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HPLC Column (hplc + column)
Selected AbstractsUse of LC,MS,MS for the rapid, specific and sensitive quality control measurement of carrier in a PET radioligand: [18F]FECNTJOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 13 2005H. Umesha Shetty Abstract In the production of radioligands for imaging low concentrations of target proteins (e.g. receptors or transporters) in human subjects with positron emission tomography, control of specific radioactivity is necessary for efficacy and safety. Such quality control requires a fast method to be available for measuring carrier (non-radioactive ligand) in each batch of radioligand, preceding its release for administration. Measurement is usually achieved with HPLC equipped with an ultraviolet (UV) absorbance detector. However, this method is not easily applicable to radioligands that have low UV extinction coefficients and are produced at high specific radioactivity, such as [18F] (2,-carbomethoxy-3,-(4-chlorophenyl)-8-(2-fluoroethyl)nortropane; [18F]FECNT). Here we describe a fast, specific and sensitive LC,MS,MS method for measuring carrier in [18F]FECNT preparations. Small samples of formulated [18F]FECNT plus an added internal standard (2,-carbomethoxy-3,-(4-chlorophenyl)-8-(n -propyl)nortropane; INTSTD) are rapidly eluted from a short reverse phase HPLC column into an MS probe. Following electrospray ionization, the molecular ions ([MH]+) of FECNT (m/z=326) and INTSTD (m/z=322) are isolated and energized for collision-induced dissociation. The product ions from FECNT (m/z=294) and INTSTD (m/z=290) are monitored selectively. The calibration curve for MS response is linear for FECNT concentrations in the range 2,20 pg/µl and suitable for reproducibly (RSD 5%) and rapidly (<3 min) measuring low concentrations of carrier in [18F]FECNT preparations. Copyright © 2005 John Wiley & Sons, Ltd. [source] A combined loop-SPE method for the automated preparation of [11C]doxepinJOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 4 2002R. Iwata Abstract A simple and versatile loop-solid phase extraction (SPE) method was developed for the automated preparation of [11C]doxepin, a histamine H1 receptor antagonist, from [11C]methyl triflate ([11C]MeOTf). This labeling agent was passed through a Teflon or Tefzel loop coated internally with a film of the precursor solution. The reaction products were then flushed from the loop to a short SPE column, where they were concentrated and then injected onto a semi-preparative HPLC column simply by switching an injection valve. By applying this combined loop-SPE technique the whole procedure turned out to be easily automated. The formation of [11C]methylated doxepin ([11C]methyldoxepin) was observed and the ratio of doxepin to methyldoxepin was found to be clearly correlated with the mass ratio of nordoxepin to MeOTf. This observation highlights the importance of [11C]MeOTf specific activity in the [11C]methylation of secondary amines. Using this method, [11C]Doxepin was prepared in over 40% radiochemical yield from high specific activity [11C]MeOTf. Copyright © 2002 John Wiley & Sons, Ltd. [source] Measurement of caffeine and five of the major metabolites in urine by high-performance liquid chromatography/tandem mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 3 2005Allan Weimann Abstract Analysis of caffeine and its metabolites is of interest with respect to caffeine exposure, for kinetic and metabolism studies and for opportunistic in vivo estimation of drug metabolizing enzyme activity in humans and animals. For the latter, analysis is usually done by high-performance liquid chromatography (HPLC) with UV detection. However, this method is close to the detection limit for certain of the metabolites and requires very long chromatography, 30,60 min. We have developed a fast method for the quantification of caffeine and its metabolites 1-methylxanthine, 1-methyluric acid, 1,7-dimethyluric acid, 5-acetylamino-6-amino-3-methyluracil (AAMU) and 5-acetylamino-6-formylamino-3-methyluracil (AFMU) by HPLC tandem mass spectrometry (MS/MS) in urine that requires only its dilution with buffer and centrifugation before injection into the HPLC/MS/MS system. The chromatography lasts 7 min and is followed by 4.5 min for re-equilibration of the HPLC column, giving a total analysis time of 11.5 min. The method provides a great sensitivity improvement with detection limits for all analytes ,25 nM in real samples. Also, the analysis provides much improvement in capacity to ,125 samples per 24 h. Intra- and inter-day coefficients of variation of a single analysis are <6.5% for all the analytes. The inter-day coefficient of variation of duplicate analyses is <4.8% for all analytes. The method is automated, including automated integration, and it is fast, robust and suitable for large-scale investigations in humans and animals. Copyright © 2005 John Wiley & Sons, Ltd. [source] HPLC,SPE,NMR hyphenation in natural products research: optimization of analysis of Croton membranaceus extract,MAGNETIC RESONANCE IN CHEMISTRY, Issue 9 2005Maja Lambert Abstract The HPLC,SPE,NMR technique was used for the analysis of a root-bark extract of Croton membranaceus. The components of the extract were separated on an analytical-size reversed-phase HPLC column, the chromatographic peaks were trapped on SPE (solid-phase extraction) cartridges after post-column dilution of the eluate with water and the compounds were eluted from the cartridges with acetonitrile- d3 into a 30 µl 600 MHz NMR probe in a fully automated procedure. The trapping efficiency of scopoletin (1), the major extract constituent, was much higher on a GP (general phase, a polystyrene-type polymer) SPE phase than on a C18 phase. Thus, under the conditions used, up to 100 µg of scopoletin per cartridge could be accumulated linearly after repeated trappings. The maximum achievable NMR signal-to-noise ratio using the GP cartridges was at least four times higher than that achievable with the C18 cartridges. It was shown that excessively long T1 relaxation times may compromise experiments in which acetonitrile- d3 is used as the cartridge eluent. Nevertheless, the sensitivity gain provided by the HPLC,SPE,NMR technique through repeated peak trappings allowed the acquisition of good-quality proton-detected 2D NMR spectra without the need for solvent suppression. Copyright © 2005 John Wiley & Sons, Ltd. [source] Identification of isomeric tropane alkaloids from Schizanthus grahamii by HPLC-NMR with loop storage and HPLC-UV-MS/SPE-NMR using a cryogenic flow probePHYTOCHEMICAL ANALYSIS, Issue 2 2006Stefan Bieri Abstract Two fully automated HPLC-NMR methods are reported and compared for the structure elucidation of four isomeric tropane alkaloids from the stem-bark of an endemic Chilean plant, Schizanthus grahamii Gill. (Solanaceae). The first approach interfaced a conventional HPLC column to NMR by means of a loop storage unit. After elution with a mobile phase consisting of deuterated water and standard protonated organic solvents, the separated analytes were momentarily stored in a loop cassette and then transferred one-at-a-time to the NMR flow probe for measurements. The second strategy combined HPLC with parallel ion-trap MS detection and NMR spectroscopy using an integrated solid-phase extraction (SPE) unit for post-column analyte trapping. The SPE cartridges were dried under a gentle stream of nitrogen and analytes were sequentially eluted and directed to a cryogenically cooled flow-probe with an NMR-friendly solvent. The structures of the four isomeric alkaloids, 3, -senecioyloxy-7, -hydroxytropane, 3, -hydroxy-7, -angeloyloxytropane, 3, -hydroxy-7, -tigloyloxytropane and 3, -hydroxy-7, -senecioyloxytropane, were unambiguously determined by combining NMR assignments with MS data. Copyright © 2005 John Wiley & Sons, Ltd. [source] HPLC-UV assay for monitoring total and unbound mycophenolic acid concentrations in childrenBIOMEDICAL CHROMATOGRAPHY, Issue 1 2009L. Zeng Abstract A simple, accurate and sensitive HPLC method was developed for measuring total and unbound mycophenolic acid (MPA) in human plasma. Total MPA was extracted by protein precipitation and ultrafiltration was used to assess unbound MPA concentrations. The supernatant (20 µL) or ultrafiltrate (100 µL) was injected onto a C18 HPLC column with a mobile phase of 0.05 m sodium phosphate buffer (pH 2.31),acetonitrile (55:45, v/v for total MPA; 50:50 for unbound MPA) with UV detection at 254 nm. The extraction recovery was over 93% and reproducible. The assay was linear over the concentration range of 0.07,50 mg/L for total MPA and 4,1500 µg/L for unbound MPA. Intra- and inter-day assay reproducibility was less than 10%. Detection limits were 0.04 mg/L and 2 µg/L for total and unbound MPA, respectively. The assay utility was established in samples collected from five paediatric bone marrow transplant recipients who were receiving intravenous doses of mycophenolate mofetil. In these patients MPA concentrations ranged from 0.07 to 7.83 mg/L and unbound drug concentrations ranged from 2.1 to 107.5 µg/L. This method can be effectively applied to MPA pharmacokinetics in paediatric patients. Copyright © 2008 John Wiley & Sons, Ltd. [source] A rapid and simple HPLC-UV method for the determination of inhibition characteristics of farnesyl transferase inhibitorsBIOMEDICAL CHROMATOGRAPHY, Issue 2 2006Natalie M. G. M. Appels Abstract Ras