Biomedical subjects

L Puignou

Publications and source records attributed to L Puignou.

36 records · Page 2Linked to original sources

PubMed2001

Determination of free and total sulfate and phosphate in glycosaminoglycans by column-switching high-performance size-exclusion and ion chromatography and single-column ion chromatography.

Analytical procedures for the determination of free and total sulfate and phosphate in glycosaminoglycans by high-performance liquid chromatography were studied. A column-switching method coupling high-performance size-exclusion chromatography (HPSEC) and ion chromatography (IC) is proposed for the determination of free anions. Good run-to-run and day-to-day precision values (RSD) of < 4.7% were obtained for both anions. Total anion contents were determined after wet acid hydrolysis with nitric acid-hydrogen peroxide (5 + 1) by single-column IC and ICP-AES elemental analysis in order to validate the results. Recoveries ranging from 94.6 to 99.0% for sulfate and from 80.8 to 94.0% for phosphate were obtained. Both HPSEC-IC and single-column IC methods were applied to the analysis of a low molecular mass heparin, a non-fractionated heparin and a chondroitin 4-sulfate. From the free and total sulfate determinations, the content of linked sulfur was calculated and ranged from 5.1 to 12.2% m/m.

Journal Article↗

PubMed2000

Separation of heteroaromatic amines in food products.

In recent years, many studies have dealt with the role of certain heteroaromatic amines (HAs) as mutagenic compounds, and their occurrence in foodstuffs. Here we examine the determination of HAs, focusing on the analytical strategies for their extraction and preconcentration from several matrices. We summarise the properties of heteroaromatic amines and the main drawbacks involved in their analysis, and then concentrate on the separation procedures, sorbents and solvents used in the sample treatment. We discuss the requirements of the analytical techniques and the strategies most frequently followed to achieve accurate results.

Amines↗

PubMed2000

Determination of ebrotidine and its metabolites by capillary electrophoresis with UV and mass spectrometry detection.

This study describes the application of capillary electrophoresis (CE) to the analysis of ebrotidine and its metabolites as an alternative analytical technique to liquid chromatography. Comparison between UV-diode array spectroscopy and mass spectrometry (MS) using an ion-trap system with electrospray ionization as detection systems has been performed. The quality parameters of the UV detection method were established, obtaining linear calibration curves over the range studied (8-200 mg ml(-1)), limits of detection between 3.4 and 4.3 microg ml(-1), and run-to-run and day-to-day precision lower than 14%. For these compounds the protonated species [M+H]+ and, in some cases, sodium adducts were observed in the MS spectra. Using MS coupled to CE, limits of detection were between 0.5 and 2.6 microg ml(-1).

Benzenesulfonates↗

PubMed2000

Comparison of different commercial solid-phase extraction cartridges used to extract heterocyclic amines from a lyophilised meat extract.

Heterocyclic amines are a group of potent mutagenic compounds which are generated when muscle meat is cooked. Since they are possible human carcinogens, these mutagens have received considerable attention in recent years, and several analytical techniques have been developed for their quantification. Although the purification step is one of the most important, there are a great number of variables influencing the recovery of the amines, especially when real samples are analysed. In this work we studied the influence of sample spiking mode on the recoveries. Furthermore, on the basis of a previously developed clean-up method, the effect of changing commercial source and structure of the sorbents used in two solid-phase extraction steps was examined. This purification method was applied to the quantification of the heterocyclic aromatic amines present in a lyophilised meat extract by means of liquid chromatography-mass spectrometry.

Amines↗

PubMed2000

Analysis of glycosaminoglycan monosaccharides by capillary electrophoresis using indirect laser-induced fluorescence detection.

Two methods for monosaccharide analysis by capillary electrophoresis (CE) using counterelectroosmotic and coelectroosmotic modes with indirect laser-induced fluorescence detection were optimised and compared. A mixture of seven glycosaminoglycan-derived hexoses was separated in alkaline fluorescein-based electrolytes and detected in both counterelectroosmotic and coelectroosmotic conditions. The fluorescein concentration and pH of the background electrolyte, and the influence of the reversal of electroosmotic flow by addition of hexadimethrine bromide on the separation were studied. Coelectroosmotic CE conditions provided better resolution and limits of detection. A 10(-6) M fluorescein solution at pH 12.25 containing 0.0005% (w/v) hexadimethrine bromide was used as background electrolyte. Quality parameters such as run-to-run, day-to-day precision and limits of detection were calculated, and better figures of merit were obtained for the coelectrooosmotic conditions than for the counterelectroosmotic mode. The coelectroosmotic method was applied to the quantitation of the hexosamine contents in glycosaminoglycans after acid hydrolysis. The method proved to be suitable for the determination of dermatan sulfate in heparin down to 2% (w/w).

