A new HPLC-DAD-MS/MS method for the simultaneous determination of major compounds in the crude extract of Lychnophora salicifolia Mart. (Brazilian arnicao) leaves: Application to chemical variability evaluation

Lychnophora salicifolia Mart., which occurs in the Brazilian Cerrado in the states of Bahia and Minas Gerais as well as in the southeast of the state of Goias, is the most widely distributed and also the most polymorphic species of the genus. This plant is popularly known to have anti-inflammatory and analgesic activities. In this work, we have studied the variation in terms of polar metabolites of ninety-three Lychnophora salicifolia Mart, specimens collected from different regions of the Brazilian Cerrado. Identification of the constituents of this mixture was carried out by analysis of the UV spectra and MS data after chromatographic separation. Twenty substances were identified, including chlorogenic acid derivatives, a flavonoid C-glucoside, and other sesquiterpenes. The analytical method was validated, and the reliability and credibility of the results was ensured for the purposes of this study. The concentration range required for analysis of content variability within the analyzed group of specimens was covered with appropriate values of limits of detection and quantitation, as well as satisfactory precision and recovery. A quantitative variability was observed among specimens collected from the same location, but on average they were similar from a chemical viewpoint. In relation to the study involving specimens from different locations, there were both qualitative and quantitative differences among plants collected from different regions of Brazil. Statistical analysis revealed that there is a correlation between geographical localization and polar metabolites profile for specimens collected from different locations. This is evidence that the pattern of metabolites concentration depends on the geographical distribution of the specimens. (C) 2012 Elsevier B.V. All rights reserved.

DETERMINATION OF KINETIC AND THERMODYNAMIC PARAMETERS OF L-CYSTEINE ADSORPTION ONTO GOLD BY THE QCM TECHNIQUE

DETERMINATION OF KINETIC AND THERMODYNAMIC PARAMETERS OF L-CYSTEINE ADSORPTION ONTO GOLD BY THE QCM TECHNIQUE. This article discusses the adsorption kinetics of a L-cysteine monolayer onto a gold surface by means of information obtained through the QCM technique. The results indicate that the adsorption process is rapid and follows the Langmuir isotherm, in which adsorption and desorption are considered. From these measurements the following parameter values were obtained: k(d) = (4.2 +/- 0.4) x 10(-3) s(-1), k(a) = 75 +/- 6 M-1 s(-1), K-eq=(1.8 +/- 0.3) x 10(4) M-1 and Delta G(ads) = -(5.8 +/- 0.2) kcal mol(-1).

A simple and precise conductometric method for the determination of losartan in pharmaceutical products

Losartan is an antihypertensive agent that lost its patent protection in 2010, and, consequently, it has been available in generic form. The latter motivated the search for a rapid and precise alternative method. Here, a simple conductometric titration in aqueous medium is described for the losartan analysis in pharmaceutical formulations. The first step of the titration occurs with the protonation of losartan producing a white precipitate and resulting in a slow increase in conductivity. When the protonation stage is complete, a sharp increase in conductivity occurs which was determined to be due to the presence of excess of acid. The titrimetric method was applied to the determination of losartan in pharmaceutical products and the results are comparable with values obtained using a chromatographic method recommended by the United States Pharmacopoeia. The relative standard deviation for successive measurements of a 125 mg L-1 (2.71x10(-4) mol L-1) losartan solution was approximately 2%. Recovery study in tablet samples ranged between 99 and 102.4%. The procedure is fast, simple, and represents an attractive alternative for losartan quantification in routine analysis. In addition, it avoids organic solvents, minimizes the risk of exposure to the operator, and the waste treatment is easier compared to classical chromatographic methods.

Flow-injection spectrophotometric determination of captopril in pharmaceutical formulations using a new solid-phase reactor containing AgSCN immobilized in a polyurethane resin

A simple flow-injection analysis procedure was developed for determining captopril in pharmaceutical formulations employing a novel solid-phase reactor containing silver thiocyanate immobilized in a castor oil derivative polyurethane resin. The method was based on silver mercaptide formation between the captopril and Ag(I) in the solid-phase reactor. During such a reaction, the SCN- anion was released and reacted with Fe3+, which generated the FeSCN2+ complex that was continuously monitored at 480 nm. The analytical curve was linear in the captopril concentration range from 3.0 x 10(-4) mol L-1 to 1.1 x 10(-3) mol L-1 with a detection limit of 8.0 x 10(-5) mol L-1. Recoveries between 97.5% and 103% and a relative standard deviation of 2% for a solution containing 6.0 x 10(-4) mol L-1 captopril (n = 12) were obtained. The sample throughput was 40 h(-1) and the results obtained for captopril in pharmaceutical formulations using this procedure and those obtained using a pharmacopoeia procedure were in agreement at a 95% confidence level.

