Simple method for determination of cocaine and main metabolites in urine by CE coupled to MS

in this work, a simple method for the simultaneous determination of cocaine (COC) and five COC metabolites (benzoylecgonine, cocaethylene (CET), anhydroecgonine, anhydroecgonine methyl ester and ecgonine methyl ester) in human urine using CE coupled to MS via electrospray ionization (CE-ESI-MS) was developed and validated. Formic acid at 1 mol/L concentration was used as electrolyte whereas formic acid at 0.05 mol/L concentration in 1:1 methanol:water composed the coaxial sheath liquid at the ESI nozzle. The developed method presented good linearity in the dynamic range from 250 ng/mL to 5000 ng/mL (coefficient of determination greater than 0.98 for all compounds). LODs (signal-to-noise ratio of 3) were 100 ng/mL for COC and CET and 250 ng/mL for the other studied metabolites whereas LOQ`s (signal-to-noise ratio of 10) were 250 ng/mL for COC and CET and 500 ng/mL for all other compounds. Intra-day precision and recovery tests estimated at three different concentration levels (500, 1500 and 5000 ng/mL) provided RSD lower than 10% (except anhydroecgonine, 18% RSD) and recoveries from 83-109% for all analytes. The method was successfully applied to real cases. For the positive urine samples, the presence of COC and its` metabolites was further confirmed by MS/MS experiments.

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.

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.

Fabrication and integration of planar electrodes for contactless conductivity detection on polyester-toner electrophoresis microchips

In this report, we describe the microfabrication and integration of planar electrodes for contactless conductivity detection on polyester-toner (PT) electrophoresis microchips using toner masks. Planar electrodes were fabricated by three simple steps: (i) drawing and laser-printing the electrode geometry on polyester films, (ii) sputtering deposition onto substrates, and (iii) removal of toner layer by a lift-off process. The polyester film with anchored electrodes was integrated to PT electrophoresis microchannels by lamination at 120 degrees C in less than 1 min. The electrodes were designed in an antiparallel configuration with 750 mu m width and 750 gm gap between them. The best results were recorded with a frequency of 400 kHz and 10 V-PP using a sinusoidal wave. The analytical performance of the proposed microchip was evaluated by electrophoretic separation of potassium, sodium and lithium in 150 mu m wide x 6 mu m deep microchannels. Under an electric field of 250 V/cm the analytes were successfully separated in less than 90 s with efficiencies ranging from 7000 to 13 000 plates. The detection limits (S/N = 3) found for K+, Na+, and Li+ were 3.1, 4.3, and 7.2 mu mol/L, respectively. Besides the low-cost and instrumental simplicity, the integrated PT chip eliminates the problem of manual alignment and gluing of the electrodes, permitting more robustness and better reproducibility, therefore, more suitable for mass production of electrophoresis microchips.

Multimilligram enantioresolution of sulfoxide proton pump inhibitors by liquid chromatography on polysaccharide-based chiral stationary phase

The enantiomers of sulfoxide proton pump inhibitors - omeprazole, lansoprazole, rabeprazole and Ro 18-5364 - were enantiomerically separated by liquid chromatography at multimilligram scale on a poly saccharide-based chiral stationary phase using normal and polar organic conditions as mobile phase. The values of the recovery and production rate were significant for each enantiomer; better results were achieved using a solid-phase injection system. However, this system was applied just for the enantionteric separation of omeprazole to demonstrate the applicability of this injection mode at milligram scale. The chiroptical characterization of the compounds was performed using a polarimeter and a circular dichroism detector. The higher enantiomeric purity obtained for the isolated enantiomers suggests that the methods here described should be considered as a simple and rapid way to obtain enantiomeric pure standards for analytical purpose. (C) 2007 Elsevier B.V. All rights reserved.