Electrophoresis, Capillary↗

PubMed2000

Determination of heterocyclic aromatic amines in meat extracts by liquid chromatography-ion-trap atmospheric pressure chemical ionization mass spectrometry.

When protein-rich foods are processed under normal cooking conditions, heterocyclic aromatic amines (HAAs) can be generated at a few parts per billion level. In this work, we have analyzed the HAAs present in a lyophilized meat extract by means of a simplified solid-phase extraction procedure. All the analytes were collected in a single extract with recoveries in the range of 45.6-75.2%, so the analysis time has been greatly reduced. Problems derived from the less exhaustive purification of the extract have been solved by using MS(ion trap) detection. The RSD for quantification ranged from 2.1% to 5.1% for run-to-run precision and from 5.2% to 11% for day-to-day precision. The limits of detection for standard solutions ranged from 20 to 150 pg injected. For the meat extract analyzed limits of detection from 0.9 to 11.2 ng g(-1) were obtained. Results of the quantification are in agreement with those obtained using different clean-up procedures.

Amines↗

PubMed2000

New carrier electrolytes for the separation of chlorophenols by capillary electrophoresis.

Optimum conditions for the separation of positional isomers of chlorophenols by capillary zone electrophoresis (CZE) were established. The behavior of five volatile electrolytes (L-cysteic acid, 3-amino-1-propanesulfonic acid, aminomethanesulfonic acid, diethylmalonic acid, and ammonium acetate) was compared. The best performance based on low electrophoretic current and high separation efficiency was obtained for diethylmalonic acid as working electrolyte. The influence of pH on the separation, using both uncoated fused-silica capillaries and modified capillaries (NaAMPS from EKT) with anionic coating, was discussed. Moreover, the effect of electrolyte concentration and applied voltage using fused-silica capillaries was studied. The optimum CZE conditions that allowed the separation of 16 chlorophenols were 20 kV, 30 mM diethylmaIonic acid, pH 7.25, and uncoated fused-silica capillary. Figures of merit such as run-to-run and day-to-day precision, linearity, and limits of detection were calculated.

Chlorophenols↗

PubMed2000

Potentiality of proton nuclear magnetic resonance and multivariate calibration methods for the determination of dermatan sulfate contamination in heparin samples.

A 1H NMR method for the quantification of dermatan sulfate impurities in heparin industrial samples is proposed. The method is based on the analysis of 1H NMR spectral data by multivariate calibration. The 1H NMR spectra of heparin and dermatan sulfate standards showed characteristic profiles. Thus, differences in the methyl peaks of acetamido groups of heparin and dermatan sulfate were greatly advantageous for the analysis. Other hydrogens of the sugar ring were also relevant in this study. Thus, the determination of dermatan sulfate by multivariate calibration depended on all these differences. Partial least squares regression (PLS) was chosen as the calibration method. In addition, a data standardization procedure was developed in order that 1H NMR spectra registered with different instruments operating under different measurement conditions were comparable. The quantification of dermatan sulfate in the samples was satisfactory, with an overall prediction error of 6%.

Calibration↗

PubMed1999

Evaluation of different clean-up procedures for the analysis of heterocyclic aromatic amines in a lyophilized meat extract.

Along with other mutagenic and carcinogenic contaminants in foods such as aflatoxins, and polycyclic aromatic hydrocarbons, heterocyclic aromatic amines (HAAs) have received considerable attention in recent years. A major drawback in the analysis of HAAs in foods is their very low level of concentration (0.1 50 ng g-1) as well as matrix interferences. Solid-phase extraction (SPE), forming an integral part of chromatographic analysis, is one of the procedures currently used for the extraction and purification of HAAs in food samples. In this paper a comparative study of several SPE procedures for HAAs determination was performed. Recoveries of the heterocyclic amines in the analysis of both a simple matrix such as a standard methanolic solution and a contaminated meat extract were established. HAAs were determined by HPLC analysis with photodiode-array detection (DAD) of the purified extracts, and the adequacy of different clean-up procedures for the analysis of a contaminated meat extract was discussed.

Animals↗

PubMed1998

Use of reversed polarity and a pressure gradient in the analysis of disaccharide composition of heparin by capillary electrophoresis.

