Biomedical subjects

J Barbosa

Publications and source records attributed to J Barbosa.

At least 37 records · Page 2Linked to original sources

PubMed2001

Prediction of retention behaviour and evaluation of pka values of peptides and quinolones in liquid chromatography.

The present paper examines the effect of the solute ionisation on the retention behaviour in liquid chromatography of a series of peptide and quinolone compounds of biological interest, using acetonitrile-water media as mobile phases and a polymeric-based stationary phase. Polymeric columns with polystyrene-divinylbenzene (PS-DVB) polymer show advantages over silica-based reversed-phase packings since the former are stable in a wide pH range. (s)(s)pKa values have been evaluated using chromatographic data in acetonitrile-water mixtures with acetonitrile percentages of 30, 35, 40 and 50% (v/v) for quinolones and 12.5 and 20% (v/v) for peptides. The quinolones show great retention on PS-DVB phase stationary. It was thus necessary to work with a higher acetonitrile content in the mobile phase than for the less retained peptides. The pH values were measured in the hydroorganic mixtures, used as mobile phases, instead of in water and account was taken of the effect of activity coefficients. The derived equations permit the chromatographic determination of (s)(s)pKa. values of the peptides and quinolones in acetonitrile-water mixtures by fitting it to the experimental data in a nonlinear least-square procedure and also permit the prediction of the effect of (s)(s)pH on their chromatographic behaviour. We have also compared the obtained (s)(s)pKa values with those previously obtained in acetonitrile-water mixtures from potentiometric measurements.

Amino Acid Sequence↗

PubMed2001

Discovery of heterocyclic ureas as a new class of raf kinase inhibitors: identification of a second generation lead by a combinatorial chemistry approach.

Heterocyclic ureas, such as N-3-thienyl N'-aryl ureas, have been identified as novel inhibitors of raf kinase, a key mediator in the ras signal transduction pathway. Structure-activity relationships were established, and the potency of the screening hit was improved 10-fold to IC(50)=1.7 microM. A combinatorial synthesis approach enabled the identification of a breakthrough lead (IC(50)=0.54 microM) for a second generation series of heterocyclic urea raf kinase inhibitors.

Proto-Oncogene Proteins c-raf↗

PubMed2001

Determination of residues of enrofloxacin and its metabolite ciprofloxacin in biological materials by capillary electrophoresis.

A method for the analysis of enrofloxacin and ciprofloxacin in chicken muscle using marbofloxacin as internal standard is proposed. Clean-up and pre-concentration of the samples are effected by solid-phase extraction and determination is carried out by capillary electrophoresis using a photodiode array detector. The calibration graphs are linear for enrofloxacin and ciprofloxacin from 10 to 300 microg/kg. The method recoveries for enrofloxacin and ciprofloxacin are 74 and 54%, respectively. The limit of detection for the two compounds is lower than 25 microg/kg, which allows the detection of positive muscle samples at the required maximum residue limits.

Animals↗

PubMed2001

Electrophoretic behavior of peptides in capillary electrophoresis influence of ionic strength and pH in aqueous-organic media.

Through correct pH, pKa and activity coefficients values, a model describing the effect of pH on electrophoretic mobility of substances has been applied to a series of peptides in water and in acetonitrile-water mixtures. The derived equations permit prediction of the optimum pH for the electrophoretic separation from only a few experimental values and they also permit determination of pKa values of analytes in the aqueous-organic media employed. Furthermore, the electrophoretic resolution between pairs of substances can be predicted, in order to evaluate electrophoretic separations of the studied peptides.

Electrophoresis, Capillary↗

PubMed2001

Prediction of electrophoretic behaviour of a series of quinolones in aqueous methanol.

Quinolones are a family of antibacterial agents used in human and veterinary clinics. The examination of protonation equilibria is essential because their antibacterial activity is pH-dependent. In this work, dissociation constants of quinolones in MeOH-water mixtures were obtained using capillary electrophoresis. The method is based on a model that relates electrophoretic mobility of the solute with pH. The effect of pH, pKa and activity coefficient on electrophoretic behaviour was considered. Standard pH values for buffer solutions were previously determined in MeOH-water mixtures, and the pH can thus be measured in these media as in water. This model is also used to obtain the optimum conditions for the separation of a series of substances because it allows one to predict the resolution between adjacent peaks from a few experimental data.

