Ultrasensitive Simultaneous Quantification of 1,N(2)-Etheno-2 `-deoxyguanosine and 1,N(2-)Propano-2 `-deoxyguanosine in DNA by an Online Liquid Chromatography-Electrospray Tandem Mass Spectrometry Assay

Exocyclic DNA adducts produced by exogenous and endogenous compounds are emerging as potential tools to study a variety of human diseases and air pollution exposure. A highly sensitive method involving online reverse-phase high performance liquid chromatography with electrospray tandem mass spectrometry detection in the multiple reaction monitoring mode and employing stable isotope-labeled internal standards was developed for the simultaneous quantification of 1,N(2)-etheno-2`-deoxyguanosine (1,N(2)-epsilon dGuo) and 1,N(2)-propano-2`-deoxyguanosine (1,N(2)-propanodGuo) in DNA. This methodology permits direct online quantification of 2`-deoxyguanosine and ca. 500 amol of adducts in 100 mu g of hydrolyzed DNA M the same analysis. Using the newly developed technique, accurate determinations of 1,N(2)-etheno-2`-deoxyguanosine and 1,N2-propano-2`-deoxyguanosine levels in DNA extracts of human cultured cells (4.01 +/- 0.32 1,N(2)-epsilon dGuo/10(8) dGuo and 3.43 +/- 0.33 1,N(2)-propanodGuo/10(8) dGuo) and rat tissue (liver, 2.47 +/- 0.61 1,N(2)-epsilon dGuo/10(8) dGuo and 4.61 +/- 0.69 1,N(2)-propanodGuo/108 dGuo; brain, 2.96 +/- 1.43,N(2)-epsilon dGuo/10(8) dGuo and 5.66 +/- 3.70 1,N(2)-propanoclGuo/10(8) dGuo; and lung, 0,87 +/- 0.34 1,N(2)-edGuo/ 10(8) dGuo and 2.25 +/- 1.72 1,N(2)-propanodGuo/10(8) dGuo) were performed. The method described herein can be used to study the biological significance of exocyclic DNA adducts through the quantification of different adducts in humans and experimental an with pathological conditions and after air pollution exposure.

Quantification of N-acetylcysteine in pharmaceuticals using cobalt phthalocyanine modified graphite electrodes

Flow injection analysis (FIA) with amperometric detection was employed for the quantification of N-acetylcysteine (NAC) in pharmaceutical formulations, utilizing an ordinary pyrolytic graphite (OPG) electrode modified with cobalt phthalocyanine (CoPc). Cyclic voltammetry was used in preliminary studies to establish the best conditions for NAC analysis. In FIA-amperometric experiments the OPG-CoPc electrode exhibited sharp and reproducible current peaks over a wide linear working range (5.0 x 10(-5)-1.0 x 10(-3) mol L(-1)) in 0.1 mol L(-1) NaOH solution. High sensitivity (130 mA mol(-1) cm(2)) and a low detection limit (9.0 x 10(-7) mol L(-1)) were achieved using the sensor. The repeatability (R.S.D.%) for 13 successive flow injections of a solution containing 5.0 x 10(-4) mol L(-1) NAC was 1.1%. The new procedure was applied in analyses of commercial pharmaceutical products and the results were in excellent agreement with those obtained using the official titrimetric method. The proposed amperometric method is highly suitable for quality control analyses of NAC in pharmaceuticals since it is rapid, precise and requires much less work than the recommended titrimetric method. (C) 2010 Elsevier B.V. All rights reserved.