PDIA3, HSPA5 and viementin, proteins identified by 2-DE in the valvular tissue, are the target antigens of peripheral and heart infiltrating T cells from chronic rheumatic heart disease patients

Rheumatic fever (RF) is a post-infectious autoimmune disease due to sequel of group A streptococcus (GAS) pharyngitis. Rheumatic heart disease (RHD), the major manifestation of RF, is characterized by inflammation of heart valves and myocardium. Molecular mimicry between GAS antigens and host proteins has been shown at B and T cell level. However the identification of the autoantigens recognized by B and T cells within the inflammatory microenvironment of heart tissue in patients with RHD is still incompletely elucidated. In the present study, we used two-dimensional gel electrophoresis (2-DE) and mass spectrometry to identify valvular tissue proteins target of T cells from chronic RHD patients. We could identify three proteins recognized by heart infiltrating and peripheral T cells as protein disulfide isomerase ER-60 precursor (PDIA3), 78 kD glucose-regulated protein precursor (HSPA5) and vimentin, with coverage of 45%, 43 and 34%, respectively. These proteins were recognized in a proliferation assay by peripheral and heart infiltrating T cells from RHD patients suggesting that they may be involved in the autoimmune reactions that leads to valve damage. We also observed that several other proteins isolated by 2-DE but not identified by mass spectrometry were also recognized by T cells. The identified cardiac proteins are likely relevant antigens involved in T cell-mediated autoimmune responses in RF/RHD that may contribute to the development of RHD

Proteome analysis of schizophrenia patients Wernicke's area reveals an energy metabolism dysregulation

Background: Schizophrenia is likely to be a consequence of DNA alterations that, together with environmental factors, will lead to protein expression differences and the ultimate establishment of the illness. The superior temporal gyrus is implicated in schizophrenia and executes functions such as the processing of speech, language skills and sound processing. Methods: We performed an individual comparative proteome analysis using two-dimensional gel electrophoresis of 9 schizophrenia and 6 healthy control patients' left posterior superior temporal gyrus (Wernicke's area - BA22p) identifying by mass spectrometry several protein expression alterations that could be related to the disease. Results: Our analysis revealed 11 downregulated and 14 upregulated proteins, most of them related to energy metabolism. Whereas many of the identified proteins have been previously implicated in schizophrenia, such as fructose-bisphosphate aldolase C, creatine kinase and neuron-specific enolase, new putative disease markers were also identified such as dihydrolipoyl dehydrogenase, tropomyosin 3, breast cancer metastasis-suppressor 1, heterogeneous nuclear ribonucleoproteins C1/C2 and phosphate carrier protein, mitochondrial precursor. Besides, the differential expression of peroxiredoxin 6 (PRDX6) and glial fibrillary acidic protein (GFAP) were confirmed by western blot in schizophrenia prefrontal cortex. Conclusion: Our data supports a dysregulation of energy metabolism in schizophrenia as well as suggests new markers that may contribute to a better understanding of this complex disease.

Method for cadmium and lead longitudinal profiles determination in hair by solid sampling graphite furnace atomic absorption spectrometry

This paper describes methods for the direct determination of Cd and Pb in hair segments (c.a. 5 mm similar to 80 mu g) by solid sampling graphite furnace atomic absorption spectrometry, becoming possible longitudinal profiles in a single strand of hair. To distinguish endogenous and exogenous content. strands of hair were washed by using two different procedures: IAEA protocol (acetone + water + acetone) and the combination of IAEA protocol with HCl washing (acetone + water + acetone + 0.1 mol l(-1) HCl). The concentration of Cd and Pb increased from the root Until the tip of hair washed according to IAEA protocol. However, when the strand of hair was washed using the combination of IAEA protocol and 0.1 mol l(-1) HCl, Cd concentrations decreased in all segments, and Pb concentrations decreased drastically near to the root (5 to 12 mm) and was systematically higher ill the end. The proposed method showed to be useful to assess the temporal variation to Cd and Pb exposure and call be Used for toxicological and environmental investigations. The limits of detection were 2.8 ng g(-1) for Cd and 40 ng g(-1) for Pb. The characteristic masses based oil integrated absorbance were 2.4 pg for Cd and 22 pg for Pb.

Dual contactless conductivity and amperometric detection on hybrid PDMS/glass electrophoresis microchips

A new approach for the integration of dual contactless conductivity and amperometric detection with an electrophoresis microchip system is presented. The PDMS layer with the embedded channels was reversibly sealed to a thin glass substrate (400 mu m), on top of which a palladium electrode had been previously fabricated enabling end-channel amperometric detection. The thin glass substrate served also as a physical wall between the separation channel and the sensing copper electrodes for contactless conductivity detection. The latter were not integrated in the microfluidic device, but fabricated on an independent plastic substrate allowing a simpler and more cost-effective fabrication of the chip. PDMS/glass chips with merely contactless conductivity detection were first characterized in terms of sensitivity, efficiency and reproducibility. The separation efficiency of this system was found to be similar or slightly superior to other systems reported in the literature. The simultaneous determination of ionic and electroactive species was illustrated by the separation of peroxynitrite degradation products, i.e. NO(3)(-) (non-electroactive) and NO(2)(-) (electroactive), using hybrid PDMS/glass chips with dual contactless conductivity and amperometric detection. While both ions were detected by contactless conductivity detection with good efficiency, NO(2)(-) was also simultaneously detected amperometrically with a significant enhancement in sensitivity compared to contactless conductivity detection.