Simultaneous determination of dextromethorphan, dextrorphan and doxylamine in human plasma by HPLC coupled to electrospray ionization tandem mass spectrometry: Application to a pharmacokinetic study

In the present study, a fast, sensitive and robust method to quantify dextromethorphan, dextrorphan and doxylamine in human plasma using deuterated internal standards (IS) is described. The analytes and the IS were extracted from plasma by a liquid-liquid extraction (LLE) using diethyl-ether/hexane (80/20, v/v). Extracted samples were analyzed by high performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Chromatographic separation was performed by pumping the mobile phase (acetonitrile/water/formic acid (90/9/1, v/v/v) during 4.0 min at a flow-rate of 1.5 mL min(-1) into a Phenomenex Gemini (R) C18, 5 mu m analytical column (150 x 4.6 mm id.). The calibration curve was linear over the range from 0.2 to 200 ng mL(-1) for dextromethorphan and doxylamine and 0.05 to 10 ng mL(-1) for dextrorphan. The intra-batch precision and accuracy (%CV) of the method ranged from 2.5 to 9.5%, and 88.9 to 105.1%, respectively. Method inter-batch precision (%CV) and accuracy ranged from 6.7 to 10.3%, and 92.2 to 107.1%, respectively. The run-time was for 4 min. The analytical procedure herein described was used to assess the pharmacokinetics of dextromethorphan, dextrorphan and doxylamine in healthy volunteers after a single oral dose of a formulation containing 30 mg of dextromethorphan hydrobromide and 12.5 mg of doxylamine succinate. The method has high sensitivity, specificity and allows high throughput analysis required for a pharmacokinetic study. (C) 2012 Elsevier B.V. All rights reserved.

Kaurenoic Acid from Sphagneticola trilobata Inhibits Inflammatory Pain: Effect on Cytokine Production and Activation of the NO-Cyclic GMP-Protein Kinase G-ATP-Sensitive Potassium Channel Signaling Pathway

Kaurenoic acid [ent-kaur-16-en-19-oic acid (1)] is a diterpene present in several plants including Sphagneticola trilobata. The only documented evidence for its antinociceptive effect is that it inhibits the writhing response induced by acetic acid in mice. Therefore, the analgesic effect of 1 in different models of pain and its mechanisms in mice were investigated further. Intraperitoneal and oral treatment with 1 dose-dependently inhibited inflammatory nociception induced by acetic acid. Oral treatment with 1 also inhibited overt nociception-like behavior induced by phenyl-p-benzoquinone, complete Freund's adjuvant (CFA), and both phases of the formalin test. Compound 1 also inhibited acute carrageenin- and PGE(2)-induced and chronic CFA-induced inflammatory mechanical hyperalgesia. Mechanistically, 1 inhibited the production of the hyperalgesic cytokines TNF-alpha and IL-1 beta. Furthermore, the analgesic effect of 1 was inhibited by L-NAME, ODQ, KT5823, and glybenclamide treatment, demonstrating that such activity also depends on activation of the NO-cyclic GMP-protein kinase G-ATP-sensitive potassium channel signaling pathway, respectively. These results demonstrate that 1 exhibits an analgesic effect in a consistent manner and that its mechanisms involve the inhibition of cytokine production and activation of the NO-cyclic GMP-protein lcinase G-ATP-sensitive potassium channel signaling pathway.

Determination of trace elements in bovine semen samples by inductively coupled plasma mass spectrometry and data mining techniques for identification of bovine class

