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Chromatographic Resolution (chromatographic + resolution)
Selected AbstractsDetermination of didanosine in maternal plasma, amniotic fluid, fetal and placental tissues by high-performance liquid chromatography,tandem mass spectrometryBIOMEDICAL CHROMATOGRAPHY, Issue 6-7 2006T. Nicole Clark Abstract A rapid and efficient high-performance liquid chromatography (HPLC)-tandem mass spectrometry method for the determination of didanosine concentrations in maternal rat plasma, amniotic fluid, placental and fetal tissue samples has been developed and validated. Tissue samples were homogenized in optima water and centrifuged. The supernatant was subjected to solid-phase extraction (SPE) prior to analysis. Plasma and amniotic fluid samples were extracted without pretreatment. An Agilent 1100 Series HPLC coupled with a Micromass Quattro II triple quadrupole mass spectrometer was used for all analyses. Chromatographic resolution was achieved on a Nova-Pak phenyl analytical column (2.0 × 150 mm, 4 µm particle size) equipped with a Phenomenex Security-guard phenyl guard cartridge (2.0 × 4.0 mm) using 60% methanol in 10 mm ammonium acetate buffer mobile phase for all matrices at a flow rate of 0.15 mL/min. The method yields retention times of 2.9 min for didanosine and 3.0 min for the internal standard, stavudine. Limits of detection were 1 ng/mL for all matrices. Recoveries were 70% or greater for both compounds in the different matrices. Within- and between-run precision (%RSD) and accuracy (%error) was less than 15% for all matrices. Copyright © 2006 John Wiley & Sons, Ltd. [source] Hard-modelled trilinear decomposition (HTD) for an enhanced kinetic multicomponent analysisJOURNAL OF CHEMOMETRICS, Issue 5 2002Yorck-Michael Neuhold Abstract We present a novel approach for kinetic, spectral and chromatographic resolution of trilinear data sets acquired from slow chemical reaction processes via repeated chromatographic analysis with diode array detection. The method is based on fitting rate constants of distinct chemical model reactions (hard-modelled, integrated rate laws) by a Newton,Gauss,Levenberg/Marquardt (NGL/M) optimization in combination with principal component analysis (PCA) and/or evolving factor analysis (EFA), both known as powerful methods from bilinear data analysis. We call our method hard-modelled trilinear decomposition (HTD). Compared with classical bilinear hard-modelled kinetic data analysis, the additional chromatographic resolution leads to two major advantages: (1) the differentiation of indistinguishable rate laws, as they can occur in consecutive first-order reactions; and (2) the circumvention of many problems due to rank deficiencies in the kinetic concentration profiles. In this paper we present the theoretical background of the algorithm and discuss selected chemical rate laws. Copyright © 2002 John Wiley & Sons, Ltd. [source] The influence of cytosine methylation on the chemoselectivity of benzo[a]pyrene diol epoxide-oligonucleotide adducts determined using nanoLC/MS/MSJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 8 2009James Glick Abstract Benzo[a]pyrene is a major carcinogen implicated in human lung cancer. Almost 60% of human lung cancers have a mutation in the p53 tumor suppressor gene at several specific codons. An on-line nanoLC/MS/MS method using a monolithic nanocolumn was applied to investigate the chemoselectivity of the carcinogenic diol epoxide metabolite, ( ± )-(7R,8S,9S,10R)-benzo[a]pyrene 7,8-diol 9,10-epoxide [( ± )- anti -benzo[a]pyrene diol epoxide (BPDE)], which was reacted in vitro with a synthesized 14-mer double stranded oligonucleotide (5,-ACCCG5CG7TCCG11CG13C-3,/5,-GCGCGGGCGCGGGT-3,) derived from the p53 gene. This sequence contained codons 157 and 158, which are considered mutational ,hot spots' and have also been reported as chemical ,hot spots' for the formation of BPDE-DNA adducts. In evaluating the effect of cytosine methylation on BPDE-DNA adduct binding, it was found that codon 156, containing the nucleobase G5 instead of the mutational hot spot codons 157 (G7) and 158 (G11), was the preferential chemoselective binding site for BPDE. In all permethylated cases studied, the relative ratio for adduction was found to be G5, G11 > G13 > G7. Permethylation of CpG dinucleotide sites on either the nontranscribed or complementary strand did not change the order of sequence preference but did enhance the relative adduction level of the G11 CpG site (codon 158) approximately two-fold versus the unmethylated oligomer. Permethylation of all CpG dinucleotide sites on the duplex changed the order of relative adduction to G5, G7 > G11 > G13. The three- to four-fold increase in adduction at the mutational hot spot codon 157 (G7) relative to the unmethylated or single-stranded permethylated cases suggests a possible relationship between the state of methylation and adduct formation for a particular mutation site in the p53 gene. Using