Reversed-phase Chromatographic System Research Articles

Effect of rosiglitazone on the differential expression of diabetes-associated proteins in pancreatic islets of C57Bl/6 lep/lep mice.

The insulin sensitizer drug, rosiglitazone, has been shown to have a protective effect on pancreatic islet cell structure and function in animal models of type 2 diabetes. The identification of new molecular targets associated both with islet cell dysfunction and protection is a crucial research goal. In the present study, a proteomics approach has been used to identify such targets. Obese C57Bl/6J lep/lep mice and lean littermates were given the insulin sensitizer drug BRL49653, rosiglitazone. It normalized the impaired glucose tolerance in lep/lep mice but had no significant effect on glucose tolerance in the lean mice. Pancreatic islet polypeptides were arrayed by a two-dimensional gel electrophoresis system that separated more than 2500 individual spots. Three overexpressed and six underexpressed proteins were significant (p < 0.05) between lep/lep and lean mice, and four were modulated significantly (p < 0.05) by the rosiglitazone treatment of the obese mice. The identity of these differentially expressed proteins was made using mass spectrometric analysis and provided evidence that differential expression of actin-binding proteins may be an important aspect of defective islet function. Rosiglitazone increased carboxypeptidase B expression in both lep/lep and normal mice suggesting that this might be an independent effect of rosiglitazone that contributes to improved insulin processing.

Systematic search for surrogate chromatographic models of biopartitioning processes.

A systematic approach for identifing surrogate chromatographic models for biopartitioning processes is described. The method is based on a comparison of the system constant ratios of the solvation parameter model for biopartitioning processes and a database of system constant ratios for reversed-phase liquid chromatographic and micellar electrokinetic chromatographic systems compiled from literature sources. An acceptance filter of < or = 0.2 is applied for each difference in system constant ratio for the compared systems to provide a reasonable probability of success without outputting too many systems with limited predictive properties. Surrogate chromatographic models identified for the non-specific toxicity of neutral organic compounds to the fathead minnow and the soil-water distribution constant are tested by construction of a correlation model for the characteristic property of the biological process and the chromatographic retention factors for a structurally varied group of compounds. Although these models are not the best that could be obtained based on ranking of the differences in system constant ratios the predictive ability of the correlation models is suitable for typical applications and similar to the accepted uncertainty in the measurements of the biological property. Retention factors on the immobilized artificial membrane column (IAM PC DD 2) with 10% (v/v) methanol-water as mobile phase are able to estimate non-specific toxicity to the fathead minnow with a standard error (SE) of 0.22 log units and coefficient of determination (r2) of 0.97 for 31 compounds. Retention factors on a Bakerbond DIOL column with 20% (v/v) acetonitrile-water as mobile phase are able to estimate the soil-water distribution constant with an SE of 0.38 log units and r2 = 0.88 for 59 compounds. Other potential surrogate chromatographic models are identified for non-specific toxicity to the guppy, tadpole, Vibrio fischeri and Terahymena pyriformis as well as the plant cuticle matrix-water distribution constant. On the other hand reversed-phase chromatographic systems seem poorly suited for estimating intestinal absorption and the blood-brain distribution constant.

A relatively simple and rapid multi-component method for analysis of steroid profiles in blood, fecal and liver samples.

Various methods of steroid analysis were assessed using radiolabeled steroids and thin layer chromatography. Three reversed phase chromatography systems were evaluated for separation and recovery of steroids extracted from blood, liver tissue and feces. The use of different numbers of Sep-Pak C(18) cartridges for the purification of steroid extracts was examined and steroid recoveries were measured and compared. The results indicated that recoveries were best when 4-6 cartridges were used. Rapid and slow procedures of enzymatic hydrolysis and acidic solvolysis of steroid conjugates were compared. A new and relatively rapid method for analysis of steroid profiles in liver, blood and fecal samples was developed. Assessment of this method showed that steroid recoveries were improved compared to existing methods with percentage recoveries of 64.1-82.5 for liver samples, 55.2-75 for blood samples, and 65.1-76.3 for fecal samples.

APP transgenic mice Tg2576 accumulate Abeta peptides that are distinct from the chemically modified and insoluble peptides deposited in Alzheimer's disease senile plaques.