Electrophoresis, Capillary↗

PubMed2001

Retention behaviour of peptides, quinolones, diuretics and peptide hormones in liquid chromatography. Influence of ionic strength and pH on chromatographic retention.

Peptides, quinolones, diuretics and peptide hormones are important substances of biomedical interest. In this work a model describing the effect of pH on retention in liquid chromatography (LC) is established and tested for these compounds using an octadecylsilica column. The suggested model uses the pH value measured in the hydro-organic mixture used as mobile phase instead of the pH value in water and takes into account the effect of the activity coefficients. The proposed equations permit the prediction of the pH optimum using a minimum number of measurements and also permit the determination of the acidity constants of the compounds considered in the medium used as mobile phase. Moreover, these equations can be combined with the previously derived equations, that relate the retention with the solvent composition of the mobile phase, to establish a general model that relates the elution behaviour of the solute with the significant mobile phase properties: composition, pH and ionic strength.

Chromatography, Liquid↗

PubMed2001

Migration behavior of therapeutic peptide hormones: prediction of optimal separation by capillary electrophoresis.

A general equation that relates electrophoretic mobility of polyprotic peptide substances and pH of the running electrolytes is established, taking into account the species in solution and the activity coefficients. Modelling electrophoretic mobility as a function of pH can be simultaneously used for determination of ionization constants and selection of the optimum pH for separation of mixtures of the modelled compounds. The proposed relationships allow an important reduction of the experimental data needed for development of new separation methods. The accuracy of the proposed equations is verified by modelling the migration behavior of a heterogeneous series of polyprotic amphoteric peptide hormones. By calculating the values of predicted resolutions, selection of the optimum pH to perform separation of their mixtures becomes a rapid and simple process.

Electrophoresis, Capillary↗

PubMed2001

Determination and characterization of diuretics in human urine by liquid chromatography coupled to pneumatically assisted electrospray ionization mass spectrometry.

The aim of this work was to develop a method for the characterization and determination of diuretics in human urine samples by liquid chromatography (LC) coupled to pneumatically assisted electrospray ionization (ES) mass spectrometry (MS). The diuretics studied were substances forbidden by the IOC such as trichlormethiazide, furosemide, canrenoic acid, benzthiazide, bendroflumethiazide, bumetanide, etacrynic acid and spironolactone. For this purpose, the operational parameters of electrospray, such as counter electrode voltage, capillary voltage, sample cone voltage and source temperature, were optimized in order to obtain the best signal stability and the highest sensitivity for the greatest number of diuretic agents. The optimized separation method was successfully coupled with the MS system to analyze the above-mentioned diuretics extracted from spiked urine samples by a liquid extraction and clean-up procedure at basic pH, using ethyl acetate as solvent and the salting-out effect (NaCl). The mass spectra obtained provide adequate information for identification purposes. Positive urine samples obtained from athletes were also analyzed. The presence of these substances in human urine was confirmed by this method, making LC/ES-MS an analytical tool to be considered in the area of antidoping control.

Chromatography, Liquid↗

PubMed2001

Investigation of synthetic peptide hormones by liquid chromatography coupled to pneumatically assisted electrospray ionization mass spectrometry: analysis of a synthesis crude of peptide triptorelin.

Triptorelin, a synthetic peptide hormone used in the treatment of prostate cancer by means of reduction in the action of male hormone testosterone, is studied here. The synthetic procedure commonly results in unwanted side products that require extensive purification and characterization of the synthesis mixture. The chromatographic separation of triptorelin from the crude mixture was developed by applying the linear solvation energy relationship (LSER) methodology previously developed, to optimize the composition of the mobile phase in order to avoid lengthy empirical optimization procedures. Electrospray ionization mass spectrometry coupled to liquid chromatography (LC/ES-MS) was used to obtain reliable information on the inevitable side products. The knowledge of the identity of these impurities allows fast optimization of the synthetic procedure and also the therapeutic use of triptorelin peptide hormone.

Amino Acid Sequence↗

PubMed2001

Evaluation of electrophoretic method versus chromatographic, potentiometric and absorptiometric methodologies for determing pK(a) values of quinolones in hydroorganic mixtures.