this method, only 125 ng (30 pmol) of adducted oligonucleotide was analyzed with minimal sample cleanup and high chromatographic resolution of positional isomers in a single chromatographic run. Copyright © 2009 John Wiley & Sons, Ltd. [source] GC-MS analysis of multiply derivatized opioids in urineJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 8 2007Bud-Gen Chen Abstract Opiates such as hydrocodone, hydromorphone, oxycodone, noroxycodone, and oxymorphone reportedly may interfere with the analysis of morphine and codeine. The analysis of these compounds themselves also is an important issue. Thus, double derivatization approaches utilizing methoxyamine and hydroxylamine to first form oxime products with keto-opiates, followed by the derivatization with trimethylsilyl (TMS) or propionyl groups, have been developed for the simultaneous analysis of these compounds. However, these studies have not included all compounds of interest and resulted in inadequate chromatographic resolution or significant intensity cross-contribution between the ions designating the analyte and its deuterated internal standard for certain compounds. By exploring three-step derivatization approaches with the combination of various derivatization groups and orders, this study concluded that application of methoxyimino/propionyl/TMS groups, in the order listed, facilitated the simultaneous analysis of eight opiates (morphine, 6-acetylmorphine, hydromorphone, oxymorphone, codeine, hydrocodone, oxycodone and noroxycodone) in urine samples, achieving satisfactory limits of quantitation and detection. In addition, the adapted approach resulted in two usable products for morphine and codeine providing alternatives, should interferences render any of these products non-usable. Copyright © 2007 John Wiley & Sons, Ltd. [source] Thin-layer chromatography/electrospray ionization triple-quadrupole linear ion trap mass spectrometry system: analysis of rhodamine dyes separated on reversed-phase C8 plates ,JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 7 2005Michael J. Ford Abstract The direct analysis of separated rhodamine dyes on reversed-phase C8 thin-layer chromatography plates using a surface sampling/electrospray emitter probe coupled with a triple-quadrupole linear ion trap mass spectrometer is presented. This report represents continuing work to advance the performance metrics and utility of this basic surface sampling electrospray mass spectrometry system for the analysis of thin-layer chromatography plates. Experimental results examining the role of sampling probe spray end configuration on liquid aspiration rate and gas-phase ion signal generated are discussed. The detection figures-of-merit afforded by full-scan, automated product ion and selected reaction monitoring modes of operation were examined. The effect of different eluting solvents on mass spectrum signal levels with the reversed-phase C8 plate was investigated. The combined effect of eluting solvent flow-rate and development lane surface scan rate on preservation of chromatographic resolution was also studied. Analysis of chromatographically separated red pen ink extracts from eight different pens using selected reaction monitoring demonstrated the potential of this surface sampling electrospray mass spectrometry system for targeted compound analysis with real samples. Copyright © 2005 John Wiley & Sons, Ltd. [source] Alkylated poly(styrene-divinylbenzene) monolithic columns for ,-HPLC and CEC separation of phenolic acidsJOURNAL OF SEPARATION SCIENCE, JSS, Issue 17 2007Zdenka Ku, erová Abstract Macroporous poly(styrene-divinylbenzene) monolithic columns were prepared in fused silica capillaries of 100 ,m id by in-situ copolymerization of styrene with divinylbenzene in the presence of propan-1-ol and formamide as the porogen system. The monoliths were subsequently alkylated with linear alkyl C-18 groups via Friedel-Crafts reaction to improve the retention and chromatographic resolution of strongly polar phenolic acids. A new thermally initiated grafting procedure was developed in order to shorten the time of the alkylation process. The grafting procedure was optimized with respect to the reaction temperature, time, the grafting reactant concentration, and the solvent used. The type of solvent and the grafting temperature are the most significant factors affecting the hydrodynamic properties, porosity, and efficiency of the columns. While the equivalent particle diameter of the grafted column increased, the capillary-like flow-through pore diameter decreased in comparison to non-alkylated monoliths. The hydrodynamic permeability of the monolith decreased, but the monolithic column still permitted fast ,-HPLC separations. [source] High-performance liquid chromatographic resolution of 1-(1,4-benzodioxane-2-formyl)- piperazine enantiomers after chiral derivatizationJOURNAL OF SEPARATION SCIENCE, JSS, Issue 2 2005Zhiqiong Chen Abstract Chiral separation of racemic mixtures