Development and validation of a high-performance liquid chromatography method for quantification of egonol and homoegonol in Styrax species

Styrax camporum Pohl, known in Brazil as estoraque do campo or cuia de brejo, has been used in the treatment of gastrointestinal diseases. The therapeutic action of S. camporum has been attributed to the ethyl acetate fraction, although the chemical composition of this fraction has not yet been analyzed. In this study, a high-performance liquid chromatography photodiode array detection (HPLC-PAD) method for analysis of Brazilian Styrax species has been developed. The compounds egonol (1) and homoegonol (2) were found to be present in all the samples investigated by HPLC. These compounds were isolated by open column chromatography followed by preparative TLC, and were identified by 1H NMR. Compounds 1 and 2 were thus proposed as phytochemical markers for Styrax, owing to their biological properties and presence in other Styrax species. The developed method has been validated and successfully applied for quantification of 1 and 2 in S. camporum dried leaves and crude ethanolic extracts from S. ferrugineus and S. pohlii aerial parts. Copyright (c) 2011 John Wiley & Sons, Ltd.

Leishmanicidal and cholinesterase inhibiting activities of phenolic compounds of Dimorphandra gardneriana and Platymiscium floribundum, native plants from Caatinga biome

Nos últimos anos, o Ministério da Saúde do Brasil e a Organização Mundial da Saúde tem apoiado a investigação de novas tecnologias que possam contribuir para a vigilância, novos tratamentos e controle da leishmaniose visceral no país. Assim, o objetivo deste trabalho foi isolar compostos de plantas do bioma Caatinga, e investigar a toxicidade destes compostos contra as formas promastigotas e amastigotas de Leishmania infantum chagasi, principal parasita responsável pela leishmaniose visceral na América do Sul, e avaliar a sua capacidade para inibir a enzima acetil-colinesterase (AChE). Após a exposição aos compostos em estudo, foram realizados testes utilizando a forma promastigota que expressa luciferase e ELISA in situ para medir a viabilidade das formas promastigotas e amastigota, respectivamente. O ensaio colorimétrico MTT foi realizado para determinar a toxicidade destas substâncias utilizando células monocíticas murina RAW 264.7. Todos os compostos foram testados in vitro para as sua propriedade anti-colinesterásica. Um cumarina, escoparona, foi isolada a partir de hastes de Platymiscium floribundum, e os flavonóides, rutina e quercetina, foram isolados a partir de grãos de Dimorphandra gardneriana. Estes compostos foram purificados, utilizando cromatografia em coluna gel eluída com solventes orgânicos em misturas de polaridade crescente, e identificados por análise espectral. Nos ensaios leishmanicidas, os compostos fenólicos mostraram eficácia contra as formas extracelulares promastigotas, com EC50 para escoporona de 21.4µg/mL e para quercetina e rutina 26 e 30.3µg/mL, respectivamente. Os flavonóides apresentaram resultados comparáveis à droga controle, a anfotericina B, contra as formas amastigotas com EC50 para quercetina e rutina de 10.6 e 43.3µg/mL, respectivamente. Os compostos inibiram a enzima AChE com halos de inibição variando de 0,8 a 0,6cm, indicando um possível mecanismo de ação para a atividade leishmanicida.

Detection of isometric, dsRNA-containing viral particles in Colletotrichum gloeosporioides isolated from cashew tree

Fungi are disease-causing agents in plants and affect crops of economic importance. One control method is to induce resistance in the host by using biological control with hypovirulent phytopathogenic fungi. Here, we report the detection of a mycovirus in a strain of Colletotrichum gloeosporioides causing anthracnose of cashew tree. The strain C. gloeosporioides URM 4903 was isolated from a cashew tree (Anacardium occidentale) in Igarassu, PE, Brazil. After nucleic acid extraction and electrophoresis, the band corresponding to a possible double-stranded RNA (dsRNA) was purified by cellulose column chromatography. Nine extrachromosomal bands were obtained. Enzymatic digestion with DNAse I and Nuclease S1 had no effect on these bands, indicating their dsRNA nature. Transmission electron microscopic examination of extracts from this strain showed the presence of isometric particles (30-35 nm in diameter). These data strongly suggest the infection of this C. gloeosporioides strain by a dsRNA mycovirus. Once the hypovirulence of this strain is confirmed, the strain may be used for the biological control of cashew anthracnose.

