Structural matching of 2D electrophoresis gels using deformed graphs

2D electrophoresis is a well-known method for protein separation which is extremely useful in the field of proteomics. Each spot in the image represents a protein accumulation and the goal is to perform a differential analysis between pairs of images to study changes in protein content. It is thus necessary to register two images by finding spot correspondences. Although it may seem a simple task, generally, the manual processing of this kind of images is very cumbersome, especially when strong variations between corresponding sets of spots are expected (e.g. strong non-linear deformations and outliers). In order to solve this problem, this paper proposes a new quadratic assignment formulation together with a correspondence estimation algorithm based on graph matching which takes into account the structural information between the detected spots. Each image is represented by a graph and the task is to find a maximum common subgraph. Successful experimental results using real data are presented, including an extensive comparative performance evaluation with ground-truth data. (C) 2010 Elsevier B.V. All rights reserved.

Fully electrochemical hyphenated flow system for preconcentration, cleanup, stripping, capillary electrophoresis with stacking and contactless conductivity detection of trace heavy metals

Successful coupling of electrochemical preconcentration (EPC) to capillary electrophoresis (CE) with contactless conductivity detection (C(4)D) is reported for the first time. The EPC-CE interface comprises a dual glassy carbon electrode (GCE) block, a spacer and an upper block with flow inlet and outlet, pseudo-reference electrode and a fitting for the CE silica column, consisting of an orifice perpendicular to the surface of a glassy carbon electrode with a bushing inside to ensure a tight press fit. The end of the capillary in contact with the GCE is slant polished, thus defining a reproducible distance from the electrode surface to the column bore. First results with EPC-CE-C(4)D are very promising, as revealed by enrichment factors of two orders of magnitude for Tl, Cu, Pb and Cd ion peak area signals. Detection limits for 10 min deposition time fall around 20 nmol L(-1) with linear calibration curves over a wide range. Besides preconcentration, easy matrix exchange between accumulation and stripping/injection favors procedures like sample cleanup and optimization of pH, ionic strength and complexing power. This was demonstrated for highly saline samples by using a low conductivity buffer for stripping/injection to improve separation and promote field-enhanced sample stacking during electromigration along the capillary. (C) 2010 Elsevier B.V. All rights reserved.

Total Trans Fatty Acid Analysis in Spreadable Cheese by Capillary Zone Electrophoresis

An alternative method for determination of total trans fatty acids expressed as elaidic acid by capillary zone electrophoresis (CZE) under indirect UV detection at 224 nm within an analysis time of 7.5 min was developed. The optimized running electrolyte includes 15.0 mmol L(-1) KH(2)PO(4)/Na(2)HPO(4) buffer (pH similar to 7.0), 4.0 mmol L(-1) SDBS, 8.0 mmol L(-1) Brij35, 45%v/v ACN, 8% methanol, and 1.5% v/v n-octanol. Baseline separation of the critical pair C18-9cis/C18:1-9t: with a resolution higher than 1.5 was achieved using C15:0 as the internal standard. The optimum capillary electrophoresis (CE) conditions for the background electrolyte were established with the aid of Raman spectroscopy and experiments of a 3(2) factorial design. After response factor (R(F)) calculations, the CE method was applied to total trans fatty acid (TTFA) analysis in a hydrogenated vegetable fat (HVF) sample, and compared with the American Oil Chemists` Society (AOCS) official method by gas chromatography (GC). The methods were compared with an independent sample t test, and no significant difference was found between CE and GC methods within the 95% confidence interval for six genuine replicates of TTFA analysis (p-value > 0.05). The CE method was applied to TTFA analysis in a spreadable cheese sample. Satisfactory results were obtained, indicating that the optimized methodology can be used for trans fatty acid determination for these samples.

Optimization of a method for determination of phenolic acids in exotic fruits by capillary electrophoresis