The amyloid (Abeta) peptides generated in Hsiao's APP Tg2576 transgenic (Tg) mice are physically and chemically distinct from those characteristic of Alzheimer's disease (AD). Transgenic mouse Abeta peptides were purified using sequential size-exclusion and reverse-phase chromatographic systems and subjected to amino acid sequencing and mass spectrometry analyses. The mouse Abeta peptides lacked the extensive N-terminal degradations, posttranslational modifications, and cross-linkages abundant in the stable Abeta peptide deposits observed in AD. Truncated Abeta molecules appear to be generated in vivo by hydrolysis at multiple sites rather than by post-mortem C-terminal degradation. In contrast to AD amyloid cores, the Tg mice peptides were soluble in Tris-SDS-EDTA solutions, revealing both monomeric and SDS-stable oligomeric species of Abeta. In contrast to our report on Novartis Pharma APP23 Tg mice [Kuo et al. (2001) J. Biol. Chem. 276, 12991], which maintain high levels of soluble Abeta early on with later development of extensive vascular amyloid, Tg2576 mice exhibited an age-related elevation of soluble Abeta with relatively limited vascular amyloid deposition. The transgenic mouse levels of carboxy-terminal (CT) APP fragments were nearly 10-fold greater than those of human brains, and this condition may contribute to the unique pathology observed in these animals. Immunization of transgenic mice may act to prevent the pathological effects of betaAPP overproduction by binding CT molecules or halting their processing to toxic forms, in addition to having any effects on Abeta itself. Thus, differences in disease evolution and biochemistry must be considered when using transgenic animals to evaluate drugs or therapeutic interventions intended to reduce the Abeta burden in Alzheimer's disease.

Hydrophobic displacement chromatography of proteins.

Displacement chromatography of proteins was successfully carried out in both hydrophobic interaction and reversed-phase chromatographic systems using low-molecular weight displacers. The displacers employed for hydrophobic displacement chromatography were water soluble, charged molecules containing several short alkyl and/or aryl groups. Spectroscopy was employed to verify the absence of structural changes to the proteins displaced on these hydrophobic supports. Displacement chromatography on a reversed-phase material was employed to purify a growth factor protein from its closely related variants, demonstrating the high resolutions that can be achieved by hydrophobic displacement chromatography. This process combines the high-resolution/high-throughput characteristics of displacement chromatography with the unique selectivity of these hydrophobic supports and offers the chromatographic engineer a powerful tool for the preparative purification of proteins.

Reversed-phase high-performance liquid chromatography of ionogenic compounds: comparison of retention models

Two kinds of retention models describing a behaviour of ionogenic substances in reversed-phase chromatographic systems were compared. Model A utilises a concept of limiting retention factors and is especially suitable for the prediction of retention of compounds co-existing in several forms in mobile phase. An effect of the concentration of organic modifier (e.g., methanol) on the magnitudes of the limiting retention factors and equilibrium constants (dissociation constants of the separated substances) can be expressed with the aid of various, more or less sophisticated, relationships. A stoichiometric displacement model (model B) in its original form simply relates the analyte retention to the content of organic modifier in the mobile phase. In this work, it was modified to also express an effect of the mobile phase pH introducing side equilibria (acid–base) into the model. Both models predict a sigmoidal dependence of the analyte retention factor on the mobile phase pH in accordance with experimental data, and allow, among others, to estimate dissociation constants from those data. Experimental dependencies between the analyte retention and the concentration of methanol in the mobile phase comply well with model A, whereas the stoichiometric displacement model could be used only in a limited range of the methanol concentrations.

Thermodynamic properties for the solute transfer from the mobile to the stationary phase in reversed phase liquid chromatography obtained by squalane-impregnated C 18 bonded phase

We have devised a reversed phase chromatographic system which secured a simple retention mechanism and showed reproducible solute retention over a long period of time. We used a squalane impregnated C 18 phase in a thermostated system with extreme care, and obtained retention data of some selected solutes (benzene, toluene, ethylbenzene, phenol, and acetophenone) at 25, 30, 35, 40, 45, and 50°C in aqueous methanol eluents. The van’t Hoff plots were nicely linear, thus we calculated dependable enthalpies and entropies of solute transfer from the mobile to the stationary phase based on three independent retention measurements on different days (or weeks). We observed that the solute transfer from the mobile to the stationary phase was enthalpically favorable and entropically unfavorable in general except for highly aqueous mobile phases. The enthalpic contribution to the overall solute transfer free energy was found generally more important than the entropic contribution. The cavity formation effect was found the major factor that governs the solute distribution between the mobile and stationary phases for methanol-rich mobile phases while the hydrophobic effect became significant in highly aqueous mobile phases. Comparison of our data with the literature data leaded to the conclusion that C 18 stationary phases with a high ligand density generally follow a partition mechanism while a C 18 phase with a low ligand density follows an adsorption-like mechanism.