proteins play an important role in the development of cancer. Farnesyl transferase inhibitors (FTIs) block the first obligatory post-translational step for activation, prenylation, of Ras proteins. To find new potent FTIs, rapid enzyme activity assays are required to reduce FTI development time. Most assays to date are based on radioactive labelled substrates. We developed a new, in vitro, farnesyl transferase assay based on gradient chromatography coupled to UV detection. Unfarnesylated and farnesylated H-Ras proteins were resolved on a C18 wide-pore HPLC column and their concentrations were determined with use of a calibration curve of unfarnesylated H-Ras. The assay was used to investigate inhibition characteristics of FTIs. The IC50 values of the FTIs L778,123 and SCH66336 were 4.2 nm and 78 µm, respectively. This assay could support the screening and development of FTIs to obtain rapid insights into their inhibitory properties. Copyright © 2005 John Wiley & Sons, Ltd. [source] Simultaneous determination of four antiepileptic drugs in serum by high-performance liquid chromatographyBIOMEDICAL CHROMATOGRAPHY, Issue 1 2002H. Levert A high-performance liquid chromatographic method has been developed for the simultaneous determination of oxcarbazepine, 10-hydroxycarbamazepine, epoxycarbamazepine, carbamazepine, phenobarbital and phenytoin. After protein precipitation by acetonitrile, the supernatant was analysed on a C18 reversed-phase HPLC column. Antiepileptic drugs and oxazepam (internal standard) were detected by ultraviolet absorbance at 240,nm. Linearity was established for the whole concentration range for each compound. Quantitation limits of oxcarbazepine, 10-hydroxycarbamazepine, epoxycarbamazepine, carbamazepine, phenobarbital and phenytoin were 0.58, 3.5, 2.35, 0.66, 1.02 and 3.13,µg/mL, respectively, and mean recoveries added to serum were 105.15, 84.76, 94,45, 96.52, 98.62 and 95.08%, respectively. This method has been used for the simultaneous determination of steady-state serum concentration of antiepileptic drugs in patients treated by one or more anticonvulsive treatment. Copyright © 2001 John Wiley & Sons, Ltd. [source] Enantiomeric separation of mineralocorticoid receptor (hMR) antagonists using the Chiralcel® OJ-H HPLC column with novel polar cosolvent eluent systemsCHIRALITY, Issue 6 2006V. Scott Sharp Abstract This study demonstrates the increased versatility of the Chiralcel® OJ-H stationary phase when using various alcohol/acetonitrile mobile phases. This chiral stationary phase has traditionally been employed in the normal phase mode and more recently with neat alcohols as eluents. Selected isomeric human mineralocorticoid receptor (hMR) antagonist pharmaceutical candidates and synthetic intermediates were separated using the Chiralcel® OJ-H HPLC column with novel polar cosolvent eluent systems. The capacity factors, resolution, and selectivity of the chiral separations were assessed while varying the alcohol/acetonitrile composition and alcohol identity. The mixed polar eluents provide separations that are nearly always superior to both the traditional hexane-rich and single-alcohol "polar organic" eluents for the compounds tested in this article. Chirality, 2006. © 2006 Wiley-Liss, Inc. [source] Chiral stationary phase covalently bound with a chiral pseudo-18-crown-6 ether for enantiomer separation of amino compounds using a normal mobile phaseCHIRALITY, Issue 3 2005Keiji Hirose Abstract In order to apply the excellent chiral recognition ability of chiral pseudo-18-crown-6 ethers that we developed to chiral separation, we prepared a chiral stationary phase (CSP) by immobilizing a chiral pseudo-18-crown-6-type host on 3-aminopropyl silica gel. A chiral column was prepared by the slurry-packing method in a stainless steel HPLC column. A liquid chromatography system using this CSP combined with the detection by mass spectrometry was used for enantiomer separation of amino compounds. A normal mobile phase can be used on this CSP as opposed to conventional dynamic coating-type CSPs. Enantiomers of 18 common natural amino acids were efficiently separated. The chiral separation observed for amino acid methyl esters, amino alcohols, and lipophilic amines was fair using this HPLC system. In view of the correlation between the enantiomer selectivity observed in chromatography and the complexion in solution, the chiral recognition in host,guest interactions might contribute to this enantiomer separation. Chirality 17:142,148, 2005. © 2005 Wiley-Liss, Inc. [source] Chiral stationary phases for separation of intermedine and lycopsamine enantiomers from Symphytum uplandicumJOURNAL OF SEPARATION SCIENCE, JSS, Issue 2 2010Rahul S. Pawar Abstract Enantioseparation of the