Biomedical subjects

Manuel Silva

Publications and source records attributed to Manuel Silva.

14 recordsLinked to original sources

PubMed2007

MEKC: an update focusing on practical aspects.

This paper reviews recent methodological and instrumental advances in MEKC. Improvements in sensitivity arising from the use of on-line sample concentration (sweeping, stacking, and combination of both protocols) and derivatization (in-capillary reactions and coupling with flow-injection systems) and improvements in resolution obtained by changing the composition of the BGE (e.g., with organic modifiers, ionic liquids, nonionic and zwitterionic surfactants, mixed micelles, and vesicles) or using coated capillaries are discussed in detail. In addition, MS and LIF spectroscopy are examined in relation to their advantages and restrictions as applied to MEKC analysis. Some thoughts on potential future directions are also expressed.

Chromatography, Micellar Electrokinetic Capillary↗

PubMed2006

Determination of amikacin in body fluid by high-performance liquid-chromatography with chemiluminescence detection.

A simple and sensitive method was developed for the quantification of amikacin in human plasma and urine samples. The method involves centrifugation of body fluid plasma after dilution with an ethanol/sodium carbonate mixture, and then an aliquot of the supernatant is directly injected into the chromatograph. After separation on a reversed-phase C18 column (runtime 20 min), aminoglycoside is detected on the basis of its complex formation reaction with Cu(II), the catalyst of the luminol/hydrogen peroxide chemiluminescence system. Using a volume of 500 microl biological sample, linearity is established over the concentration range 0.15-2.0 microg/ml and the limit of detection (LOD) is ca. 50 microg/l in plasma or urine. The intra-day and inter-day precision (measured by relative standard deviation, R.S.D.%) are always less than 9%, and relative recoveries are found to be over 92%.

Amikacin↗

PubMed2006

Rapid and sensitive determination of aminoglycoside antibiotics in water samples using a strong cation-exchange chromatography non-derivatisation method with chemiluminescence detection.

A simple and sensitive chemiluminescence (CL) detection approach was developed for the direct analysis of aminoglycoside antibiotics in water samples following strong cation-exchange chromatographic (SCX-HPLC) separation. This detection system, which eliminates the need for sample derivatisation, is based on the inhibitory effect of aminoglycosides on the CL reaction between luminol and hydrogen peroxide catalysed by copper(II). As the operational and chemical variables that affect the CL signal were optimised, ionic strength and the Triton X-100 micelles turned out to be the keys to obtain maximum CL efficiency. Aminoglycosides were successfully separated in 10 min on a SCX column using a mobile phase consisting of an aqueous solution containing 5.0 x 10(-3)mol/l sodium acetate and 0.65 mol/l sodium chloride at pH 6.1. Sample volumes of 50 ml were preconcentrated by passage through a weakly acidic IRC-50 exchange column. Limits of detection from 0.7 to 10 microg/l and relative standard deviations from 2.7 to 5.4% were thus obtained. The proposed method surpasses other chromatographic alternatives in terms of the limit of detection, sample requirements for analysis and cost.

Aminoglycosides↗

PubMed2006

Trace analysis of aminoglycoside antibiotics in bovine milk by MEKC with LIF detection.

This work describes a straightforward and sensitive method for the multi-residue analysis of aminoglycoside antibiotics (kanamycin B, amikacin, neomycin B and paromomycin I) in bovine milk samples. The method involves the pre-capillary derivatization of antibiotics with sulfoindocyanine succinimidyl ester (Cy5) and their separation and determination by MEKC with LIF detection. The optimum procedure includes a derivatization step of the antibiotics at 25 degrees C for 30 min and direct injection for MEKC analysis, which is performed in about 20 min by using borate buffer (35 mM; pH 9.2) with 55 mM SDS as an anionic surfactant and 20% ACN as the organic modifier. Under these conditions, dynamic ranges of 10-500 microg/L and RSDs (within-day precision) from 3.8 to 5.3% were obtained. These results indicate that the proposed MEKC-LIF method is useful as a selective and sensitive tool for the determination of these antibiotics and surpasses other reported electrophoretic alternatives. Finally, the method was successfully applied to bovine milk samples after a simple solid-phase extraction clean-up and preconcentration procedure. The aminoglycosides were readily detected at 0.5-1.5 microg/kg levels with average recoveries ranging from 89.4 to 93.3%.

