Biomedical subjects

Emanuel Carrilho

Publications and source records attributed to Emanuel Carrilho.

18 recordsLinked to original sources

PubMed2006

Optimization of solid-phase microextraction procedures for the determination of tricyclic antidepressants and anticonvulsants in plasma samples by liquid chromatography.

Simple, sensitive, and reproducible off-line solid-phase microextraction and liquid chromatography (SPME/LC) methods are described for the determination of seven anticonvulsants and tricyclic antidepressants in human plasma. Factorial design and simplex methodology were applied in the optimization of the SPME procedure for tricyclic antidepressants analyses. Important factors in the SPME efficiency are discussed, such as the fiber coatings (both lab-made and commercial), extraction time, pH, ionic strength, influence of plasma proteins, and desorption conditions. The development of the lab-made fiber coatings, namely, octadecylsilane, aminosilane, and polyurethane, are further described and applied to anticonvulsants analyses. The investigated plasmatic range for the evaluated anticonvulsants, using CW-TPR fiber, were the following: phenylethylmalonamide (3.00-40.0 microg mL(-1)), phenobarbital (5.00-40.0 microg mL(-1)), primidone (3.00-40.0 microg mL(-1)), carbamazepine and carbamazepine-epoxide (2.00-24.0 microg mL(-1)), phenytoin (2.00-40.0 microg mL(-1)), and lamotrigine (0.50-12.0 microg mL(-1)). The antidepressants' linear plasmatic concentration ranged from 75.0 to 500 ng mL(-1) for imipramine, amitriptyline, and desipramine, and from 50.0 to 500 ng mL(-1) for nortriptyline, being in all cases, the limit of quantification represented by the lowest value. The precision (interassays) for all investigated drugs in plasma sample spiked with different concentrations of each analyte and submitted to the described procedures were lower than 15%. The off-line SPME/LC methodologies developed allow anticonvulsants and antidepressants analyses from therapeutic to toxic levels for therapeutic drug monitoring.

Amitriptyline↗

PubMed2006

Capillary electrophoresis-mass spectrometry of citrus endophytic bacteria siderophores.

CE-ESI-MS with a liquid sheath interface and IT mass analyzer was used for analysis of siderophores from different strains of Methylobacterium spp. citrus endophyte extracts. Three Methylobacterium strains were investigated according to positive bioassay tests. Bacteria cultures were grown under Fe(III) absence (siderophore producing cultures) and under Fe(III) presence (control cultures). Siderophores were extracted from culture supernatant with polystyrene resins. BGE and sheath-liquid composition were optimized, respectively, in order to assure both, best peak resolution and ESI-MS sensitivity. The best analysis conditions were obtained with 100 mmol/L ammonium bicarbonate at pH 8 as BGE and methanol:H(2)O 25:75 + 0.05% formic acid as sheath liquid. CZE-ESI-MS analysis revealed two possible siderophores, according to bacterium species, presenting M(r) of 1004.3 and 798.3 Da.

Bicarbonates↗

PubMed2006

Tandem mass spectrometry of coprogen and deferoxamine hydroxamic siderophores.

Mechanisms of fragmentation of hydroxamic siderophores are proposed comparing deuterated and nondeuterated samples. Standard siderophores (e.g. deferoxamine and coprogen) were directly injected into both ion trap and linear quadrupole mass spectrometers with electrospray ionization (ESI). Four and two fragmentation steps were carried out for deferoxamine and coprogen (analyzed by positive and negative ESI, respectively). Deferoxamine cleavages occurred in both peptide and hydroxamic bonds while the coprogen fragmentation pattern is more elaborate, since it contains Fe(III) in its structure.

Chromatography, Liquid↗

PubMed2005

In vitro monitoring of GTPase activity and enzyme kinetics studies using capillary electrophoresis.

A capillary electrophoresis (CE)-based method for the in vitro detection and monitoring of nucleotide-triphosphatase activity is described. This robust and reproducible method was used to investigate GTPase activity of a recombinant protein construct containing the catalytic domain of Human SEPT4/Bradeion beta (GST-rDGTPase). This example application demonstrates that the CE technique can replace classical radioactive methods for GTPase activity assays and may be used as a routine analytical tool. Enzyme kinetics of GST-rDGTPase was studied and yielded the following kinetic parameters: v(max) = 1.7 microM min(-1) +/- 0.1, Km = 1.0 mM +/- 0.3, and apKcat = 9 x 10(-3) s(-1). In addition the effect of co-factors such as Mg2+ and Mn2+ on the catalytic activity was investigated. The described analytical method was also shown to be useful to analyze diphosphated and triphosphated forms of other nucleotides.

Biosensing Techniques↗

PubMed2005

Electrokinetic control of fluid in plastified laser-printed poly(ethylene terephthalate)-toner microchips.