In this work, the separation of nine phenolic acids (benzoic, caffeic, chlorogenic, p-coumaric, ferulic, gallic, protocatechuic, syringic, and vanillic acid) was approached by a 32 factorial design in electrolytes consisting of sodium tetraborate buffer(STB) in the concentration range of 10-50 mmol L(-1) and methanol in the volume percentage of 5-20%. Derringer`s desirability functions combined globally were tested as response functions. An optimal electrolyte composed by 50 mmol L(-1) tetraborate buffer at pH 9.2, and 7.5% (v/v) methanol allowed baseline resolution of all phenolic acids under investigation in less than 15 min. In order to promote sample clean up, to preconcentrate the phenolic fraction and to release esterified phenolic acids from the fruit matrix, elaborate liquid-liquid extraction procedures followed by alkaline hydrolysis were performed. The proposed methodology was fully validated (linearity from 10.0 to 100 mu g mL(-1), R(2) > 0.999: LOD and LOQ from 1.32 to 3.80 mu g mL(-1) and from 4.01 to 11.5 mu g mL(-1), respectively; intra-day precision better than 2.8% CV for migration time and 5.4% CV for peak area; inter-day precision better than 4.8% CV for migration time and 4.8-11% CV for peak area: recoveries from 81% to 115%) and applied successfully to the evaluation of phenolic contents of abiu-roxo (Chrysophyllum caimito), wild mulberry growing in Brazil (Morus nigra L.) and tree tomato (Cyphomandra betacea). Values in the range of 1.50-47.3 mu g g(-1) were found, with smaller amounts occurring as free phenolic acids. (C) 2009 Elsevier B.V. All rights reserved.

Multivariant optimization, validation, and application of capillary electrophoresis for simultaneous determination of polyphenols and phenolic acids in Brazilian wines

A method for the simultaneous determination of the stilbene resveratrol, four phenolic acids (syringic, coumaric, caffeic, and gallic acids), and five flavonoids (catechin, rutin, kaempferol, myricetin, and quercetin) in wine by CE was developed and validated. The CE electrolyte composition and instrumental conditions were optimized using 2(7-3) factorial design and response surface analysis, showing sodium tetraborate, MeOH, and their interaction as the most influential variables. The optimal electrophoretic conditions, minimizing the chromatographic resolution statistic values, consisted of 17 mmol/L sodium tetraborate with 20% methanol as electrolyte, constant voltage of 25 kV, hydrodynamic injection at 50 mbar for 3 s, and temperature of 25 degrees C. The R(2) values for linearity varied from 0.994 to 0.999; LOD and LOQ were 0.1 to 0.3 mg/L and 0.4 to 0.8 mg/L, respectively. The RSDs for migration time and peak area obtained from ten consecutive injections were less than 2% and recoveries varied from 97 to 102%. The method was applied to 23 samples of inexpensive Brazilian wines, showing wide compositional variation.

Detection of adulterations in processed coffee with cereals and coffee husks using capillary zone electrophoresis

The proposed method for the identification of adulteration was based on the controlled acid hydrolysis of xylan and starch present in some vegetable adulterants, followed by the analysis of the resulting xylose and glucose, which are the monosaccharides that compose, respectively, the two polysaccharides. The acid hydrolysis with HCl increases the ionic strength of the sample, which impairs the electrophoretic separation. Thus, a neutralization step based on anion exchange resin was necessary. The best separations were obtained in NaOH 80 mmol/L, CTAB 0.5 mmol/L, and methanol 30% v/v. Because of the high value of pH, monosaccharides are separated as anionic species in such running electrolyte. The LOQ for both monosaccharides was 0.2 g for 100 g of dry matter, which conforms to the tolerable limits.

Detection of Paraquat in Oral Fluid, Plasma, and Urine by Capillary Electrophoresis for Diagnosis of Acute Poisoning

FAPESP

Determination of sorbate and benzoate in beverage samples by capillary electrophoresis - Optimization of the method with inspection of ionic mobilities

The aim of this study was to develop a fast capillary electrophoresis method for the determination of benzoate and sorbate ions in commercial beverages. In the method development the pH and constituents of the background electrolyte were selected using the effective mobility versus pH curves. As the high resolution obtained experimentally for sorbate and benzoate in the studies presented in the literature is not in agreement with that expected from the ionic mobility values published, a procedure to determine these values was carried out. The salicylate ion was used as the internal standard. The background electrolyte was composed of 25 mmol L(-1) tris(hydroxymethyl)aminomethane and 12.5 mmol L(-1) 2-hydroxyisobutyric acid, atpH 8.1.Separation was conducted in a fused-silica capillary(32 cm total length and 8.5 cm effective length, 50 mu m I.D.), with short-end injection configuration and direct UV detection at 200 nm for benzoate and salicylate and 254 nm for sorbate ions. The run time was only 28 s. A few figures of merit of the proposed method include: good linearity (R(2) > 0.999), limit of detection of 0.9 and 0.3 mg L(-1) for benzoate and sorbate, respectively, inter-day precision better than 2.7% (n =9) and recovery in the range 97.9-105%. Beverage samples were prepared by simple dilution with deionized water (1:11, v/v). Concentrations in the range of 197-401 mg L(-1) for benzoate and 28-144 mg L(-1) for sorbate were found in soft drinks and tea. (c) 2008 Elsevier B.V. All rights reserved.