Rapid prototyping of polymeric electrophoresis microchips with integrated copper electrodes for contactless conductivity detection

A simple and easy approach to produce polymeric microchips with integrated copper electrodes for capacitively coupled contactless conductivity detection (CD) is described. Copper electrodes were fabricated using a printed circuit board (PCB) as an inexpensive thin-layer of metal. The electrode layout was first drawn and laser printed on a wax paper sheet. The toner layer deposited on the paper sheet was thermally transferred to the PCB surface working as a mask for wet chemical etching of the copper layer. After the etching step, the toner was removed with an acetonitrile-dampened cotton. A poly(ethylene terephthalate) (PET) film coated with a thin thermo-sensitive adhesive layer was used to laminate the PCB plate providing an insulator layer of the electrodes to perform CID measurements. Electrophoresis microchannels were fabricated in poly(dimethylsiloxane) (PDMS) by soft lithography and reversibly sealed against the PET film. These hybrid PDMS/PET chips exhibited a stable electroosmotic mobility of 4.25 +/- 0.04 x 10(-4) V cm(-2) s(-1), at pH 6.1, over fifty runs. Efficiencies ranging from 1127 to 1690 theoretical plates were obtained for inorganic cations.

Preliminary studies of laser-induced breakdown spectrometry for the determination of Ba, Cd, Cr and Pb in toys

The performance of laser-induced breakdown spectrometry (LIBS) for the determination of Ba, Cd, Cr and Pb in toys has been evaluated by using a Nd:YAG laser operating at 1064 nm and an Echelle spectrometer with intensified charge-coupled device detector. Samples were purchased in different cities of Sao Paulo State market and analyzed directly without sample preparation. Laser-induced breakdown spectrometry experimental conditions (number of pulses, delay time. integration time gate and pulse energy) were optimized by using a Doehlert design. Laser-induced breakdown spectrometry signals correlated reasonably well with inductively coupled plasma optical emission spectrometry (ICP OES) concentrations after microwave-assisted acid digestion of selected samples. Thermal analysis was used for polymer identification and scanning electron microscopy to Visualize differences in crater geometry of different polymers employed for toy fabrication. Results indicate that laser-induced breakdown spectrometry can be proposed as a rapid screening method for investigation of potentially toxic elements in toys. The unique application of laser-induced breakdown spectrometry for identification of contaminants in successive layers of ink and polymer is also demonstrated. (C) 2009 Elsevier B.V. All rights reserved.

Total and inorganic mercury determination in fish tissue by flow injection cold vapour atomic fluorescence spectrometry

A simple and reliable method for Hg determination in fish samples has been developed. Lyophilised fish tissue samples were extracted in a 25% (w/v) tetramethylammonium hydroxide (TMAH) solution; the extracts were then analysed by FI-CVAFS. This method can be used to determine total and inorganic Hg, using the same FI manifold. For total Hg determination, a 0.1% (w/v) KMnO(4) solution was added to the FI manifold at the sample zone, followed by the addition of a 0.5% (w/v) SnCl(2) solution, whereas inorganic Hg was determined by adding a 0.1% (w/v) L-cysteine solution followed by a 1.0% (w/v) SnCl(2) solution to the FI system. The organic fraction was determined as the difference between total and inorganic Hg. Sample preparation, reagent consumption and parameters that can influence the FI-CVAFS performance were also evaluated. The limit of detection for this method is 3.7 ng g(-1) for total Hg and 4.3 ng g(-1) for inorganic Hg. The relative standard deviation for a 1.0 mu gL(-1) CH(3)Hg standard solution (n = 20) was 1.1%, and 1.3% for a 1.0 mu gL(-1) Hg(2+) standard solution (n = 20). Accuracy was assessed by the analysis of Certified Reference Material (dogfish: DORM-2, NRCC). Recoveries of 99.1% for total Hg and 93.9% inorganic Hg were obtained. Mercury losses were not observed when sample solutions were re-analysed after a seven day period of storage at 4 degrees C.