A capillary electrophoresis method with reversed polarity, combining both the application of a voltage and a pressure gradient between the buffer vials, was developed for the analysis of eight heparin-derived delta-disaccharides obtained by enzymatic depolymerization. A 60 mM formic acid buffer at pH 3.40 was selected as running electrolyte, with an applied voltage of -15 kV and an over-imposed pressure gradient (3.45.10(-3) MPa) for 6 min from inlet to outlet starting at 20 min. Figures of merit such as run-to-run and day-to-day precision, and limits of detection were established. The electrophoretic method was applied to the analysis of depolymerization products of different kinds of heparins. The composition of the depolymerization buffer was selected in order to reduce baseline distortions in the electrophoretic separation, thus a buffer solution containing 20 mM Tris, 50 mM sodium chloride, and 3 mM calcium chloride at pH 7.10 was used. Percentages of molar disaccharide compositions for unfractionated heparins from porcine, bovine and ovine intestinal mucosa, and bovine lung were determined. In addition, low-molecular-mass heparins from bovine and porcine intestinal mucosa were analysed as well.

Animals↗

PubMed1997

Liquid chromatography-atmospheric-pressure chemical ionization mass spectrometry as a routine method for the analysis of mutagenic amines in beef extracts.

A liquid chromatography-mass spectrometry (LC-MS) method using atmospheric-pressure chemical ionisation as interface was developed for the simultaneous determination of 14 heterocyclic aromatic amines and related compounds in beef extracts. The separation was performed on a conventional C18 column using a binary mobile phase composed of acetonitrile and 50 mM ammonium acetate at pH 5.7, and elution was carried out in gradient mode. Several parameters influencing the mass spectra were optimized, and the effect of the variation of cone voltage on the mass spectra was studied. The [M+H]+ ions and some fragments produced in the source were observed in the mass spectra when several extraction voltages were applied. Quality parameters (run-to-run and day-to-day reproducibility, intervals of linearity, and limits of detection) were studied in the optimum working conditions. The method was used to analyze the heterocyclic amines present in a commercial beef extract. Therefore, a solid-phase extraction clean-up procedure was performed prior the LC-MS analysis due to the complexity of the sample and the compounds Glu-P-1, Harman, Norharman and A alpha C were identified in the samples at ppb levels and successfully confirmed using in-source fragmentation.

Amines↗

PubMed1997

Liquid chromatography-electrospray mass spectrometry with in-source fragmentation for the identification and quantification of fourteen mutagenic amines in beef extracts.

Conditions for the liquid chromatography-electrospray mass spectrometric (LC-ES-MS) determination of the fourteen most abundant heterocyclic amines and related compounds that can be produced in thermally processed foods were established. The simultaneous separation of all HAs on an octadecylsilane microbore column using a gradient elution with 5 mM ammonium acetate, pH 4.0, and acetonitrile was optimized. The mass spectra obtained at different extraction voltages were studied. The highest extraction voltages provided structural information for the characterization of these amines. Good detection limits comparable with those of LC and electrochemical detection were obtained. The method was applied to the analysis of these mutagens in beef extracts after a solid-phase extraction clean-up and in-source fragmentation had been applied for confirmation purposes. The levels of several heterocyclic amines measured were estimated to be in the range of 7.8-135.2 ng g-1.

Amines↗

PubMed1996

Solid-phase extraction for the selective isolation of polycyclic aromatic hydrocarbons, azaarenes and heterocyclic aromatic amines in charcoal-grilled meat.

A method for the simultaneous analysis of 12 mutagenic and/or carcinogenic compounds is described; these substances belong to three different chemical groups: polycyclic aromatic hydrocarbons (PAHs), azaarenes, i.e., nitrogen-containing polycyclic aromatic hydrocarbons (PANHs), and heterocyclic aromatic amines (HAAs). The selective enrichment procedure includes coupling of solid-phase extraction (SPE) steps using diatomaceous earth, propylsulfonic acid, silica gel and octadecylsilane columns. The eluted fractions were analysed by high-performance liquid chromatography with UV and electrochemical detection. Levels measured were estimated to be 4-19 ng g-1. Peak confirmation was carried out by GC-MS for both PAHs and PANHs, and by LC with a photodiode array detector for HAAs. The method was applied to the analysis of charcoal-grilled meat and was judged to be generally applicable for detection of these mutagens at the ppb level in processed foods.

Amines↗

PubMed1996

Determination of heterocyclic amines by pneumatically assisted electrospray liquid chromatography-mass spectrometry.