The reproductive performance of cattle may be influenced by several factors, but mineral imbalances are crucial in terms of direct effects on reproduction. Several studies have shown that elements such as calcium, copper, iron, magnesium, selenium, and zinc are essential for reproduction and can prevent oxidative stress. However, toxic elements such as lead, nickel, and arsenic can have adverse effects on reproduction. In this paper, we applied a simple and fast method of multi-element analysis to bovine semen samples from Zebu and European classes used in reproduction programs and artificial insemination. Samples were analyzed by inductively coupled plasma spectrometry (ICP-MS) using aqueous medium calibration and the samples were diluted in a proportion of 1:50 in a solution containing 0.01% (vol/vol) Triton X-100 and 0.5% (vol/vol) nitric acid. Rhodium, iridium, and yttrium were used as the internal standards for ICP-MS analysis. To develop a reliable method of tracing the class of bovine semen, we used data mining techniques that make it possible to classify unknown samples after checking the differentiation of known-class samples. Based on the determination of 15 elements in 41 samples of bovine semen, 3 machine-learning tools for classification were applied to determine cattle class. Our results demonstrate the potential of support vector machine (SVM), multilayer perceptron (MLP), and random forest (RF) chemometric tools to identify cattle class. Moreover, the selection tools made it possible to reduce the number of chemical elements needed from 15 to just 8.

Caffeine Determination at a Carbon Fiber Ultramicroelectrodes by Fast-Scan Cyclic Voltammetry

Caffeine determination using a fast-scan voltammetric procedure at a carbon fiber ultramicroelectrode (CF-UME) is described. The CF-UME was submitted to electrochemical pretreatment. Parameters such as number of acquisition cycles, scan rate, potential window, and the electrochemical surface pretreatment were optimized. Using the optimized conditions, it was possible to achieve a LDR from 10.0 up to 200 mu mol L-1, with a LOD of 3.33 mu mol L-1. The method has been applied in the determination of caffeine in commercial samples, with errors of 1.0-3.5% in relation to the label values and recoveries of 97-114% within the linear range.

Platinum nanoparticles incorporated in silsesquioxane for use in LbL films for the simultaneous detection of dopamine and ascorbic acid

We describe the preparation of platinum nanoparticles (PtNPs) using the 3-n-propylpyridinium silsesquioxane chloride (SiPy+Cl-) as a nanoreactor and stabilizer. The formation of PtNPs was monitored by UV-Vis spectroscopy by measuring the decrease in the intensity of the band at 375 nm, which is attributed to the electronic absorption of PtCl62- ions. TEM images of Pt-SiPy+Cl- nanohybrid indicated an average size of 3-40 nm for PtNPs. The Pt-SiPy+Cl- was used as a polycation in the preparation of layer-by-layer films (LbL) on a glass substrate coated with fluorine-doped tin oxide (FTO) alternating with the polyanion poly(vinyl sulfonic acid) (PVS). The films were electrochemically tested in sulfuric acid to confirm the deposition of Pt-SiPy+Cl- onto the LbL films, observing the adsorption and desorption of hydrogen (E-pa = 0.1 V) and by the redox process of formation for PtO with E-pa = 1.3 V and E-pc = 0.65 V. FTIR and Raman spectra confirmed the presence of the PVS and Pt-SiPy+Cl- in the LbL films. A linear increase in the absorbance in the UV-Vis spectra of the Pt-SiPy+Cl- at 258 nm (pi -> pi* transition of the pyridine groups) with a number of Pt-SiPy+Cl-/PVS or PVS/SiPy+Cl- bilayers (R = 0.992) was observed. These LbL films were tested for the determination of dopamine (DA) in the presence of ascorbic acid (AA) with a detection limit (DL) on the order of 2.6 x 10(-6) mol L-1 and a quantification limit (QL) of 8.6 x 10(-6) mol L-1. The films exhibited a good repeatability and reproducibility, providing a potential difference of 550 mV for the oxidation of DA with AA interferent.

Regulation of neurogenesis and gliogenesis of retinoic acid-induced P19 embryonal carcinoma cells by P2X2 and P2X7 receptors studied by RNA interference

Embryonic carcinoma cells are widely used models for studying the mechanisms of proliferation and differentiation occurring during early embryogenesis. We have now investigated how down-regulation of P2X2 and P2X7 receptor expression by RNA interference (RNAi) affects neural differentiation and phenotype specification of P19 embryonal carcinoma cells. Wild-type P19 embryonal carcinoma cells or cells stably expressing shRNAs targeting P2X2 or P2X7 receptor expression were induced to differentiate into neurons and glial cells in the presence of retinoic acid. Silencing of P2X2 receptor expression along differentiation promoted cell proliferation and an increase in the percentage of cells expressing glial-specific GFAP, while the presence of beta-3 tubulin-positive cells diminished at the same time. Proliferation induction in the presence of stable anti-P2X2 receptor RNAi points at a mechanism where glial proliferation is favored over growth arrest of progenitor cells which would allow neuronal maturation. Differently from the P2X2 receptor, inhibition of P2X7 receptor expression during neural differentiation of P19 cells resulted in a decrease in cell proliferation and GFAP expression, suggesting the need of functional P2X7 receptors for the progress of gliogenesis. The results obtained in this study indicate the importance of purinergic signaling for cell fate determination during neural differentiation, with P2X2 and P2X7 receptors promoting neurogenesis and gliogenesis, respectively. The shRNAs down-regulating P2X2 or P2X7 receptor gene expression, developed during this work, present useful tools for studying mechanisms of neural differentiation in other stem cell models. (C) 2012 ISDN. Published by Elsevier Ltd. All rights reserved.

