Issue 30 2007Mikko Ora Dr. Abstract To obtain detailed data on the kinetics of hydrolytic reactions of triester-like nucleoside 5,- O -aryl- N -alkylphosphoramidates, potential prodrugs of antiviral nucleoside monophosphates, the hydrolysis of diastereomeric (RP/SP) thymidine 5,-{O -phenyl- N -[(1S)-2-oxo-2-methoxy-1-methylethyl]phosphoramidate} (3), a phosphoramidate derived from the methyl ester of L -alanine, has been followed by reversed-phase HPLC over the range from H0=0 to pH,8 at 90,°C. According to the time-dependent product distributions, the hydrolysis of 3 proceeds at pH<4 by two parallel routes, namely by nucleophilic displacement of the alaninyl ester moiety by a water molecule and by hydrolysis of the carboxylic ester linkage that allows intramolecular attack of the carboxy group on the phosphorus atom, thereby resulting in the departure of either thymidine or phenol without marked accumulation of any intermediates. Both routes represent about half of the overall disappearance of 3. The departure of phenol eventually leads to the formation of thymidine 5,-phosphate. At pH>5, the predominant reaction is hydrolysis of the carboxylic ester linkage followed by intramolecular displacement of a phenoxide ion by the carboxylate ion and hydrolysis of the resulting cyclic mixed anhydride into an acyclic diester-like thymidine 5,-phosphoramidate. The latter product accumulated quantitatively without any indication of further decomposition. Hydroxide-ion-catalyzed POPh bond cleavage of the starting material 3 occurred as a side reaction. Comparative measurements with thymidine 5,-{N -[(1S)-2-oxo-2-methoxy-1-methylethyl]phosphoramidate} (4) revealed that, under acidic conditions, this diester-like compound is hydrolyzed by PN bond cleavage three orders of magnitude more rapidly than the triester-like 3. At pH>5, the stability order is reversed, with 3 being hydrolyzed six times as rapidly as 4. Mechanisms of the partial reactions are discussed. [source] Structure Elucidation and Phytotoxicity of Ecdysteroids from Chenopodium albumCHEMISTRY & BIODIVERSITY, Issue 4 2005Marina DellaGreca The leaves of Chenopodium album were infused in H2O/MeOH. The extract treated with cold acetone gave heavy precipitation, which was removed by centrifugation. Solid material was fractionated into acidic and neutral fractions. The acidic material was subjected to different silica-gel column chromatographies, and then it was purified by reversed-phase HPLC to afford four known ecdysteroids and the new 3,,14, -dihydroxy-5, -pregn-7-ene-2,6,20-trione that were characterized by extensive spectroscopic investigation, especially by 1D- and 2D-NMR. Their effects on germination and growth of Lactuca sativa L. have been studied. The results are reported as percentage differences of germination, root elongation and shoot elongation, from the control at concentrations ranging from 10,4 to 10,7,M. [source] Enantioselective chromatography of alkyl derivatives of 5-ethyl-5-phenyl-2-thiobarbituric acid studied by semiempirical AM1 methodCHIRALITY, Issue 8 2002Krzysztof Zborowski Abstract Complexation of alkyl derivatives of 5-ethyl-5-phenyl-2-thiobarbituric acid (2-thiophenobarbital) enantiomers by ,-cyclodextrin was investigated by the AM1 method. The inclusion complexes of ,-cyclodextrin with neutral and anionic forms of these enantiomers have been modeled and energetically optimized. The chiral discrimination of enantiomers was analyzed in terms of differences in the interaction energies. The calculated interaction energies between each enantiomer of the investigated 2-thiobarbiturates and ,-cyclodextrin confirm the ability of ,-cyclodextrin to act as a mobile phase additive in reversed-phase HPLC to separate enantiomers by liquid chromatography and rationalize their order of elution. Chirality 14:632,637, 2002. © 2002 Wiley-Liss, Inc. [source] | |||||||||||||