The advantages of using capillary electrophoresis (CE) over other methodologies for determining pK(a) values of drugs in hydroorganic media are discussed. The focus of the discussion based upon the pK(a) values of a series of quinolones determined in acetonitrile (MeCN)--water mixtures by CE, liquid chromatography, potentiometric, and spectrophotometric methods.

4-Quinolones↗

PubMed2001

Trafficking of green fluorescent protein tagged-vesicular acetylcholine transporter to varicosities in a cholinergic cell line.

Synaptic vesicle proteins are suggested to travel from the trans-Golgi network to active zones via tubulovesicular organelles, but the participation of different populations of endosomes in trafficking remains a matter of debate. Therefore, we generated a green fluorescent protein (GFP)-tagged version of the vesicular acetylcholine transporter (VAChT) and studied the localization of VAChT in organelles in the cell body and varicosities of living cholinergic cells. GFP-VAChT is distributed to both early and recycling endosomes in the cell body and is also observed to accumulate in endocytic organelles within varicosities of SN56 cells. GFP-VAChT positive organelles in varicosities are localized close to plasma membrane and are labeled with FM4-64 and GFP-Rab5, markers of endocytic vesicles and early endosomes, respectively. A GFP-VAChT mutant lacking a dileucine endocytosis motif (leucine residues 485 and 486 changed to alanine residues) accumulated at the plasma membrane in SN56 cells. This endocytosis-defective GFP-VAChT mutant is localized primarily at the somal plasma membrane and exhibits reduced neuritic targeting. Furthermore, the VAChT mutant did not accumulate in varicosities, as did VAChT. Our data suggest that clathrin-mediated internalization of VAChT to endosomes at the cell body might be involved in proper sorting and trafficking of VAChT to varicosities. We conclude that genesis of competent cholinergic secretory vesicles depends on multiple interactions of VAChT with endocytic proteins.

Acetylcholine↗

PubMed2001

Analytical methodology for the detection of benzodiazepine consumption in opioid-dependent subjects.

Benzodiazepines are frequently abused by heroin users, but not all compounds have shown the same abuse liability. We developed an analytical method that was able to detect various benzodiazepine compounds in a single run. Enzymatically hydrolyzed urine underwent a liquid-liquid extraction procedure with chloroform/isopropanol (9:1) at pH 8-9 followed by a solid-liquid clean-up (Bond Elut TCA C18) to obtain appropriate extracts for HPLC analysis. Mobile phase composition was optimized by means of the linear solvation energy relationship methodology based on Reichardt's normalized solvatochromic parameter (E(T)N). The method was validated for the simultaneous detection and quantitation of alprazolam, alpha-hydroxyalprazolam, oxazepam, 7-aminoflunitrazepam, flunitrazepam, nordiazepam, lorazepam, and diazepam. The prevalence of benzodiazepine consumption in 229 opioid-dependent subjects on methadone-maintenance treatment was 48%. Oxazepam and nordiazepam were the benzodiazepines most frequently recoved in urine samples. The prevalence of alprazolam use (40%) was higher than that of flunitrazepam (10%).

Adult↗

PubMed2000

Analysis of a peptide hormone mixture of therapeutic interest by liquid chromatography coupled to high-flow pneumatically assisted electrospray mass spectrometry.

High-flow pneumatically assisted electrospray ionization mass spectrometry (ESI-MS) has been extensively used for the characterization and determination of peptides and peptide hormones available for biomedical research and therapeutic applications. The aim of this study was to optimize a method of characterization and determination of a mixture of peptide hormones with therapeutic interest by liquid chromatography (LC) coupled to ESI-MS. In this work the linear solvation energy relationship methodology was used in order to optimize the mobile phase to be used in the LC separation of the peptide hormone series and the operational parameters of the source and analyzer of ESI were also optimized to obtain the best signal stability and the highest sensitivity. To validate the proposed method for peptide hormone analysis, quality parameters were determined and satisfactory results were obtained. Likewise, the method detection limit was picomole level for most of the peptides employing selected-ion monitoring of the [M+nH]n+ ions.