Catalytic ethanolysis of soybean oil with immobilized lipase from Candida antarctica and (1)H NMR and GC quantification of the ethyl esters (biodiesel) produced

The catalytic ethanolysis of soybean oil with commercial immobilized lipase type B from Candida antarctica to yield ethyl esters (biodiesel) has been investigated. Transesterification was monitored with respect to the following parameters: quantity of biocatalyst, reaction time, amount of water added and turnover of lipase. The highest yields of biodiesel (87% by (1)H NMR; 82.9% by GC) were obtained after a reaction time of 24 h at 32 degrees C in the presence of lipase equivalent to 5.0% (w/w) of the amount of soybean oil present. The production of ethyl esters by enzymatic ethanolysis was not influenced by the addition of water up to 4.0% (v/v) of the alcohol indicating that it is possible to use hydrated ethanol in the production of biodiesel catalyzed by lipase. The immobilized enzyme showed high stability under moderate reaction conditions and retained its activity after five production cycles. The (1)H NMR methodology elaborated for the quantification of biodiesel in unpurified reaction mixtures showed good correlations between the signal areas of peaks associated with the alpha-methylene groups of the ethyl esters and those of the triacyl-glycerides in residual soybean oil. Monoacylglycerides, diacylglycerides and triglycerides could also be detected and quantified in the crude biodiesel using (1)H NMR spectroscopic and GC-FID chromatographic methods. The biodiesel production by enzymatic catalysis was promising. In this case, was produced a low concentration of glycerol (0.74%) and easily removed by water extraction. (C) 2010 Elsevier B.V. All rights reserved.

Development of an Analytical Methodology for Quantification of Strong Acid in Diesel Oil

Many factors can affect the quality of diesel oil, in particular the degradation processes that are directly related to some organosulfur compounds. During the degradation process, these compounds are oxidized into their corresponding sulfonic acids, generating a strong acid content during the process. p-Toluene sulfonic acid analysis was performed using the linear sweep voltammetry technique with a platinum ultramicroelectrode in aqueous solution containing 3 mol L(-1) potassium chloride. An extraction step was introduced prior to the voltammetric detection in order to avoid the adsorption of organic molecules, which inhibit the electrochemical response. The extraction step promoted the transference of sulfonic acid from the diesel oil to an aqueous phase. The method was accurate and reproducible, with detection and quantification limits of 5 ppm and 15 ppm, respectively. Recovery of sulfonic acid was around 90%.

Quantification of isoorientin and total flavonoids in Passiflora edulis fruit pulp by HPLC-UV/DAD

A method is reported for the quantification of isoorientin (using a standard addition method) and total flavonoids (expressed as rutin, using the external standard method) in passion fruit pulp (Passiflora edulis Sims f. flavicarpa Degener, Passifloraceae). Extraction of flavonoids was optimized by experimental design methodology, and quantitative analysis was performed by high-performance liquid chromatography with photo-diode array detection (HPLC-UV/DAD). The method was developed and validated according to ICH requirements for specificity, linearity, accuracy, precision (repeatability and intermediate precision). LOD and LOQ. Rutin was chosen as standard for the quantification of total flavonoids in order to propose a HPLC method feasible for routine analysis of the flavonoids in the passion fruit pulp. The passion fruit pulp contained 16.226 +/- 0.050 mg L(-1) of isoorientin and 158.037 +/- 0.602 mg L(-1) of total flavonoid, suggesting that P. edulis fruits may be comparable with other flavonoid food sources such as orange juice or sugarcane juice. (C) 2010 Elsevier B.V. All rights reserved.

FURTHER INSIGHTS TOWARD VITAMIN C DETERMINATION AND STABILITY. PROPOSAL OF A NEW QUANTIFICATION METHOD

This work aimed at an evaluation of the classical iodine method for quantification of vitamin C (L-ascorbic acid) in fruit juices, as well as at a search into the stability of this so popular vitamin under different conditions of pH, temperature and light exposition, in addition to a proposal of a new quantification method. Our results point to the persistent reversibility of the blue color of the starch-triiodide complex at the end point when using the classical iodine titration, and the overestimation of the true vitamin concentration in fruit juices. A new quantification method is proposed in order to overcome this problem. Surprising conclusions were obtained regarding the controversial stability of L-ascorbic acid toward atmospheric oxygen, at low pH, even in fruit juice and at room temperature, showing that the major problem concerned with aging of fruit juices is proliferation of microorganisms rather than expontaneous oxidation of L-ascorbic acid.