Aminoglycosides↗

PubMed2005

Rapid determination of aniline metabolites of chlorpropham in potatoes by micellar electrokinetic chromatography using negative-charged mixed micelles and laser-induced fluorescence detection.

A rapid, reliable method has been developed for the multi-residue analysis of aniline metabolites of chlorpropham in potato samples. The method involves the precolumn derivatization of aniline metabolites with 5-(4,6-dichloro-s-triazin-2-ylamino) fluorescein (DTAF) and their subsequent separation and determination by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection (MEKC-LIF). The optimum procedure includes a derivatization step of the aniline metabolites (3-chloroaniline, 3-chloro-4-hydroxyaniline and 3-chloro-4-methoxyaniline) at 40 degrees C for 40 min and a 5-fold dilution prior to MEKC analysis, which is conducted within about 7 min using negative-charged mixed micelles (SDS/Triton X-100) in the running buffer. Under these conditions, the DTAF-anilines were readily detected at 0.3-3.1 microg/L level with a precision of 4.8-6.4%. These results indicate that negative-charged mixed surfactant MEKC-LIF is useful as a selective, rapid, and sensitive tool for the determination of these anilines and surpasses other electrophoretic alternatives based on the use of fluorescein-isothiocyanate (FITC) as label reagent. Finally, the potato matrix showed no significant effects on the derivatization and determination of these analytes, since the analytical figures of merit for the real samples were similar to those obtained in aqueous solutions, and the average recovery at fortification levels of 10-250 microg/kg was over 97%.

Aminophenols↗

PubMed2005

Rapid and sensitive determination of phosphorus-containing amino acid herbicides in soil samples by capillary zone electrophoresis with diode laser-induced fluorescence detection.

A straightforward and sensitive method has been developed for the analysis of phosphorus-containing amino acid herbicides (glufosinate and aminomethylphosphonic acid, the major metabolite of glyphosate) in soil samples. For this purpose, the analytical features of two indocyanine fluorescent dyes, sulfoindocyanine succinimidyl ester (Cy5) and 1-ethyl-1-[5-(N-succinimidyl-oxycarbonyl)pentyl]-3,3,3,3-tetramethyl-indodicarbocyanine chloride, as labeling reagents for the determination of these herbicides by CZE with diode LIF detection were investigated. Practical aspects related to the labeling chemistry and CZE separation showed that the two probes behave similarly, Cy5 being the best choice for the determination of these herbicides on account of its higher sensitivity. The optimum procedure includes a derivatization step of the pesticides at 25 degrees C for 30 min and direct injection to CZE analysis, which is conducted within about 14 min using ACN in the running buffer. The lowest detectable analyte concentration ranged from 0.025 to 0.18 microg/L with a precision of 3.6-5.4%. These results indicate that indocyanine fluorescence dyes are useful as rapid and sensitive labels for the determination of these herbicides when compared with typical fluorescein dyes such as FITC and 5-(4,6-dichloro-s-triazin-2-ylamino) fluorescein, because they provide faster labeling reactions even at room temperature and the excess of reagent practically does not interfere the determination. Finally, the Cy5 method was successfully applied to soil samples without a preliminary clean-up procedure, and the herbicides were measured without any interference from coexisting substances. The recoveries of these compounds in these samples at fortification levels of 100-500 ng/g were 90-93%.

Aminobutyrates↗

PubMed2003

Monitoring some phenoxyl-type N-methylcarbamate pesticide residues in fruit juices using high-performance liquid chromatography with peroxyoxalate-chemiluminescence detection.

A systematic investigation of optimal conditions for determining three of the most common phenoxyl-type N-methylcarbamate pesticides (carbaryl, carbofuran and propoxur) in fruit juices by HPLC with peroxyoxalate-chemiluminescence detection is described. The required pre-column hydrolysis of pesticides and derivatization of their hydrolytic metabolites with dansyl chloride was simultaneously carried out in a short time thanks to the micellar catalytic effect provided by cetyltrimethyl ammonium bromide micelles on the hydrolysis step. The liquid chromatographic separation of the dansylated phenols was performed on a reversed-phase C18 column with isocratic elution. The analytes were detected by using an integrated derivatization chemiluminescence detection unit based on the bis(2,4,6-trichlorophenyl)oxalate-hydrogen peroxide system. Fruit juice samples containing 4.0-1500 microg/l pesticides were analysed with a precision of ca. 6.5%. After contamination of the fruit juice samples, average recovery > 93% at fortification levels of 10-100 microg/l was obtained.