The application of plastified laser-printed poly(ethylene terephthalate)(PET)-toner microchips to capillary electrophoresis was investigated. Electroosmotic flow was observed in the direction of the cathode for the buffer system studied (phosphate, pH 3-10). Average electroosmotic mobilities of 1.71 x 10(-4) to 4.35 x 10(-4) cm(2) V(-1) s(-1) were observed from pH 3 to 10. This variation suggests that silica fillers in the toner and on the surface of the polymer dominate the zeta potential of the material, which is also confirmed by XPS measurements. Dopamine and catechol were used as model analytes for microchip electrophoresis in combination with electrochemical detection. Results show that these two analytes can be efficiently separated and detected electrochemically with the plastified laser-printed PET-toner microchips.

Journal Article↗

PubMed2005

Determination of the dissociation constants (pKa) of secondary and tertiary amines in organic media by capillary electrophoresis and their role in the electrophoretic mobility order inversion.

Non-aqueous capillary electrophoresis (NACE) may provide a selectivity enhancement in separations since the analyte dissociation constants (pKa) in organic media are different from those in aqueous solutions. In this work, we have studied the inversion in mobility order observed in the separation of tertiary (imipramine (IMI) and amitryptiline (AMI)) and secondary amines (desipramine (DES) and nortryptiline (NOR)) in water, methanol, and acetonitrile. We have determined the pKa values in those solvents and the variation of dissociation constants with the temperature. From these data, and applying the Van't Hoff equation, we have calculated the thermodynamic parameters deltaH and deltaS. The pKa values found in methanol for DES, NOR, IMI, and AMI were 10.80, 10.79, 10.38, and 10.33, respectively. On the other hand, in acetonitrile an opposite relation was found since the values were 20.60, 20.67, 20.74, and 20.81 for DES, NOR, IMI, and AMI. This is the reason why a migration order inversion is observed in NACE for these solvents. The thermodynamic parameters were evaluated and presented a tendency that can be correlated with that observed for pKa values.

Amines↗

PubMed2005

Simplex maximization of the correlation coefficient for DNA sizing analysis by capillary electrophoresis.

The separation of DNA molecules in polymeric solution by capillary electrophoresis involves the optimization of several variables, such as polymer solution concentration, electric field separation, temperature, etc. The optimization of each variable individually usually is a time-consuming process and the results may reach a false optimum point. Chemometric methods are suitable to be applied in such cases in which a number of variables can be optimized simultaneously. The simplex is a chemometric method that can perform such a task easily and efficiently. In this study, a simplex method was carried out to maximize the correlation coefficient (r(2)) of a logarithmic plot of mobility (mu) vs. base pair (bp), which was obtained from the separation of DNA fragments of size between 75 and 4072 bp. The simplex showed three vertexes with r(2) > 0.98 and the vertex 21 showing the highest resolution. For the fragments between 201 and 2036 bp, the r(2) increased to 0.992 with and relative standard deviation (RSD) lower than 0.2% (inter- and intra-day variation). The precision of the method in determining the size of a PCR DNA fragment was carried out using a 1 kbp DNA ladder. With the addition of an internal standard to the sample, the precision could be further improved.

Chemical Fractionation↗

PubMed2005

Nonaqueous capillary electrophoresis in coated capillaries: an interesting alternative for proteomic applications.

This work brings together some contributions for the use of nonaqueous media for proteomic analysis, for both capillary electrophoresis (CE) separation and the preparation of tryptic digests. First, a ternary nonaqueous buffer consisting of 60/30/10 v/v methanol/acetonitrile/acetic acid with 12.5 mmol/L ammonium acetate was optimized for CE separation of the tryptic digest of lysozyme. Lysozyme was chosen as a model system for the protein digestion, which has also been prepared in an organic-rich medium with methanol/50 mmol/L NH(4)HCO(3), pH 8.0 (60/40 v/v). The separation results were compared to in silico (PeptideCutter program) digestion conditions, and high-efficiency peak separation (18 peaks) was obtained in 20 min with an electric field of 350 V/cm. In addition, we have evaluated the stability of a coated capillary with poly-N,N-dimethylacrylamide (60/30 cm total/effective length and 75 microm ID) for over 100 runs of tryptic digest with the nonaqueous background electrolyte solvent system. The migration times for ten selected peptide peaks presented 3-7% relative standard deviation.

Buffers↗

PubMed2005

Growth and siderophore production of Xylella fastidiosa under iron-limited conditions.