Taenia crassiceps cysticerci: Characterization of the 14-kDa glycoprotein with homologies to antigens from Taenia solium cysticerci

Glycoproteins from the total vesicular fluid of Taenia crassiceps (VF-Tc) were prepared using three different purification methods, consisting of ConA-lectin affinity chromatography (ConA-Tc), preparative electrophoresis (SDS-PAGE) (14gp-Tc), and monoclonal antibody immunoaffinity chromatography (18/14-Tc). The complex composition represented by the VF-Tc and ConA-Tc antigens revealed peptides ranging from 101 - to 14-kDa and from 92- to 12-kDa, respectively. Immunoblotting using lectins confirmed glucose/mannose (glc/man) residues in the 18- and 14-kDa peptides, which are considered specific and immunodominant for the diagnosis of cysticercosis, and indicated that these fractions are glycoproteins. Serum antibodies from a patient with neurocysticercosis that reacted to the 14gp band from T. crassiceps (Tc) were eluted from immunoblotting membranes and showed reactivity to 14gp from Taenia solium. In order to determine the similar peptide sequence, the N-terminal amino acid was determined and analyzed with sequences available in public databases. This sequence revealed partial homology between T. crassiceps and T solium peptides. In addition, mass spectrometry along with theoretical M(r) and pI of the 14gp-Tc point suggested a close relationship to some peptides of a 150-kDa protein complex of the T solium previously described. The identification of these common immunogenic sites will contribute to future efforts to develop recombinant antigens and synthetic peptides for immunological assays. (C) 2009 Elsevier Inc. All rights reserved.

Simultaneous Determination of Choline Citrate and Acetylmethionine in Injectable Solutions by High Performance Liquid Chromatography

Choline citrate (CC) and acetylmethionine (AM) are lipotropic drugs used in several pharmaceutical formulations. The objective of this research was to develop and validate a high performance liquid chromatographic (HPLC) method for simultaneous determination of CC and AM in injectable solutions, aiming its application in routine analysis for quality control of these pharmaceutical formulations. The method was validated using a Shim-Pack (R) C18 (250 x 4.6 mm, 5 mu m) column. The mobile phase was constituted of 25 mM potassium phosphate buffer solution, pH 5.7, adjusted with 10 % orthophosphoric acid, acetonitrile and methanol (88:10:2, v/v/v). The flow rate was 1.1 mL.min(-1) and the UV detection was made at 210 nm. The analyses were made at room temperature (25 +/- 1 degrees C). The method is precise, selective, accurate and robust, and was successfully applied for simultaneous quantitative determination of CC and AM in injectables.

Visualizing inhibition of fatty acid synthase through mass spectrometric analysis of mitochondria from melanoma cells

Fatty acid synthase (FASN) is the metabolic enzyme responsible for the endogenous synthesis of the saturated long-chain fatty acid palmitate. In contrast to most normal cells, FASN is overexpressed in a variety of human cancers including cutaneous melanoma, in which its levels of expression are associated with a poor prognosis and depth of invasion. Recently, we have demonstrated the mitochondrial involvement in FASN inhibition-induced apoptosis in melanoma cells. Herein we compare, via electrospray ionization mass spectrometry (ESI-MS), free fatty acids (FFA) composition of mitochondria isolated from control (EtOH-treated cells) and Orlistat-treated B16-F10 mouse melanoma cells. Principal component analysis (PCA) was applied to the ESI-MS data and found to separate the two groups of samples. Mitochondria from control cells showed predominance of six ions, that is, those of m/z 157 (Pelargonic, 9:0), 255 (Palmitic, 16:0), 281 (Oleic, 18:1), 311 (Arachidic, 20:0), 327 (Docosahexaenoic, 22:6) and 339 (Behenic, 22:0). In contrast, FASN inhibition with Orlistat changes significantly mitochondrial FFA composition by reducing synthesis of palmitic acid, and its elongation and unsaturation products, such as arachidic and behenic acids, and oleic acid, respectively. ESI-MS of mitochondria isolated from Orlistat-treated cells presented therefore three major ions of m/z 157 (Pelargonic, 9:0), 193 (unknown) and 199 (Lauric, 12:0). These findings demonstrate therefore that FASN inhibition by Orlistat induces significant changes in the FFA composition of mitochondria. Copyright (C) 2011 John Wiley & Sons, Ltd.