Beverages↗

PubMed2002

Analytical potential of fluorescein analogues for ultrasensitive determinations of phosphorus-containing amino acid herbicides by micellar electrokinetic chromatography with laser-induced fluorescence detection.

The analytical potential of three fluorescein analogues, fluorescein isothiocyanate isomer I (FITC), 5-(4,6-dichlorotriazinylamino) fluorescein (DTAF) and 5(6)-carboxyfluorescein N-succinimidyl ester (CFSE), as labelling reagents for the ultrasensitive determination of phosphorus-containing amino acid herbicides (glufosinate and glyphosate) and aminomethylphosphonic acid (the major metabolite of glyphosate) by nonionic surfactant micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection was investigated. Practical aspects related to label chemistry and MEKC separation showed that DTAF is the best choice for the determination of these herbicides; in addition, the most important features of these reagents for the derivatization of amino compounds are discussed. The optimum procedure includes a derivatization step of the herbicides at 40 degrees C with DTAF for 1 h and a 2-fold dilution prior to MEKC analysis, which is conducted within about 10 min using Brij-35 in the running buffer. This nonionic surfactant improves the selectivity and therefore the sensitivity of the method at low analyte concentrations by shifting the interfering peaks of the DTAF excess. The lowest detectable analyte concentration ranged from 0.06 to 0.16 microg/L with a precision of 2.1-3.2%. These results indicate that nonionic surfactant MEKC-LIF is useful as a selective, rapid and sensitive tool for the determination of these herbicides showing a great potential for their analysis in environmental samples without previous enrichment steps. The proposed method surpasses other chromatographic alternatives in terms of limit of detection and sample requirements for the analysis.

Amino Acids↗

PubMed2002

In-capillary derivatization and analysis of amino acids, amino phosphonic acid-herbicides and biogenic amines by capillary electrophoresis with laser-induced fluorescence detection.

This paper describes a general approach for the in-capillary derivatization of amino compounds and the subsequent sensitive determination of the derivatives by micellar electrokinetic chromatography (MEKC) or capillary zone electrophoresis (CZE) with laser-induced fluorescence (LIF) detection. Amino acids, biogenic amines and amino phosphonic acid-herbicides were chosen as model analytes to evaluate the analytical potential of this approach. Fulfilment of the in-capillary reaction of the analytes using LIF detection hinged on the excellent labeling chemistry of 5-(4,6-dichloro-s-triazin-2-ylamino)fluorescein (DTAF) and the good resolution achieved in the separation of derivatized analytes. Careful optimization of the electrophoretic conditions in the mixing step of this protocol allowed the determination of amino acids, biogenic amines and phosphorus-containing amino acid-herbicides with concentration limits of detection at the nug/L level and relative standard deviations from 3.5 to 5.8%. The whole analysis is carried out within 20 min, resulting in a very simple, fast and practical approach for the fully automated analysis of amino acids and related compounds in low-volume and low-concentration samples.

Amino Acids↗

PubMed2002

Micellar electrokinetic chromatography: current developments and future.

This review highlights recent methodological and instrumental advances in micellar electrokinetic chromatography (MEKC). Enhancements in sensitivity and selectivity of the technique through the use of on-line preconcentration approaches (stacking and sweeping) and nonconventional pseudostationary phases, namely nonionic and zwitterionic surfactants, mixed micelles and polymers, are discussed in detail. Laser-induced fluorescence and mass spectrometry, as alternatives to UV-absorption detection, have been covered to evaluate their advantages and limitations when applied to analysis in an MEKC format. Some thoughts on future directions in this area such as in-capillary reactions, coated capillaries and MEKC on microchips are also presented.

Chromatography, Micellar Electrokinetic Capillary↗

PubMed2002

Enrichment of microbial cultures able to degrade 1,3-dichloro-2-propanol: a comparison between batch and continuous methods.