In this study, the production of siderophores by Xylella fastidiosa from the citrus bacteria isolate 31b9a5c (FAPESP - ONSA, Brazil) was investigated. The preliminary evidence supporting the existence of siderophore in X. fastidiosa was found during the evaluation of sequencing data generated in our lab using the BLAST-X tool, which indicated putative open reading frames (ORFs) associated with iron-binding proteins. In an iron-limited medium siderophores were detected in the supernatant of X. fastidiosa cultures. The endophytic bacterium Methylobacterium extorquens was also evaluated. Capillary electrophoresis was used to separate putative siderophores produced by X. fastidiosa. The bacterial culture supernatants of X. fastidiosa were identified negative for hydroxamate and catechol and positive for M. extorquens that secreted hydroxamate-type siderophores.

Catechols↗

PubMed2004

Validation of non-aqueous capillary electrophoresis for simultaneous determination of four tricyclic antidepressants in pharmaceutical formulations and plasma samples.

We present the validation of a method using non-aqueous capillary electrophoresis (NACE) for quantitative analysis of four tricyclic antidepressants (TADs) in pharmaceutical formulations and plasma. The method presented high resolution allowing the separation of the TADs in 4.3 min at optimized conditions: 50 mM ammonium acetate, 1 M acetic acid in acetonitrile, capillary with 48 cm in length, 40 cm to the detector, and voltage of 30 kV. Acceptable precision (relative standard deviation R.S.D.14.1% from plasma samples) and linearity were achieved using the internal standard (IS) method. The limits of quantification determined for plasma, after liquid-liquid extraction (LLE), were between 30 and 50 ng ml-1. These values are beyond the plasmatic therapeutic concentration. Our results were found comparable or better than those described in the literature for high performance liquid chromatography (HPLC)-based methods.

Antidepressive Agents, Tricyclic↗

PubMed2004

Electrophoresis microchip fabricated by a direct-printing process with end-channel amperometric detection.

We describe the development of an electrophoresis microchip fabricated by a direct-printing process, based on lamination of printed polyester films with end-channel amperometric detection. The channel structures are defined by polyester (base and cover) and by a toner layer (walls). The polyester-toner devices presented an electroosmotic flow (EOF) magnitude of approximately 10(-5) cm2 V(-1) s(-1), which is generated by a polymeric mixture of the toner and polyester composition. The microelectrodes used for detection were produced combining this laser-printer technology to compact discs. The performance of this device was evaluated by amperometric detection of iodide and ascorbate. The detection limits found were 500 nmol.L(-1) (135 amol) and 1.8 micromol.L(-1) (486 amol) for iodide and ascorbate, respectively.

Ascorbic Acid↗

PubMed2004

Semiquantitative determination of Alicyclobacillus acidoterrestris in orange juice by reverse-transcriptase polymerase chain reaction and capillary electrophoresis--laser induced fluorescence using microchip technology.

The semiquantitative detection of Alicyclobacillus acidoterrrestris in orange juice by reverse-transcriptase polymerase chain reaction (RT-PCR) with a linear dynamic range of 2 x 10(5)-2 colony forming units (CFU)/mL in terms of cell count is described. Separation, detection, and quantification of the RT-PCR products were accomplished using the Agilent 2100 bioanalyzer in conjunction with the DNA 1000 LabChip kit. After 0 and 12 h of enrichment, it was possible to generate a linear standard curve between the amount of cells and amplicon concentration of RT-PCR and PCR products. Using this method, cell diminution was verified in samples of orange juice treated with a natural inhibitor (Sapindus saponaria), determining the persistence of viable cells. Semiquantitative RT-PCR using the Agilent 2100 bioanalyzer is a potentially useful approach for rapid in vitro determination of A. acidoterrestris and monitoring of inhibitor susceptibility for the orange juice-producing industry.

Bacillus↗

PubMed2003

Applications of capillary electrophoresis with laser-induced fluorescence for analysis of dGMP-BPDE adduct.

DNA adducts are thought to be crucial to the initiation of mutational and carcinogenic processes. Polycyclic aromatic hydrocarbons (PAHs) have been identified as one major source of carcinogenic risk since they can bind to DNA thus forming an adduct. Quantification of this adduct is important because it may correlate to the risk for cancer development. In this study, the adduct formed between 2'-deoxyguanosine 5'-monophosphate and benzo[ a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) was analyzed by capillary electrophoresis. Both capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) modes with laser-induced fluorescence detection were used for the separation and analysis of DNA adducts. The exploration of capillary electrophoresis in several modes provided different separation mechanisms in which the stereochemical forms of the adduct could be separated. The best result obtained was using a coated fused-silica capillary in Tris-TAPS buffer, which provided high sensitivity with a detection limit of 2.5x10(-9) mol L(-1). MECC separation of the BPDE adduct, although less sensitive, provided an efficient enantioselective separation option.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed2003

Multiplexed DNA sizing by capillary electrophoresis using entangled polymer solutions and diode array detection.