is of the greatest importance to the pharmaceutical industry, as the isomers of a given racemate may exhibit substantially different pharmacological effects, not to mention possibly differing toxicity behaviour. A novel chiral separation method is developed for the determination of 1-(1,4-benzodioxane-2-formyl)piperazine (BFP) enantiomers. The indirect resolution is performed by applying precolumn derivatization with the chiral reagent 2,3,4,6-tetra- O -acetyl-,-D-glucopyranosyl isothiocyanate (GITC). The resulting diastereoisomers are separated on a reversed-phase ODS column with methanol-potassium dihydrogen phosphate (0.02mol/L, 50:50) as mobile phase. UV detection is at 250 nm. The effect of mobile phase composition upon resolution and analysis time is investigated. Two diastereoisomers show nearly base-line separation under optimal chromatographic conditions. The presented study provides a simple and accurate method for the enantiomeric quality control and the optical purity assay of BFP. [source] A general precursor ion-like scanning mode on quadrupole-TOF instruments compatible with chromatographic separationPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 1 2006Ricarda Niggeweg Abstract MS protein identification and quantitation are key proteomic techniques in biological research. Besides identification of proteins, MS is used increasingly to characterize secondary protein modifications. This often requires trimming the analytical strategy to a specific type of modification. Direct analysis of protein modifications in proteomic samples is often hampered by the limited dynamic range of current analytical tools. Here we present a fast, sensitive, multiplexed precursor ion scanning mode , implemented on a quadrupole-TOF instrument , that allows the specific detection of any modified peptide or molecule that reveals itself by a specific fragment ion or pattern of fragment ions within a complex proteomic sample. The high mass accuracy of the TOF mass spectrometer is available for the marker ion specificity and the precursor ion mass determination. The method is compatible with chromatographic separation. Fragment ions and intact molecular ions are acquired quasi-simultaneously by continuously switching the collision energy between elevated and low levels. Using this technique many secondary modifications can be analyzed in parallel; however, the number of peptides carrying a specific modification that can be analyzed successfully is limited by the chromatographic resolution or, more generally, by the depth of the resolved time domain. [source] Utility of porous graphitic carbon stationary phase in quantitative liquid chromatography/tandem mass spectrometry bioanalysis: quantitation of diastereomers in plasmaRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 12 2006Yuan-Qing Xia A major challenge in selecting an appropriate stationary phase for diastereomeric separation is that it is difficult to predict which of the commercially available stationary phases could achieve the required liquid chromatographic (LC) separation. This work describes the selection and evaluation of a porous graphitic carbon (PGC) column coupled with tandem mass spectrometry (MS/MS) for the simultaneous quantitation of an experimental drug candidate (I), its two diastereomeric metabolites (II and III), and its demethylated metabolite (IV) in rat plasma. In addition, we investigated the PGC column for the separation of another drug candidate (VI), its two diastereomeric metabolites (VII and VIII) and its ketone metabolite (IX). The PGC column showed excellent chromatographic resolution for the two diastereomers II and III, as well as for VII and VIII. In contrast, the required resolution for the diastereomers II and III could not be achieved using silica-bonded C18, C30, phenyl, perfluorinated, polar embedded and polar end-capped phases. The PGC column showed ruggedness with excellent reproducibility of retention times, peak symmetry and response over a period of more than 400 injections of a plasma acetonitrile-precipitation extract. Excellent accuracy and precision were achieved, with accuracy of 94,108% and intra- and inter-run precision within 9%. This work indicates that PGC is a valuable addition to the repertoire of LC columns used for quantitative LC/MS/MS bioanalysis, especially where the separation and quantitation of diastereomeric analytes is involved. Copyright © 2006 John Wiley & Sons, Ltd. [source] Increasing throughput and information content for in vitro drug metabolism experiments using ultra-performance liquid chromatography coupled to a quadrupole time-of-flight mass spectrometerRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 6 2005Jose Castro-Perez The field of drug metabolism has been revolutionized by liquid chromatography/mass spectrometry (LC/MS) applications with new technologies such as triple quadrupoles, ion traps and time-of-flight (ToF) instrumentation. Over the years, these developments have often