Direct detection of underivatized chitooligosaccharides produced through chitinase action using capillary zone electrophoresis

Capillary electrophoresis with capacitively coupled contactless conductivity detection was successfully used to quantify N-acetylglucosamine and five N-acetyl-chitooligosaccharides (C2-C6) produced after reaction with a purified chitinase (TmChi) from Tenebrio molitor (Coleoptera). No derivatization process was necessary. The separation was developed using 10 mM NaOH with 10% (v/v) acetonitrile as background electrolyte and homemade equipment with a system that avoids the harmful effect of electrolysis. The limit of detection for all oligosaccharides was ca. 3 mu M, and the results indicated that the larger the oligosaccharide, the higher the sensitivity. Analysis of the chitooligosaccharides produced revealed that TmChi has an endolytic cleavage pattern with C5 as the best substrate (higher catalytic efficiency k(cat)/K-M) releasing C2 and C3. (c) 2007 Elsevier Inc. All rights reserved.

Investigating the formation and the properties of monoalkyl carbonates in aqueous medium using capillary electrophoresis with capacitively coupled contactless conductivity detection

Although alkyl carbonic acids (ACAs) and their salts are referred to as instable species in aqueous medium, we demonstrate that a monoalkyl carbonate (MAC) can in fact be easily formed from bicarbonate and an alcohol even in the presence of a high amount of water. A CE system with two capacitively coupled contactless conductivity detectors (C(4)Ds) was used to obtain different parameters about these species and their reactions. Based on the mobilities obtained for a series of alcohols ranging from 1 to 5 carbons, the coefficients of diffusion and the hydrodynamic radii were calculated. When compared with the equivalent carboxylates, MACs have radii systematically smaller. Although the precise pK(a) values of the ACAs could not be obtained, because of the fast decomposition in acid medium, it was possible, for the first time, to show that they are below 4.0. This result suggests that the acidity of an ACA is quite similar to the first hydrogen of H(2)CO(3). Using a new approach to indirectly calibrate the C(4)D, the kinetic constants and the equilibrium constants of formation were also obtained. The results suggest that the increase in the chain length makes the MACs less stable and more inert.

Determination of fluoroacetate and fluoride in blood serum by capillary zone electrophoresis using capacitively coupled contactless conductivity detection

Fluoroacetate is a highly toxic species naturally found in plants and in commercial products (compound 1080) for population control of several undesirable animal species. However, it is non-selective and toxic to many other animals including humans, and thus its detection is very important for forensic purposes. This paper presents a sensitive and fast method for the determination of fluoroacetate in blood serum using capillary electrophoresis with capacitively coupled contactless conductivity detection. Serum blood samples were treated with ethanol to remove proteins. The samples were analyzed in BGE containing 15 mmol/L histidine and 30 mmol/L gluconic acid (pH 3.85). The calibration curve was linear up to 75 mu mol/L (R(2) = 0.9995 for N = 12). The detection limit in the blood serum was 0.15 mg/kg, which is smaller than the lethal dose for humans and other animals. Fluoride, a metabolite of the fluoroacetate defluorination, could also be detected for levels greater than 20 mu mol/L, when polybrene was used for reversion of the EOF. CTAB and didecyldimethylammonium bromide are not useful for this task because of the severe reduction of the fluoride level. However, no interference was observed for fluoroacetate.

Determination of ciclopirox olamine in pharmaceutical products by capillary electrophoresis with capacitively coupled contactless conductivity detection

This paper describes the determination of ciclopirox olamine in pharmaceutical formulations using capillary electrophoresis with capacitively coupled contactless conductivity detection. In an alkaline medium, ciclopirox olamine is converted into an anionic species and its detection is possible in capillary electrophoresis with capacitively coupled contactless conductivity detection without an electroosmotic flow modifier, because it is a low-mobility species. A linear working range from 2.64 to 264 mu g/mL in sodium hydroxide electrolyte as well as low detection limit (0.39 mu g/mL) and a good repeatability (RSD = 3.4% for 264 mu g/mL ciclopirox solution (n = 10)) were achieved. It was also possible to determine olamine in its cationic form when acetic acid was used as the electrolyte solution. The results obtained include a linear range from 26.4 to 184.8 mu g/mL and a detection limit of 2.6 mu g/mL olamine. The proposed methods were applied to the analysis of commercial pharmaceutical products and the results were compared with the values indicated by the manufacturer as well as those obtained using a titrimetric method recommended by American Pharmacopoeia.