Electrospray ionization mass spectrometry was applied to the study of the amines IQ, Trp-P-1, Trp-P-2, PhIP and A alpha C and the co-mutagens harman and norharman. The results obtained on a triple quadrupole mass spectrometer equipped with a pneumatically assisted electrospray source are reported. The chromatographic conditions were optimized with a reversed-phase column (1 mm I.D.) using acetonitrile-5 mM ammonium acetate (pH 6.7) (50:50) as the mobile phase at a flow-rate of 50 microliters min-1. Different parameters influencing the mass spectra were investigated. For these compounds [M + H]+ in the positive-ion mode and also some fragments produced through collisionally activated decomposition in the interface were observed. Detection limits of 5.4-44 pg were obtained for standard solutions of these amines. Analysis of a meat extract was performed by HPLC-MS using single-ion monitoring after a solid-phase extraction clean-up.

Amines↗

PubMed1996

Isolation by solid-phase extraction and liquid chromatographic determination of mutagenic amines in beef extracts.

A solid-phase extraction method was successfully optimized for the isolation and preconcentration of five mutagenic amines, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole, 3-amino-1-methyl-5H-pyrido[4,3-b]indole, 2-amino-9H-pyrido[2,3-b]indole, 2-amino-3-methyl-9H-pyrido[2,3-b]indole and 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine, and two co-mutagens, 1-methyl-9H-pyrido[4,3-b]indole and 9H-pyrido[4,3-b]indole. Coupling of diatomaceous earth, propylsulphonyl silica gel, and octadecylsilane cartridges was used to separate selectively the imidazopyridine and indolpyridine derivatives from those of quinoxaline and quinoline. A method based on this sample preparation was applied to the determination of twelve heterocyclic amines and related substances in a commercial beef extract using HPLC with electrochemical and fluorescence detection. Good recovery values were obtained, ranging between 55 and 99%. The co-mutagens 1-methyl-9H-pyrido[4,3-b]indole (harman) and 9H-pyrido[4,3-b]indole (norharman) were found in the beef extract at levels of 110 and 53 ng g-1, respectively, and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and 2-amino-9H-pyrido[2,3-b]indole (A alpha C) were tentatively identified.

Amines↗

PubMed1988

Kinetic-spectrophotometric method for the assay of copper(II) in human serum by catalytic oxidation of salicylic acid.

A very sensitive kinetic spectrophotometric method for the determination of copper(II) concentrations as low as 0.07 ng ml(-1) is described. This method is based on the oxidation of salicylic acid by hydrogen peroxide in ammoniacal medium, catalysed by copper(II) ion. The figures of merit of the procedure and the results of a study of interferences are given. The method is applied to the assay of copper in human blood serum.

Journal Article↗

PubMedDate not supplied

Determination of viable yeast cells by gravitational field-flow fractionation with fluorescence detection.

Vinification processing is largely related to yeast performance and depends on the initial cell viability. To optimize the quality of wine fermentation, control of the yeast quality is mandatory. The present paper describes a new method using gravitational field flow fractionation (GrFFF) with fluorescence detection for the determination of yeast cell viability before the fermentation process. A GrFFF calibration procedure was developed using commercial yeast to prepare standards of viable cells and propidium iodide (PI) as fluorescent probe for nonviable cells. The suitability of the new method was tested with several commercial yeast strains with a g/L content ranging from 1 to 3. The validation of the method was performed by comparing GrFFF viability values with those obtained using Coulter counter and flow cytometry techniques.

Cell Separation↗

PubMedDate not supplied

Use of fluorescent probes for determination of yeast cell viability by gravitational field-flow fractionation.

The quality of wine greatly depends on the features of the yeast used in its production, and yeast cell viability is one of the most important quality control issues to consider in this regard. In the first steps of winemaking, the use of a low-cost and simple methodology for monitoring the cell viability of yeast inoculates is of paramount importance. Gravitational field-flow fractionation is a useful technique for the determination of cell viability because it provides gentle experimental conditions, although the proper use of fluorophore probes as biomass indicators is required. In this paper the use of different fluorescent probes such as carboxyfluorescein diacetate (cFDA), calcein-AM, and SYTO-13 were considered as viability biomarkers. Calceina-AM allowed the establishment of a direct GrFFF method to determine cell viability, with a limit of detection of 5.0 x 10(4) viable cell/mL. SYTO-13 could be used as biomass indicator with a limit of detection of 3.5 x 10(4) total cells/mL. The suitability of the procedure was tested with three commercial yeast samples, and the results were compared with those obtained using standard techniques.

Calibration↗