Validation of the Deardorff model for estimating energy balance components for a sugarcane crop

The quantification of the available energy in the environment is important because it determines photosynthesis, evapotranspiration and, therefore, the final yield of crops. Instruments for measuring the energy balance are costly and indirect estimation alternatives are desirable. This study assessed the Deardorff's model performance during a cycle of a sugarcane crop in Piracicaba, State of São Paulo, Brazil, in comparison to the aerodynamic method. This mechanistic model simulates the energy fluxes (sensible, latent heat and net radiation) at three levels (atmosphere, canopy and soil) using only air temperature, relative humidity and wind speed measured at a reference level above the canopy, crop leaf area index, and some pre-calibrated parameters (canopy albedo, soil emissivity, atmospheric transmissivity and hydrological characteristics of the soil). The analysis was made for different time scales, insolation conditions and seasons (spring, summer and autumn). Analyzing all data of 15 minute intervals, the model presented good performance for net radiation simulation in different insolations and seasons. The latent heat flux in the atmosphere and the sensible heat flux in the atmosphere did not present differences in comparison to data from the aerodynamic method during the autumn. The sensible heat flux in the soil was poorly simulated by the model due to the poor performance of the soil water balance method. The Deardorff's model improved in general the flux simulations in comparison to the aerodynamic method when more insolation was available in the environment.

The role of γ -aminobutyric acid (Gaba) in somatic embryogenesis of Acca sellowiana Berg. (Myrtaceae)

The γ-aminobutyric acid (Gaba) is a non-protein amino acid found in prokaryotes and eukaryotes. Its role in plant development has not been fully established. This study reports a quantification of the levels of endogenous Gaba, as well as investigation of its role in different stages of somatic embryogenesis in Acca sellowiana Berg. (Myrtaceae). Zygotic embryos were used as explants and they were inoculated into the culture medium contained different concentrations of Gaba (0,2, 4, 6, 8 and 10 µM). The highest concentrations of endogenous Gaba were detected between the third and nine days after inoculation, reaching the value of 12.77 µmol.g-1FW. High frequency of somatic embryogenesis was observed in response to 10 µM Gaba. This treatment also resulted in a large number of normal embryos, and the lowest percentage of formation of fused somatic embryos, phenotypic characteristic of most deformed embryos in all treatments. Also, all treatments promoted the formation of the somatic embryos with positive characteristics of development resumption, which however did not originate the seedlings.

Mineralogical characterization of a highly-weathered soil by the Rietveld Method

The mineralogical characterization through mineral quantification of Brazilian soils by X-ray diffraction data using the Rietveld Method is not common. A mineralogical quantification of an Acric Ferralsol from the Ponta Grossa region, state of Paraná, Brazil, was carried out using this Method with X-Ray Diffraction data to verify if this method was suitable for mineral quantification of a highly-weathered soil. The A, AB and B3 horizons were fractioned to separate the different particle sizes: clay, silt, fine sand (by Stokes Law) and coarse sand fractions (by sieving), with the procedure free of chemical treatments. X-ray Fluorescence, Inductively Coupled Plasma Atomic Emission Spectrometry, Infrared Spectroscopy and Mössbauer Spectroscopy were used in order to assist the mineral identification and quantification. The Rietveld Method enabled the quantification of the present minerals. In a general way, the quantitative mineralogical characterization by the Rietveld Method revealed that quartz, gibbsite, rutile, hematite, goethite, kaolinite and halloysite were present in the clay and silt fractions of all horizons. The silt fractions of the deeper horizons were different from the more superficial ones due to the presence of large amounts of quartz. The fine and the coarse sand fractions are constituted mainly by quartz. Therefore, a mineralogical quantification of the finer fraction (clay and silt) by the Rietveld Method was successful.