Microbial cultures able to degrade xenobiotic compounds are the key element for biological treatment of waste effluents and are obtained from enrichment processes. In this study, two common enrichment methods, suspension batch and immobilized continuous, were compared. The main selection factor was the presence of 1,3-dichloro-2-propanol (1,3-DCP) as the single carbon source. Both methods have successfully enriched microbial consortia able to degrade 1,3-DCP. When tested in batch culture, the degradation rates of 1,3-DCP by the two consortia were different, with the consortia obtained by batch enrichment presenting slightly higher rates. A preliminary morphological and biochemical analysis of the predominant colonial types present in each degrading consortia revealed the presence of different constituting strains. Three bacterial isolates capable of degrading 1,3-DCP as single strains were obtained from the batch enrichments. These strains were classified by 16S rRNA analysis as belonging to the Rhizobiaceae group. Degradation rates of 1,3-DCP were lower when single species were used, reaching 45 mg 1(-1) d(-1), as compared to 74 mg 1(-1) d(-1) of the consortia enriched on the batch method. Mutualistic interactions may explain the better performance of the enriched consortia.

Bacteria↗

PubMed2002

Sensitive determination of low molecular mass phenols by liquid chromatography with chemiluminescence detection for the determination of phenol and 4-methylphenol in urine.

A rapid, reliable method for the routine determination of phenol and 4-methylphenol in urine samples by liquid chromatography with peroxyoxalate chemiluminescence detection was developed. Phenols were first cleaned up by passing the sample through a LiChrolut EN sorbent column and then derivatized straightforwardly with dansyl chloride (15 min at room temperature) thanks to the micellar catalytic effect provided by Triton X-100 micelles. The derivatives were successfully separated in 15 min on a C18 analytical column and determined using an integrated derivatization chemiluminescence detection unit based on the bis(2,4,6-trichlorophenyl) oxalate-hydrogen peroxide system. Linear ranges from 3 to 500 microg L(-1), limits of detection at a signal-to-noise ratio of 3 from 0.3 to 0.5 microg L(-1) and relative standard deviations from 2.8 to 4.7% were obtained. The proposed method was applied to the assay of different human urine samples (healthy, smoker and petrol station worker volunteers) and free and total phenol and 4-methylphenol were determined. The proposed method surpasses other chromatographic alternatives for the determination of these phenols in terms of limit of detection and sample requirements for the analysis.

Chromatography, Liquid↗

PubMedDate not supplied

Improving the quantification of highly overlapping chromatographic peaks by using product unit neural networks modeled by an evolutionary algorithm.

This work investigates the ability of multiplicative (on the basis of product units) and sigmoidal neural models built by an evolutionary algorithm to quantify highly overlapping chromatographic peaks. To test this approach, two N-methylcarbamate pesticides, carbofuran and propoxur, were quantified using a classic peroxyoxalate chemiluminescence reaction as a detection system for chromatographic analysis. The four-parameter Weibull curve associated with the profile of the chromatographic peak estimated by the Levenberg-Marquardt method was used as input data for both models. Straightforward network topologies (one output) allowed the analytes to be quantified with great accuracy and precision. Product unit neural networks provided better information ability, smaller network architectures, and more robust models (smaller standard deviation). The reduced dimensions of the selected models enabled the derivation of simple quantification equations to transform the input variables into the output variable. These equations can be more easily interpreted from a chemical point of view than those provided by sigmoidal neural networks, and the effect of both analytes on the characteristics of chromatographic bands, namely profile, dispersion, peak height, and residence time, can be readily established.

Journal Article↗

PubMedDate not supplied

Heuristic extraction of rules in pruned artificial neural networks models used for quantifying highly overlapping chromatographic peaks.

The suitability of an approach for extracting heuristic rules from trained artificial neural networks (ANNs) pruned by a regularization method and with architectures designed by evolutionary computation for quantifying highly overlapping chromatographic peaks is demonstrated. The ANN input data are estimated by the Levenberg-Marquardt method in the form of a four-parameter Weibull curve associated with the profile of the chromatographic band. To test this approach, two N-methylcarbamate pesticides, carbofuran and propoxur, were quantified using a classic peroxyoxalate chemiluminescence reaction as a detection system for chromatographic analysis. Straightforward network topologies (one and two outputs models) allow the analytes to be quantified in concentration ratios ranging from 1:7 to 5:1 with an average standard error of prediction for the generalization test of 2.7 and 2.3% for carbofuran and propoxur, respectively. The reduced dimensions of the selected ANN architectures, especially those obtained after using heuristic rules, allowed simple quantification equations to be developed that transform the input variables into output variables. These equations can be easily interpreted from a chemical point of view to attain quantitative analytical information regarding the effect of both analytes on the characteristics of chromatographic bands, namely profile, dispersion, peak height, and residence time.

Journal Article↗