Development and validation of a sensitive solid-phase extraction and high-performance liquid chromatographic assay for the novel bio-reductive anti-tumor agent RH1 in human and mouse plasma

A HPLC assay and solid-phase extraction technique from human plasma has been developed and validated for the experimental anticancer agent, RH1 (2,5-diaziridinyl-3-hydroxymethyl-6-methyl-1,4-benzoquinone) which is currently being evaluated by the CRC phase I/II committee. A 500 mg amino propyl solid-phase extraction cartridge was used to isolate RH1 from human plasma. Analysis was performed on a reversed-phase chromatography system using a 15 cm cyanopropyl column and isocratic elution with a 10% methanol–90% water (double distilled) solution. The lower limit of quantitation for RH1 was found to be 0.00375 μg/ml (3.75 ng/ml±8.3%) in water and following extraction from plasma. Recovery of >80%(±11.9%) was achieved over a five-day validation study. This method was used to carry out pre-clinical studies in BDF mice (standard strain of hybrid mice) at three dose levels (2, 5 and 10 mg/kg of RH1 in 0.9% (w/v) saline via an intraperotoneal injection). Standard Version of PC Winnonlin pharmacokinetic modelling software was used to model the data. A none-compartmental model was used to describe the disposition of RH1 in mice plasma. RH1 was rapidly eliminated from plasma with a mean plasma clearance of 23.4 ml/min, mean volume of distribution of 321.6 ml and mean t 1/2 α and β decays of 4.8 and 9.6 min, respectively. RH1 in human and mouse whole blood and plasma was found to be stable up to 2 h.

A Molecular Dynamics Study of a Reversed-Phase Liquid Chromatography Model

We describe a molecular dynamics simulation study of a model of the reversed-phase chromatographic system. The model consists of a slab of aqueous solvent sandwiched between two walls having attached C8 hydrocarbon chains at a surface coverage of 5.09 μmol/m2 or 32.6 A°2/chain. Long-ranged Coulombic interactions are taken into account using the Ewald sum method of Rhee et al. [Phys. Rev. B 1989, 40, 36]. The density and solvent orientation profiles are computed as a function of distance from the walls. The density profiles are found to be sensitive to the treatment of the long-ranged electrostatic interactions. The presence of an organic cosolvent (methanol or acetonitrile) at 30.8 mol % has little effect on the chain structure, which is largely collapsed against the walls. We also estimate the change in residual Helmholtz free energy along the pore width for a methane solute in the acetonitrile/water system, which indicates that a substantial portion of the free energy driving force for retention occurs in an organic-rich layer of solvent adsorbed to the hydrocarbon phase.

Liquid Chromatographic and Mass Spectral Methods of Identification for the Regioisomeric 2,3- and 3,4-Methylenedioxyphenalkylamines

A series of ring and side-chain regioisomers of 3,4-methylenedioxymethamphetamine are compared by chromatographic and spectroscopic methods. Regioisomerism at the aromatic ring and the alkyl side-chain in the methylenedioxyphenalkylamines produces a variety of compounds that have very similar analytical properties. The specific identification of one of these compounds in a forensic drug sample depends on the analyst's ability to eliminate other regioisomers as possible interfering substances. The 2,3- and 3,4-regioisomers of methylenedioxymethamphetamine, N-ethyl-methylenedioxyamphetamine, 1-methylenedioxyphenyl-2-butanamine, and N-methyM-methylenedioxyphenyl-2-butanamine are synthesized from commercially available precursor chemicals. The mass spectra for the underivatized amines are very similar and do not provide sufficient information to differentiate among the side-chain or ring regioisomers. Preparation of the pentafluoropropionamides of the amines produces derivatives that show mass spectral fragmentation identifying the substituent attached to nitrogen and the number of carbons attached directly to the aromatic ring. The regioisomeric amines are well-resolved in a reversed-phase chromatographic system using a Hypersil-Elite C18 stationary phase and acidic hydroorganic mobile phases.

Assay and purity evaluation of CP-93,393-1 by reversed-phase liquid chromatography: A snapshot of current practices for liquid chromatography methods development and validation

CP-93,393-1 is a new chemical entity under development for the treatment of anxiety and depression. This paper describes the selection and validation of a chromatographic system which was engineered to: (1) ensure acceptability by world-wide regulatory agencies, (2) maximize sample throughput, (3) simplify the analysis, (4) minimize ambiguity in peak assignments, and (5) facilitate the technology-transfer process. These objectives were achieved using a single, compound-specific and purity-indicating, isocratic, reversed-phase chromatographic system that allows quantitation of CP-93,393 concomitantly with all potential impurities from the same injection. A novel experimental matrix was employed to validate the chromatographic system.