Trypanosoma cruzi benznidazole susceptibility in vitro does not predict the therapeutic outcome of human Chagas disease

Therapeutic failure of benznidazole (BZ) is widely documented in Chagas disease and has been primarily associated with variations in the drug susceptibility of Trypanosoma cruzi strains. In humans, therapeutic success has been assessed by the negativation of anti-T. cruzi antibodies, a process that may take up to 10 years. A protocol for early screening of the drug resistance of infective strains would be valuable for orienting physicians towards alternative therapies, with a combination of existing drugs or new anti-T. cruzi agents. We developed a procedure that couples the isolation of parasites by haemoculture with quantification of BZ susceptibility in the resultant epimastigote forms. BZ activity was standardized with reference strains, which showed IC50 to BZ between 7.6-32 µM. The assay was then applied to isolates from seven chronic patients prior to administration of BZ therapy. The IC50 of the strains varied from 15.6 ± 3-51.4 ± 1 µM. Comparison of BZ susceptibility of the pre-treatment isolates of patients considered cured by several criteria and of non-cured patients indicates that the assay does not predict therapeutic outcome. A two-fold increase in BZ resistance in the post-treatment isolates of two patients was verified. Based on the profile of nine microsatellite loci, sub-population selection in non-cured patients was ruled out.

Development and validation of a simple and rapid capillary zone electrophoresis method for determination of nnrti nevirapine in pharmaceutical formulations

A simple and fast capillary zone electrophoresis (CZE) method has been developed and validated for quantification of a non-nucleoside reverse transcriptase inhibitor (NNRTI) nevirapine, in pharmaceuticals. The analysis was optimized using 10 mmol L-1 sodium phosphate buffer pH 2.5, +25 kV applied voltage, hydrodynamic injection 0.5 psi for 5 s and direct UV detection at 200 µm. Diazepam (50.0 µg mL-1) was used as internal standard. Under these conditions, nevirapine was analyzed in approximately less than 2.5 min. The analytical curve presented a coefficient of correlation of 0.9994. Limits of detection and quantification were 1.4 µg mL-1 and 4.3 µg mL-1, respectively. Intra- and inter-day precision expressed as relative standard deviations were 1.4% and 1.3%, respectively and the mean recovery was 100.81%. The active pharmaceutical ingredient was subjected to hydrolysis (acid, basic and neutral) and oxidative stress conditions. No interference of degradation products and tablet excipients were observed. This method showed to be rapid, simple, precise, accurate and economical for determination of nevirapine in pharmaceuticals and it is suitable for routine quality control analysis since CE offers benefits in terms of quicker method development and significantly reduced operating costs.

Solid-phase microextraction for determination of 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone in water

Solid-phase microextraction, using on-line bis(trimethylsilyl)trifluoroacetamide derivatisation, gas chromatography, and mass spectrometry, was evaluated in the quantification of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in water samples. Fibres encompassing a wide range of polarities were used with headspace and direct immersion sampling. For the immersion procedure, various parameters affecting MX extraction, including pH, salinity, temperature, and extraction time were evaluated. The optimised method (polyacrylate fibre; 20% Na2SO4; pH 2.0; 60 min; 20 °C) was applied for reservoir chlorinated water samples-either natural or spiked with MX (50 ng L-1 and 100 ng L-1). The recovery of MX ranged from 44 to 72%. Quantification of MX in water samples was done using external standard and the selected ion monitoring mode. Correlation coefficient (0.98%), relative standard deviation (5%), limit of detection (30 ng L-1) and limit of quantification (50 ng L-1) were obtained from calibration curve.

Protein aggregation containing beta-amyloid, alpha-synuclein and hyperphosphorylated tau in cultured cells of hippocampus, substantia nigra and locus coeruleus after rotenone exposure

Background: Protein aggregates containing alpha-synuclein, beta-amyloid and hyperphosphorylated tau are commonly found during neurodegenerative processes which is often accompanied by the impairment of mitochondrial complex I respiratory chain and dysfunction of cellular systems of protein degradation. In view of this, we aimed to develop an in vitro model to study protein aggregation associated to neurodegenerative diseases using cultured cells from hippocampus, locus coeruleus and substantia nigra of newborn Lewis rats exposed to 0.5, 1, 10 and 25 nM of rotenone, which is an agricultural pesticide, for 48 hours. Results: We demonstrated that the proportion of cells in culture is approximately the same as found in the brain nuclei they were extracted from. Rotenone at 0.5 nM was able to induce alpha-synuclein and beta amyloid aggregation, as well as increased hyperphosphorylation of tau, although high concentrations of this pesticide (over 1 nM) lead cells to death before protein aggregation. We also demonstrated that the 14kDa isoform of alpha-synuclein is not present in newborn Lewis rats. Conclusion: Rotenone exposure may lead to constitutive protein aggregation in vitro, which may be of relevance to study the mechanisms involved in idiopathic neurodegeneration.