We developed a method for the analysis of multiplexed double-stranded DNA (dsDNA) samples complexed to various intercalating dyes using entangled polymer solution. A commercial single-column capillary electrophoresis (CE) instrument with diode array detection was used for multiplexed detection of DNA samples by addition of intercalating fluorescent molecules. A Phi X174HinfI and a pGEM DNA ladder (1 mg/mL) were used for the electrophoretic separation of dsDNA fragments ranging in size from 24 to 726 and 36 to 2645 bp, respectively. The results suggested that simultaneous electrophoretic separation of different DNA ladders multiplexed with different dyes could be performed in the same capillary yielding fast DNA sizing separations. CE analysis, which is often overpowered by slab gel in sample throughput, could now overcome this disadvantage by allowing multiplexed sample analysis in a fraction of the time needed for slab gel analysis. The separation efficiency of stained DNA molecules with both dyes were dramatically improved with buffers containing a large cation such as tetrapentylammonium ion (Npe(4) (+)) as the only cation in the buffer.

Bacteriophage phi X 174↗

PubMed2003

Simplex optimization of electrokinetic injection of DNA in capillary electrophoresis using dilute polymer solution.

In DNA analysis by capillary electrophoresis with polymer solutions there are many variables that can be optimized. However, electric field strength, polymer solution concentration and temperature of analysis are the most relevant ones. These are the variables most responsible for the fragment resolution and analysis time. Optimization of such parameters can be obtained simultaneously using chemometric techniques, reaching the optimum working conditions with few experiments. In this work, we have studied the influence of the sample composition and electrokinetic injection conditions in the reproducibility and the quality of the DNA separation results. A simplex optimization has been carried out and the optimum condition was reached with nine experiments.

DNA↗

PubMed2003

Identification of a metallothionein in Synechococcus by capillary electrophoresis hyphenated with inductively coupled plasma mass spectrometry.

A home-made system hyphenating capillary electrophoresis with an inductively coupled plasma mass spectrometer (CE-ICP-MS) for cadmium speciation of protein-binding and free cadmium ions in solution is presented. The CE-ICP-MS interface consisted of an acrylic block with an internal volume ca. 20 microL in which a platinum electrode, a capillary column, and a connection to an ICP nebulizer were inserted. A make-up electrolyte solution containing 50 mmol L(-1) Tris-HCl buffer solution (pH 9.0) was continuously flowed through the interface to the ICP nebulizer. The separation of free Cd ions, Cd-cysteine, and Cd bounded to metallothionein (MT) isoforms from rabbit liver was carried out by capillary electrophoresis, and the analytes were detected by ICP-MS. The feasibility to isolate metallothionein compounds extracted from the cyanobacterium Synechococcus PCC7942 was demonstrated. The Cd binding proteins were induced in Synechococcus PCC7942 and further analyzed by CE ICP-MS.

Animals↗

PubMed2002

Laser-induced fluorescence and UV detection of derivatized aldehydes in air samples using capillary electrophoresis.

In this work, two capillary zone electrophoresis methodologies using UV absorption detection (214 nm) and laser-induced fluorescence detection (He/Cd laser, 325 nm excitation, 520 nm emission) of selected aldehydes (formaldehyde, acetaldehyde, propionaldehyde and acrolein) derivatized with dansylhydrazine (DNSH, 5-dimethylaminonaphthalene-1-sulfohydrazide) were proposed and validated. The aldehydes react with DNSH to form negatively charged molecules in methanolic medium. In both methodologies, nine DNSH-derivatives, including isomers of acetaldehyde, propionaldehyde and acrolein and two impurities were baseline separated in 20 mmol l(-1) phosphate buffer at pH 7.02, in less than 9 min. The limits of detection for the UV and LIF methodologies ranged from 1.1-9.5 microg l(-1) and 0.29-5.3 microg l(-1), respectively. The applicability of both methodologies to contemplate real samples was confirmed in the analysis of aldehyde-DNSH derivatives in indoor and outdoor air samples.

Air↗

PubMed2002

A review of DNA sequencing techniques.

The four best known DNA sequencing techniques are reviewed. Important practical issues covered are read-length, speed, accuracy, throughput, cost, as well as the automation of sample handling and preparation. The methods reviewed are: (i) the Sanger method and its most important variants (enzymic methods); (ii) the Maxam & Gilbert method and other chemical methods; (iii) the Pyrosequencing method--DNA sequencing in real time by the detection of released pyrophosphate (PPi); and (iv) single molecule sequencing with exonuclease (exonuclease digestion of a single molecule composed of a single strand of fluorescently labelled deoxynucleotides). Each method is briefly described, the current literature is covered, advantages, disadvantages, and the most suitable applications of each method are discussed.

Base Sequence↗