Direct measurement of sorption/desorption kinetics in reversed-phase chromatographic systems

The role of adsorption/desorption kinetics in chromatographic band broadening was first addressed by Giddings in his earliest theoretical work. Relaxation methods (pressure-jump and temperature-jump) have been adapted to directly measure rates of solute sorption/desorption at reversed-phase chromatographic interfaces. © 1997 John Wiley & Sons, Inc. J Micro Sep 9: 185–191, 1997

Physicochemical Basis of Amino Acid Hydrophobicity Scales: Evaluation of Four New Scales of Amino Acid Hydrophobicity Coefficients Derived from RP-HPLC of Peptides

The physicochemical relationships of four new scales of amino acid hydrophobicity coefficients, derived from the reversed-phase high-performance liquid chromatographic (RP-HPLC) retention data of 1738 peptides, have been compared with 12 previously published scales of amino acid hydrobicities. Four different reversed-phase chromatographic systems were used to obtain the experimental data, including three well-characterized hydrophobic adsorbents of different n-alkyl ligand chain length (n-octadecyl (C18), n-octyl (C8), and n-butyl (C4) ligands) and two different aquo-organic eluents, namely a binary water-acetronitrile and a ternary water-acetonitrile-2-propanol elution system, both containing 0.1% trifluoroacetc acid. Significant correlations were observated from the RP-HPLC data of these peptides separated with the C18- and C8-silica absorbents and the water-acetonitrile elution system and the hydrophobicity scales based on the transter free energies of an amino acid entity from a polar to a nonpolar solvent or the statistical degree of exposure to the surrounding solvent or the statistical degree of exposure to the surrrounding solvent of an amino acid side chain within a protein structure. Conversly, lower levels of correlation were observed between the previously calculated hydrophobicity scales for the common α-amino acids and the amino acid hydrophobicity coefficient values derived from this peptide structure-retention data base with the C4 ligand or the C18 ligand in combination with the water-acetonitrile-2-propanol solvent system. The results confirm that the relative ranking and magnitude of the hydrophobicities of amino acid side chains within polypeptides are dependent upon the chemical microenvironment of the interface established between the solute, the solvent and the immunobilized hydrocarbonaceous ligands. The availability of this extensive data base of peptide structure-chromatographic retention behavior has also permitted differences in peptide- nonpolar ligand interactions to be quantified in physicochemical terms, including the propenity of amino acid side chains in environments from aqueous solutions of different hydrogen-bonding of dipolar characteristics

Correlation Between the Chemical Structures of Dialkyl Peroxides and Their Retention in Reversed-Phase High-Performance Liquid Chromatography

Abstract High-performance liquid chromatographic capacity factors were determined on LiChrosorb RP 18 10 μm stationary phase for various organic peroxides. Studies of the quantitative structure-retention relationship (QSRR) employing non-empirical structural descriptors for the investigated compounds were performed by means of multiple regression analysts Semi-empirical methods MINDO/3, MNDO, AMI, PM3 were employed to determine the structural descriptors which may influence the retention in a reversed-phase chromatographic system. Informative values of non-empirical structural descriptors determined with various semi-empirical methods have been compared.

Chromatographic Behavior of the Anthelmintic Fenbendazole and Its Major Metabolite Oxfendazole in Various Ion-Pair Liquid Chromatographic Systems

Abstract The chromatographic behavior of fenbendazole (FBZ) and oxfendazole (OFZ) in various reversed-phase liquid chromatographic (LC) systems has been investigated. The addition of negative and/or positive charged ion-pair reagents in the mobile phase has been examined, whereas the influence of mobile phase pH, mobile phase composition, and column temperature on retention and peak height has been evaluated. The observed behavior of the analytes during the various chromatographic processes has been discussed.

Comparison of reversed-phase chromatographic systems with principal component and cluster analysis

The separation of ethoxylated 1,1,3,3-tetramethylphenyl surfactants was investigated by reversed-phase liquid chromatography (RP-LC) using various supports (C 1, C 2, C 6, C 8, C 18, polyethylene-coated silica, and polyethylene-coated alumina) and with reversed-phase thin-layer chromatography (RP-TLC) using impregnated silica and alumina supports. The retention data matrix was evaluated both by principal component analysis and cluster analysis. The retention characteristics of both polyethylene-coated supports were similar to those of C 1, and the retention capacity of RP columns for surfactants increased with the increasing length of the covalently bonded hydrocarbon chain. Both multivariate mathematical statistical methods gave similar results and adequate separation was performed with the RP-LC and RP-TLC systems.