Sources of carbonaceous aerosol in the Amazon basin

The quantification of sources of carbonaceous aerosol is important to understand their atmospheric concentrations and regulating processes and to study possible effects on climate and air quality, in addition to develop mitigation strategies. In the framework of the European Integrated Project on Aerosol Cloud Climate Interactions (EUCAARI) fine (D(p) < 2.5 mu m) and coarse (2.5 mu m < Dp < 10 mu m) aerosol particles were sampled from February to June (wet season) and from August to September (dry season) 2008 in the central Amazon basin. The mass of fine particles averaged 2.4 mu g m(-3) during the wet season and 4.2 mu g m(-3) during the dry season. The average coarse aerosol mass concentration during wet and dry periods was 7.9 and 7.6 mu g m(-3), respectively. The overall chemical composition of fine and coarse mass did not show any seasonality with the largest fraction of fine and coarse aerosol mass explained by organic carbon (OC); the average OC to mass ratio was 0.4 and 0.6 in fine and coarse aerosol modes, respectively. The mass absorbing cross section of soot was determined by comparison of elemental carbon and light absorption coefficient measurements and it was equal to 4.7 m(2) g(-1) at 637 nm. Carbon aerosol sources were identified by Positive Matrix Factorization (PMF) analysis of thermograms: 44% of fine total carbon mass was assigned to biomass burning, 43% to secondary organic aerosol (SOA), and 13% to volatile species that are difficult to apportion. In the coarse mode, primary biogenic aerosol particles (PBAP) dominated the carbonaceous aerosol mass. The results confirmed the importance of PBAP in forested areas. The source apportionment results were employed to evaluate the ability of global chemistry transport models to simulate carbonaceous aerosol sources in a regional tropical background site. The comparison showed an overestimation of elemental carbon (EC) by the TM5 model during the dry season and OC both during the dry and wet periods. The overestimation was likely due to the overestimation of biomass burning emission inventories and SOA production over tropical areas.

Development and validation of a fast RP-HPLC method to determine the analogue of the thyroid hormone, 3,5,3 '-triiodothyroacetic acid (TRIAC), in polymeric nanoparticles

The objective of this work was to develop and validate a rapid Reversed-Phase High-Performance Liquid Chromatography method for the quantification of 3,5,3 '-triiodothyroacetic acid (TRIAC) in nanoparticles delivery system prepared in different polymeric matrices. Special attention was given to developing a reliable reproductive technique for the pretreatment of the samples. Chromatographic runs were performed on an Agilent 1200 Series HPLC with a RP Phenomenex (R) Gemini C18 (150 x 4, 6 mm i.d., 5 mu m) column using acetonitrile and triethylamine buffer 0.1% (TEA) (40 : 60 v/v) as a mobile phase in an isocratic elution, pH 5.6 at a flow rate of 1 ml min(-1). TRIAC was detected at a wavelength of 220 nm. The injection volume was 20 mu l and the column temperature was maintained at 35 degrees C. The validation characteristics included accuracy, precision, specificity, linearity, recovery, and robustness. The standard curve was found to have a linear relationship (r(2) - 0.9996) over the analytical range of 5-100 mu g ml(-1) . The detection and quantitation limits were 1.3 and 3.8 mu g ml(-1), respectively. The recovery and loaded TRIAC in colloidal system delivery was nearly 100% and 98%, respectively. The method was successfully applied in polycaprolactone, polyhydroxybutyrate, and polymethylmethacrylate nanoparticles.