Automated two-dimensional separation flow system with electrochemical preconcentration, stripping, capillary electrophoresis and contactless conductivity detection for trace metal ion analysis

This paper describes the automation of a fully electrochemical system for preconcentration, cleanup, separation and detection, comprising the hyphenation of a thin layer electrochemical flow cell with CE coupled with contactless conductivity detection (CE-C(4)D). Traces of heavy metal ions were extracted from the pulsed-flowing sample and accumulated on a glassy carbon working electrode by electroreduction for some minutes. Anodic stripping of the accumulated metals was synchronized with hydrodynamic injection into the capillary. The effect of the angle of the slant polished tip of the CE capillary and its orientation against the working electrode in the electrochemical preconcentration (EPC) flow cell and of the accumulation time were studied, aiming at maximum CE-C(4)D signal enhancement. After 6 min of EPC, enhancement factors close to 50 times were obtained for thallium, lead, cadmium and copper ions, and about 16 for zinc ions. Limits of detection below 25 nmol/L were estimated for all target analytes but zinc. A second separation dimension was added to the CE separation capabilities by staircase scanning of the potentiostatic deposition and/or stripping potentials of metal ions, as implemented with the EPC-CE-C(4)D flow system. A matrix exchange between the deposition and stripping steps, highly valuable for sample cleanup, can be straightforwardly programmed with the multi-pumping flow management system. The automated simultaneous determination of the traces of five accumulable heavy metals together with four non-accumulated alkaline and alkaline earth metals in a single run was demonstrated, to highlight the potentiality of the system.

Development of a fast capillary electrophoresis method to determine inorganic cations in biodiesel samples

The aim of this study was to develop a fast capillary electrophoresis method for the determination of inorganic cations (Na(+), K(+), Ca(2+), Mg(2+)) in biodiesel samples, using barium (Ba(2+)) as the internal standard. The running electrolyte was optimized through effective mobility curves in order to select the co-ion and Peakmaster software was used to determine electromigration dispersion and buffer capacity. The optimum background electrolyte was composed of 10 mmol L(-1) imidazole and 40 mmol L(-1) of acetic acid. Separation was conducted in a fused-silica capillary (32 cm total length and 23.5 cm effective length, 50 mu m I.D.), with indirect UV detection at 214 nm. The migration time was only 36 s. In order to obtain the optimized conditions for extraction, a fractional factorial experimental design was used. The variables investigated were biodiesel mass, pH, extractant volume, agitation and sonication time. The optimum conditions were: biodiesel mass of 200 mg, extractant volume of 200 mu L. and agitation of 20 min. The method is characterized by good linearity in the concentration range of 0.5-20 mg kg(-1) (r > 0.999), limit of detection was equal to 0.3 mg kg(-1), inter-day precision was equal to 1.88% and recovery in the range of 88.0-120%. The developed method was successfully applied to the determination of cations in biodiesel samples. (c) 2010 Elsevier B.V. All rights reserved.

Development of a fast capillary electrophoresis method for the determination of propranolol-Total analysis time reduction strategies

The aim of this study was to develop a fast capillary electrophoresis method for the determination of propranolol in pharmaceutical preparations. In the method development the pH and constituents of the background electrolyte were selected using the effective mobility versus pH curves. Benzylamine was used as the internal standard. The background electrolyte was composed of 60 mmol L(-1) tris(hydroxymethyl)aminomethane and 30 mmol L(-1) 2-hydroxyisobutyric acid,at pH 8.1. Separation was conducted in a fused-silica capillary (32 cm total length and 8.5 cm effective length, 50 mu m I.D.) with a short-end injection configuration and direct UV detection at 214 nm. The run time was only 14 s. Three different strategies were studied in order to develop a fast CE method with low total analysis time for propranolol analysis: low flush time (Lflush) 35 runs/h, without flush (Wflush) 52 runs/h, and Invert (switched polarity) 45 runs/h. Since the three strategies developed are statistically equivalent, Mush was selected due to the higher analytical frequency in comparison with the other methods. A few figures of merit of the proposed method include: good linearity (R(2) > 0.9999); limit of detection of 0.5 mg L(-1): inter-day precision better than 1.03% (n = 9) and recovery in the range of 95.1-104.5%. (C) 2009 Elsevier B.V. All rights reserved.