relied on the improvements to the mass spectrometer hardware and software, which has allowed users to benefit from lower levels of detection and ease-of-use. One area in which the development pace has been slower is in high-performance liquid chromatography (HPLC). In the case of metabolite identification, where there are many challenges due to the complex nature of the biological matrices and the diversity of the metabolites produced, there is a need to obtain the most accurate data possible. Reactive or toxic metabolites need to be detected and identified as early as possible in the drug discovery process, in order to reduce the very costly attrition of compounds in late-phase development. High-resolution, exact mass measurement plays a very important role in metabolite identification because it allows the elimination of false positives and the determination of non-trivial metabolites in a much faster throughput environment than any other standard current methodology available to this field. By improving the chromatographic resolution, increased peak capacity can be achieved with a reduction in the number of co-eluting species leading to superior separations. The overall enhancement in the chromatographic resolution and peak capacity is transferred into a net reduction in ion suppression leading to an improvement in the MS sensitivity. To investigate this, a number of in vitro samples were analyzed using an ultra-performance liquid chromatography (UPLC) system, with columns packed with porous 1.7,,m particles, coupled to a hybrid quadrupole time-of-flight (ToF) mass spectrometer. This technique showed very clear examples for fundamental gains in sensitivity, chromatographic resolution and speed of analysis, which are all important factors for the demands of today's HTS in discovery. Copyright © 2005 John Wiley & Sons, Ltd. [source] Direct analysis of pharmaceutical compounds in human plasma with chromatographic resolution using an alkyl-bonded silica rod columnRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 12 2001Robert Plumb Monolithic columns have been successfully used with steep gradient and high flow rates for the direct analysis of a candidate pharmaceutical compound in human plasma. The monolithic columns showed excellent robustness with nearly 300 20-µL injections of plasma (diluted 1:1 with water) being made onto one column without significant deterioration in performance. The system gave excellent sensitivity with a limit of quantification of 5,ng/mL being achieved. Unlike previous methods of direct analysis the monolithic columns showed excellent resolution even after nearly 300 plasma injections. The column performance was measured before and after the analysis of the plasma samples. Copyright © 2001 John Wiley & Sons, Ltd. [source] Selective Treatment of Cancer: Synthesis, Biological Evaluation and Structural Elucidation of Novel Analogues of the Antibiotic CC-1065 and the DuocarmycinsCHEMISTRY - A EUROPEAN JOURNAL, Issue 16 2007Abstract Novel diastereomerically pure ,- D -galactosidic prodrugs (+)- 12,a,e of the cytotoxic antibiotics CC-1065 and the duocarmycins were prepared for an antibody directed enzyme prodrug therapy (ADEPT) using 4 as a substrate via a radical cyclization to give rac - 5 and rac - 6 followed by a chromatographic resolution of the enantiomers of rac - 5, glycosidation and linkage to the DNA-binding units 10,a,e. These only slightly toxic compounds can be toxified enzymatically by an antibody,,- D -galactosidase conjugate at the surface of malignant cells to give the cytotoxic drugs, which then alkylate DNA. The new prodrugs were tested in in vitro cytotoxicity assays showing excellent QIC50 values of 4800 and 4300 for (+)- 12,a and (+)- 12,b, respectively. The absolute configuration of precursor (+)- 5 was determined by comparison of the experimental CD spectrum with the theoretically predicted CD spectra and by X-ray structure analysis. [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] Enantiomers of 2-[(Acylamino)ethyl]-1,4-benzodiazepines, Potent ligands of ,-opioid receptor: Chiral chromatographic resolution, configurational assignment, and biological activityCHIRALITY, Issue 9 2001O. Azzolina Abstract Compounds 2a and 3a,e are racemic 2-[(acylamino)ethyl]-1,4-benzodiazepines, tifluadom analogs, with high affinity and selectivity towards the ,-opioid receptor. We describe the enantiomeric separation of all compounds through liquid chromatography with chiral stationary phases, as well as the resolution of the enantiomers of the most interesting compounds, 2a and 3a, by the semipreparative column Chiralpak AD. The configuration of the resolved enantiomers was investigated: the comparative study of CD and 1H NMR spectra shows that compounds (,)- 2a and (,)- 3a have the same absolute configuration of (+)-(S)-tifluadom. A study on the stereoselective interaction with opiate receptors is reported. Chirality 13:606,612, 2001. © 2001 Wiley-Liss, Inc. [source] | ||||||||||