pyrrolizidine alkaloid isomers intermedine and lycopsamine, isolated from Symphytum uplandicum, is discussed. The separatory power of two immobilized carbohydrate-based chiral HPLC columns, Chiralpak IA and IC, in different chromatographic conditions is compared. The study demonstrated the importance of solvent and column selection while developing such chiral HPLC separation methods. The baseline HPLC separation of the two alkaloid isomers in preparatory scale is reported for the first time. The optimized separations were achieved on a Chiralpak IA column with mobile phases of ACN/methanol (80:20) and methanol/methyl- t -butyl ether (90:10), both containing 0.1% diethylamine. [source] Capillary columns in liquid chromatography: between conventional columns and microchipsJOURNAL OF SEPARATION SCIENCE, JSS, Issue 17-18 2004Yoshihiro Saito Abstract Liquid chromatography on columns with small internal diameters has been reviewed as the intermediate technique between conventional liquid chromatography and microchip separations. The development of micro column separations in the early years has been described, starting with the papers of Horváth and co-workers and Ishii and co-workers, continuing into the first part of the eighties, then making a leap in time to recent innovations with small-bore columns. Based on internal diameters a classification of the different analytical HPLC columns has been suggested. The advantages of small-bore columns have been discussed, with particular emphasis on the advantage of coupling to concentration sensitive detectors when the sample amount is limited. Open tubular columns are treated as a part of the historic background. The recent developments include a brief look into the current status of monolithic columns, the use of packed nano columns and micro columns with electrospray mass spectrometry, and the potential of two-dimensional comprehensive liquid chromatography. Finally, the coupling of sample preparation to analytical columns and the future applications of the novel technological improvements to the microchip separation methods have been discussed. [source] Evaluation of HPLC columns: A study on surface homogeneity of chemically bonded stationary phasesJOURNAL OF SEPARATION SCIENCE, JSS, Issue 3-4 2003Bogus, aw Buszewski Abstract The aim of the current work is to study the heterogeneity of the adsorbent surface on the basis of physicochemical investigations and chromatographic tests. A series of packing materials with octadecyl chains chemically bonded to a silica matrix were prepared for this purpose. The surface and structural properties of bare silica and silica-based octadecyl phases were characterized by porosimetry, elemental analysis, 29Si CP/MAS NMR, etc. The most advanced characterization methods based on adsorption microcalorimetry (heat of wetting) measurements were employed to obtain information about the heterogeneity and topography of unmodified and modified silica gel. For the chromatographic study, these phases were evaluated on the basis of the retention data under non-aqueous conditions. A test series of solutes with various chemical properties, such as pK a values, was used. It was found that heterogeneity of the packing surface results in low HPLC resolution. Use of a non-aqueous mobile phase (n -hexane) reduces analytical interference by eliminating hydrophobic interactions between alkyl ligands and the analyte. [source] Assessment of the repeatability and reproducibility of hydrogen/deuterium exchange mass spectrometry measurements,RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 23 2008William Burkitt A system to perform automated hydrogen/deuterium exchange mass spectrometry measurements was constructed using an XYZ robotic autosampler that was capable of performing solvent manipulations and a 4.7 T Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. The system included features such as the first demonstration of a ,dual column' high-performance liquid chromatography (HPLC) setup, and a novel digestion strategy. The performance of the system, in terms of the repeatability and reproducibility of the measurement of protein hydrogen/deuterium exchange, was assessed over a 2-month period. The sensitivity of the measurement of hydrogen exchange towards several parameters was assessed, which allowed their impact on the reproducibility to be discussed. The parameters assessed were the temperature of the HPLC columns and switching valves, the temperature of the quench solutions, the pH of the mobile phase, the pH of the quenched solution, the acid used in the mobile phase and the analytical column used. Copyright © 2008 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] HPLC of basic drugs using non-aqueous