Amino Acid Sequence↗

PubMed2000

Liquid chromatography-electrospray mass spectrometry of multicomponent peptide mixtures. Characterization of a mixture from the synthesis of the hormone goserelin.

In order to separate and characterize the target peptide and the side-product peptide compounds of a synthesis mixture of the peptide hormone goserelin, liquid chromatography coupled to high-flow electrospray ionization mass spectrometry (LC-ES-MS) has been used. Goserelin is an important drug with recognized therapeutical application for palliative treatment of prostatic and breast carcinomas. Stepwise solid-phase peptide synthesis commonly results in unwanted side-products associated with incomplete peptide chains. Consequently, this procedure requires extensive purification and characterization of the final synthesis mixture. The method of linear solvation energy relationships has been applied to optimize the proportion of organic modifier of the mobile phase used in the established LC method. On the other hand, ES-MS has allowed rapid and reliable identification of the target peptide and the other impurities present in the goserelin synthesis products.

Antineoplastic Agents, Hormonal↗

PubMed2000

Prediction of electrophoretic mobilities in non-aqueous capillary electrophoresis. Optimal separation of quinolones in acetonitrile-water media.

A model of electrophoretic behaviour is used to predict the optimum conditions for the separation of a series of quinolones zwitterionic substances in mixtures of acetonitrile-water up to 30% (w/w) of acetonitrile. The effect of pH, pKa, the electrophoretic mobility of protonated and anionic species, and activity coefficients on the electrophoretic behaviour of quinolones is considered. The model proposed allows the resolution between substances in acetonitrile-water mixtures to be predicted from a few experimental data and thus permits one to obtain the best experimental conditions for separation methodologies.

4-Quinolones↗

PubMed2000

Influence of pH and pKa values on electrophoretic behaviour of quinolones in aqueous and hydro-organic media.

Through correct pH measurements, pKa and activity coefficient values, a model describing their effect on electrophoretic behaviour of substances is established. The suggested model uses the pH values in the acetonitrile-water mixtures used and takes into account the effect of activity coefficients. The model permits the calculation of acidity constants of analytes in hydro-organic media and also the prediction of the effect of pH on the electrophoretic mobility. The model is tested by determining the dissociation constants of a series of nine quinolones in acetonitrile-water mixtures of 0, 5.5, 10 and 30% (w/w) acetonitrile.

4-Quinolones↗

PubMed2000

Separation and characterization of multicomponent peptide mixtures by liquid chromatography-electrospray ionization mass spectrometry. Application to crude products of the synthesis of leuprolide.

Leuprolide is a synthetic structural analogue of luteinizing hormone-releasing hormone used for the treatment of a large number of diseases related with the regulation of sexual hormones. Solid-phase peptide synthesis is used to obtain leuprolide peptidic hormone, but this synthetic procedure results in complex mixtures that need separation and characterization. Here, liquid chromatography coupled with mass spectrometry using electrospray ionization, (LC-ES-MS), was used for the separation and characterization of multicomponent peptide mixtures of crudes of synthesis of leuprolide. To optimize the LC separation process, the method of linear solvation energy relationships was applied and the powerful coupling LC-ES-MS permitted rapid and reliable characterization of the reaction product.

Amino Acid Sequence↗

PubMed2000

Separation of potentially therapeutic peptide hormones by liquid chromatography. Optimisation of the composition and pH of the mobile phase.

The aim of this work is to optimise the proportion of the organic modifier and the pH of the mobile phase, in order to separate a series of peptide hormones with therapeutic interest in the molecular mass range from 500 to 6000. The composition of the mobile phase was optimised by establishing relationships between retention parameters and either the scale of solvent polarity, or the Kamlet-Taft multiparameter solvent scale of the eluent, using linear solvation energy relationships. Likewise, linear correlations between the chromatographic retention and Reichardt's E(T)N parameter were obtained. These relationships allowed an important reduction of the experimental retention data needed for developing a given separation. In addition, a model describing the effect of the correctly measured pH of the mobile phase on retention in LC was established and tested for the series of selected peptides using an octadecylsilica column. The proposed equations permit the prediction of the optimum pH and also permit the determination of the acidity constants of the peptides in the hydro-organic mixtures using a minimum number of measurements.

Amino Acid Sequence↗