Determination of chlorine in food samples via the AlCl molecule using high-resolution continuum source molecular absorption spectrometry in a graphite furnace

Determination of chlorine using the molecular absorption of aluminum mono-chloride (AlCl) at the 261.418 nm wavelength was accomplished by high-resolution continuum source molecular absorption spectrometry using a transversely heated graphite tube furnace with an integrated platform. For the analysis. 10 mu L of the sample followed by 10 mu L of a solution containing Al-Ag-Sr modifier, (1 g L-1 each), were directly injected onto the platform. A spectral interference due to the use of Al-Ag-Sr as mixed modifier was easily corrected by the least-squares algorithm present in the spectrometer software. The pyrolysis and vaporization temperatures were 500 degrees C and 2200 degrees C, respectively. To evaluate the feasibility of a simple procedure for the determination of chlorine in food samples present in our daily lives, two different digestion methods were applied, namely (A) an acid digestion method using HNO3 only at room temperature, and (B) a digestion method with Ag, HNO3 and H2O2, where chlorine is precipitated as a low-solubility salt (AgCl), which is then dissolved with ammonia solution. The experimental results obtained with method B were in good agreement with the certified values and demonstrated that the proposed method is more accurate than method A. This is because the formation of silver chloride prevented analyte losses by volatilization. The limit of detection (LOD, 3 sigma/s) for Cl in methods A and B was 18 mu g g(-1) and 9 mu g g(-1), respectively, 1.7 and 3.3 times lower compared to published work using inductively coupled plasma optical emission spectrometry, and absolute LODs were 2.4 and 1.2 ng, respectively. (C) 2012 Elsevier B.V. All rights reserved.

Determination of decimal reduction time (D value) of chemical agents used in hospitals for disinfection purposes

Abstract Background Prior to the selection of disinfectants for low, intermediate and high (sterilizing) levels, the decimal reduction time, D-value, for the most common and persistent bacteria identified at a health care facility should be determined. Methods The D-value was determined by inoculating 100 mL of disinfecting solution with 1 mL of a bacterial suspension (104 – 105 CFU/mL for vegetative and spore forms). At regular intervals, 1 mL aliquots of this mixture were transferred to 8 mL of growth media containing a neutralizing agent, and incubated at optimal conditions for the microorganism. Results The highest D-values for various bacteria were determined for the following solutions: (i) 0.1% sodium dichloroisocyanurate (pH 7.0) – E. coli and A. calcoaceticus (D = 5.9 min); (ii) sodium hypochlorite (pH 7.0) at 0.025% for B. stearothermophilus (D = 24 min), E. coli and E. cloacae (D = 7.5 min); at 0.05% for B. stearothermophilus (D = 9.4 min) and E. coli (D = 6.1 min) and 0.1% for B. stearothermophilus (D = 3.5 min) and B. subtilis (D = 3.2 min); (iii) 2.0% glutaraldehyde (pH 7.4) – B. stearothermophilus, B. subtilis (D = 25 min) and E. coli (D = 7.1 min); (iv) 0.5% formaldehyde (pH 6.5) – B. subtilis (D = 11.8 min), B. stearothermophilus (D = 10.9 min) and A. calcoaceticus (D = 5.2 min); (v) 2.0% chlorhexidine (pH 6.2) – B. stearothermophilus (D = 9.1 min), and at 0.4% for E. cloacae (D = 8.3 min); (vi) 1.0% Minncare® (peracetic acid and hydrogen peroxide, pH 2.3) – B. stearothermophilus (D = 9.1 min) and E. coli (D = 6.7 min). Conclusions The suspension studies were an indication of the disinfectant efficacy on a surface. The data in this study reflect the formulations used and may vary from product to product. The expected effectiveness from the studied formulations showed that the tested agents can be recommended for surface disinfection as stated in present guidelines and emphasizes the importance and need to develop routine and novel programs to evaluate product utility.