Analysis of Diethylenetriamine in Water and Soil at PPB Levels by High Performance Liquid Chromatography with Fluorescence Detection

Abstract A fast and sensitive method for the determination of diethylenetriamine in water and soil by High Performance Liquid Chromatography (HPLC) with Fluorescence Detection is described. Diethylenetriamine is converted to its fluorescamine derivatives through pre-column derivatization and is separated by an isocratic reverse phase chromatographic system (Nucleosil C18) with acetonitrile and borate buffer at pH 8 as the mobile phase. Water samples at concentrations of diethylenetriamine from 20–100 μg/L were derivatized with fluorescamine and chromatographed directly through direct aqueous injections. Recoveries averaged 94 ± 7% for a population of nine samples. Soil samples at concentrations of diethylenetriamine from 0.24–1.0 μg/g were extracted with CaCl2 solution (2 M) followed by derivatization with fluorescamine. Derivatized soil extracts were then analyzed by HPLC. Recoveries averaged 21 ± 2% for a population of nine samples.

Chromatographic separation of amide diastereomers: Correlation with molecular descriptors

The retention and separation of diastereomeric amides were studied on a reversed-phase chromatographic system. Amides were prepared by coupling chiral carboxylic acids to chiral amines with ethyl chloroformate as activating agent. Both increasing and decreasing α-values (range 0.87–1.39) with increasing eluent strength were observed, and in a few instances the elution order of diastereomers was reversed. Chromatographic data were correlated by partial least-squares (PLS) analysis with different sets of theoretical molecular descriptors, obtained from molecular mechanic calculations. A three-factor PLS model for retention explained 99.2% of the variance of the 80 amides. A six-factor model for separation factors explained 97.3% of the variance of the 40 pairs of amides. Electric potential distribution spectra, and descriptors derived from such spectra, were useful for the prediction of both absolute and relative retention.

Enzyme-based biosensor as a selective detection unit in column liquid chromatography

A reagentiess enzyme electrode based on co-immobilized alcohol oxidase and horseradish peroxidase was used as the working electrode in an amperometric flow-through cell connected to a column liquid chromatographic (CLC) system for the selective detection of methanol and ethanol. The enzymes were covalently immobilized in carbon paste (graphite-phenylmethylsilicone oil) in the presence of polyethylenimine. Electrodes prepared from the enzyme-modified carbon paste were optimized with respect to their sensitivity and selectivity. Different membranes were cast or electropolymerized directly on the surface of the electrode to increase the long-term stability of the biosensor. The compatibility with the reversed-phase chromatographic system was established. A PLRP-S polymer-based separation column was used with phosphate buffer as the mobile phase. The selectivity of the enzyme electrode was also determined by injecting some easily oxidizable and possibly interfering species normally present in biological samples. The enzyme electrode was also used in an on-line system, consisting of a microdialysis probe as the sampling unit, the CLC system and the biosensor detection device, for the selective following of the ethanol produced when a paper pulp industrial waste water was ferinented with Saccharomyces cerevisiae.

Chemiluminescence detection in liquid chromatography based on photo-oxygenation involving reactive oxygen intermediates

A luminol chemiluminescence (CL) detection system for reversed-phase liquid chromatography (RPLC), based on on-line photochemical production of organic hydroperoxides is presented. Tetramethylethylene (TME) was used as a reagent for generation of hydroperoxides in a post-column photochemical reactor, which is based on photo-oxygenation induced by the analytes eluting from the column. In the detector cell CL is generated by the microperoxidase (MP-11) catalyzed reaction of the photochemically produced hydroperoxides and luminol. The experimental parameters were optimized considering the photochemical reactor and the chemiluminescence detector independently. Optimization of the parameters was directed towards detection of analytes in a gradient reversed-phase chromatographic system and thus restricted by the eluent composition used for separation. The solvent composition was not varied freely; in order to achieve compatibility of the (new) detection system and (gradient) RPLC, it is largely determinated by the eluent required to perform the chromatographic separations. The applicability of the optimized system was evaluated for some model analytes and comparisons were made to the CL detection system based on photochemical generation of dioxetanes and to UV-visible absorption detection. Mechanistic implications with respect to the photo-induced production of hydroperoxides in the phothochemical reactor were considered and the influences of some known singlet oxygen quenchers and radical chain inhibitors on the signal were measured. The detection system was applied to the detection of polar pollutants in a gradient reversed-phase chromatographic system, analyzing spiked academic solutions and tap-water samples, in the latter case combined with on-line trace enrichment techniques.