ionic eluents: evaluation of a 3,,m strong cation-exchange materialBIOMEDICAL CHROMATOGRAPHY, Issue 3 2010Phillip E. Morgan Abstract HPLC columns packed with 3,,m particle size HPLC Technology Techsphere SCX (propylsulfonic acid-modified) silica offer considerable advantages over 5,,m SCX packings in the analysis of basic drugs using 100% methanol eluents containing an ionic modifier such as ammonium perchlorate. The basic drugs studied included clozapine and norclozapine, olanzapine, quinine and quinidine, and amitriptyline, nortriptyline, imipramine and desipramine. The 3,,m column was not only more efficient for a given column length compared with 5,,m materials, but also elution times were less, a phenomenon observed in reversed-phase systems. The high efficiencies and excellent peak shapes obtained with the 3,,m SCX-modified packing together with the relatively low back-pressures attained show that such materials deserve serious consideration by laboratories involved in the analysis of basic drugs. Manufacturers should offer such packings as a matter of routine. Alternative ionic modifiers such as ammonium acetate are available for use with mass spectrometric detection if required. Copyright © 2009 John Wiley & Sons, Ltd. [source] Fluorous "racemic" mixture synthesis: Polysaccharide-based chiral columns for simultaneous demix and enantioseparation of racemic fluorous tagged compoundsCHIRALITY, Issue 3-4 2008Takayuki Tonoi Abstract The proof of concept experiments of fluorous "racemic" mixture synthesis (FRMS) is shown using polysaccharide-based chiral stationary phases. The mixture of racemic O -benzoylmandelate derivatives bearing different lengths of fluorous cleavable tags undergoes sequential reactions to provide individual derivatives as well as their enantiomers resolved on polysaccharide-based chiral HPLC columns (DAICEL CHIRALCEL® and CHIRALPAK® series). Chirality, 2008. © 2008 Wiley-Liss, Inc. [source] Resolution, configurational assignment, and enantiopharmacology at glutamate receptors of 2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA) and demethyl-ACPA,CHIRALITY, Issue 9 2001Tommy N. Johansen Abstract We have previously described (RS)-2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA) as a potent agonist at the (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor subtype of (S)-glutamic acid (Glu) receptors. We now report the chromatographic resolution of ACPA and (RS)-2-amino-3-(3-carboxy-4-isoxazolyl)propionic acid (demethyl-ACPA) using a Sumichiral OA-5000 column. The configuration of the enantiomers of both compounds have been assigned based on X-ray crystallographic analyses, supported by circular dichroism spectra and elution orders on chiral HPLC columns. Furthermore, the enantiopharmacology of ACPA and demethyl-ACPA was investigated using radioligand binding and cortical wedge electrophysiological assay systems and cloned metabotropic Glu receptors. (S)-ACPA showed high affinity in AMPA binding (IC50 = 0.025 ,M), low affinity in kainic acid binding (IC50 = 3.6 ,M), and potent AMPA receptor agonist activity on cortical neurons (EC50 = 0.25 ,M), whereas (R)-ACPA was essentially inactive. Like (S)-ACPA, (S)-demethyl-ACPA displayed high AMPA receptor affinity (IC50 = 0.039 ,M), but was found to be a relatively weak AMPA receptor agonist (EC50 = 12 ,M). The stereoselectivity observed for demethyl-ACPA was high when based on AMPA receptor affinity (eudismic ratio = 250), but low when based on electrophysiological activity (eudismic ratio = 10). (R)-Demethyl-ACPA also possessed a weak NMDA receptor antagonist activity (IC50 = 220 ,M). Among the enantiomers tested, only (S)-demethyl-ACPA showed activity at metabotropic receptors, being a weak antagonist at the mGlu2 receptor subtype (KB = 148 ,M). Chirality 13:523,532, 2001. © 2001 Wiley-Liss, Inc. [source] Synthesis of 4-(7-diethylaminocoumarin-3-yl)benzeneisocyanate (DACB-NCO): A highly sensitive fluorescent derivatization reagent for alcohols in high-performance liquid chromatography,JOURNAL OF HETEROCYCLIC CHEMISTRY, Issue 2 2001Haruko Takechi 4-(7-Diethylaminocoumarin-3-yl)benzeneisocyanate (DACB-NCO) was synthesized as a new fluorescent derivatization reagent for alcohols for use in high-performance liquid chromatography (hplc). Saturated alcohols (C6 -C22) were derivatized in good yields into the corresponding fluorescent DACB-carbamic esters by treating with DACB-NCO. The DACB-carbamic esters of these alcohols were clearly separated on a reversed-phase hplc column (Inertsil ODS-2, mobile phase: methanol-water, excitation wavelength 402 nm; emission wavelength 488 nm). The detection limit (S/N = 3) of cetyl alcohol, as a test compound, was 5 fmol/10 ,l. [source] | ||||||||||||||||||||||