An alternative method for the simultaneous determination of copper and lead for quality control of sugar cane spirit using a nanotube-based sensor

The development of an electroanalytical method for simultaneous determination of copper and lead ions in sugar cane spirit (cachaça) using carbon paste electrode modified with ascorbic acid and carbon nanotubes (CPE-AaCNT) is described. Squarewave voltammetry (SWV) with anodic stripping was employed, and this technique was optimized with respect to the following parameters: frequency (50 Hz), amplitude (100 mV) and scan increment (9 mV). The analytical curves were linear in the range from 0.0900 to 7.00 mg L- 1 for lead and copper. The limits of detection were 48.5 and 23.9 µg L- 1 for lead and copper, respectively. The developed method was applied to the simultaneous determination of copper and lead in five commercial samples of sugar cane spirit. The results were in good agreement with those obtained by F AAS/GF AAS (flame atomic absorption spectrometry/graphite furnace atomic absorption spectrometry) and showed that CPE-AaCNT can be successfully employed in the simultaneous determination of these metals in real sugar cane spirit samples.

Monitoring and determination of fungi and mycotoxins in stored Brazil nuts

Brazil nut (Bertholletia excelsa) is an important commodity from the Brazilian Amazon, and approximately 37,000 tons (3.36 × 10⁷ kg) of Brazil nuts are harvested each year. However, substantial nut contamination by Aspergillus section Flavi occurs, with subsequent production of mycotoxins. In this context, the objective of the present investigation was to evaluate the presence of fungi and mycotoxins (aflatoxins and cyclopiazonic acid) in 110 stored samples of cultivated Brazil nut (55 samples of nuts and 55 samples of shells) collected monthly for 11 months in Itacoatiara, State of Amazonas, Brazil. The samples were inoculated in duplicate onto Aspergillus flavus and Aspergillus parasiticus agar and potato dextrose agar for the detection of fungi, and the presence of mycotoxins was determined by high-performance liquid chromatography. The most prevalent fungi in nuts and shells were Aspergillus spp., Fusarium spp., and Penicillium spp. A polyphasic approach was used for identification of Aspergillus species. Aflatoxins and cyclopiazonic acid were not detected in any of the samples analyzed. The low water activity of the substrate was a determinant factor for the presence of fungi and the absence of aflatoxin in Brazil nut samples. The high frequency of isolation of aflatoxigenic Aspergillus section Flavi strains, mainly A. flavus, and their persistence during storage increase the chances of aflatoxin production on these substrates and indicates the need for good management practices to prevent mycotoxin contamination in Brazil nuts.

Structure determination of proteins and peptides in solution: simulation, chirality and NMR studies

The study of protein fold is a central problem in life science, leading in the last years to several attempts for improving our knowledge of the protein structures. In this thesis this challenging problem is tackled by means of molecular dynamics, chirality and NMR studies. In the last decades, many algorithms were designed for the protein secondary structure assignment, which reveals the local protein shape adopted by segments of amino acids. In this regard, the use of local chirality for the protein secondary structure assignment was demonstreted, trying to correlate as well the propensity of a given amino acid for a particular secondary structure. The protein fold can be studied also by Nuclear Magnetic Resonance (NMR) investigations, finding the average structure adopted from a protein. In this context, the effect of Residual Dipolar Couplings (RDCs) in the structure refinement was shown, revealing a strong improvement of structure resolution. A wide extent of this thesis is devoted to the study of avian prion protein. Prion protein is the main responsible of a vast class of neurodegenerative diseases, known as Bovine Spongiform Encephalopathy (BSE), present in mammals, but not in avian species and it is caused from the conversion of cellular prion protein to the pathogenic misfolded isoform, accumulating in the brain in form of amiloyd plaques. In particular, the N-terminal region, namely the initial part of the protein, is quite different between mammal and avian species but both of them contain multimeric sequences called Repeats, octameric in mammals and hexameric in avians. However, such repeat regions show differences in the contained amino acids, in particular only avian hexarepeats contain tyrosine residues. The chirality analysis of avian prion protein configurations obtained from molecular dynamics reveals a high stiffness of the avian protein, which tends to preserve its regular secondary structure. This is due to the presence of prolines, histidines and especially tyrosines, which form a hydrogen bond network in the hexarepeat region, only possible in the avian protein, and thus probably hampering the aggregation.