Herbicides in the upper Poxim River, Sergipe, and the risk of contamination of water resources

Increased agricultural activity in watershed areas has been causing concern over contamination by herbicides in agricultural areas. The problem becomes more important when contamination can affect water for human consumption, as happens with water from the Poxim river, which supplies the city of Aracaju, capital of the State of Sergipe. The aim of this study was to evaluate the risk of contamination by herbicides to both surface and groundwater in the upper sub-basin of the Poxim River, and to detect the presence of the active ingredients Diuron and Ametrine up-river from the sugar-cane plantations. Risk analysis was carried out using criteria from the Environmental Protection Agency (EPA), the GUS index, and the GOSS method. It was observed that several active ingredients are at risk of leaching, demonstrating the importance of monitoring the river to control both the quality of water and the frequency and volume of herbicides used in the region. Based on the results, monitoring was carried out bi-monthly from July 2009 to July 2010 at two sampling points. Water samples were analyzed in the laboratory, where the presence of Diuron and Ametrine was noted. Water quality in the Sub-basin of the Rio Poxim is being influenced by the use of herbicides in the region. There was an increase in herbicide concentration in the surface water during the rainy season, possibly caused by soil runoff.

Leishmanicidal activity in vitro of Musa paradisiaca L. and Spondias mombin L. fractions

Visceral leishmaniasis (VL) is a zoonotic disease characterized by infection of mononuclear phagocytes by Leishmania chagasi. The primary vector is Lutzomyia longipalpis and the dog is the main domestic reservoir. The control and current treatment of dogs using synthetic drugs have not shown effectiveness in reducing the incidence of disease in man. In attempt to find new compounds with leishmanicidal action, plant secondary metabolites have been studied in search of treatments of VL. This study aimed to evaluate the leishmanicidal activity of Musa paradisiaca (banana tree) and Spondias mombin (cajazeira) chemical constituents on promastigotes and amastigotes of L. chagasi. Phytochemical analysis by column chromatography was performed on ethanol extracts of two plants and fractions were isolated. Thin layer chromatography was used to compare the fractions and for isolation the substances to be used in vitro tests. The in vitro tests on promastigotes of L chagasi used the MTT colorimetric method and the method of ELISA in situ was used against amastigotes besides the cytotoxicity in RAW 264.7 cells. Of the eight fractions tested, Sm1 and Sm2 from S. mombin had no action against promastigotes, but had good activity against amastigotes. The fractions Mp1 e Mp4 of M. paradisiaca were very cytotoxic to RAW 264.7 cells. The best result was obtained with the fraction Sm3 from S. mombin with IC50 of 11.26 mu g/ml against promastigotes and amastigotes of 0.27 mu g/ml. The fraction Sm3 characterized as tannic acid showed the best results against both forms of Leishmania being a good candidate for evaluation in in vivo tests. (C) 2012 Published by Elsevier B.V.

A comparative study of the purification of betanin

Betanin is a natural pigment with antioxidant properties used as a food colourant. This work describes the spectrophotometric and chromatographic quantification of betanin (2S/15S) and its epimer isobetanin (2S/15R) in fresh beetroot juice, food-grade beetroot powder and betanin standard diluted in dextrin. Absorption spectra of all three samples were deconvoluted using a mixed three-function model. Food-grade beetroot powder has the largest amount of violet-red impurities, probably formed during processing. The purification of betanin from these complex matrices was carried out by seven different methods. Ion exchange chromatography was the most efficient method for the purification of betanin from all samples; however, fractions contain high amounts of salt. Reversed-phase HPLC as well as reversed-phase column chromatography also produced good results at a much faster rate. The longer retention time of isobetanin when compared to betanin in reversed-phase conditions has been investigated by means of quantum-mechanical methods. (C) 2011 Elsevier Ltd. All rights reserved.

Herbicidas no alto rio Poxim, Sergipe e os riscos de contaminação dos recursos hídricos

A crescente atividade agrícola em áreas de mananciais tem causado preocupações quanto à contaminação por herbicidas em áreas agrícolas. O problema se torna mais importante quando a contaminação pode afetar a água para uso humano, como ocorre com a água do Rio Poxim, que abastece a cidade de Aracaju, capital do Estado de Sergipe. O objetivo do estudo foi avaliar o risco de contaminação de águas superficiais e subterrâneas por herbicidas no alto da Sub-bacia do Rio Poxim e detectar a presença dos princípios ativos Diuron e Ametrina, à montante das plantações de cana-de-açúcar. A análise de risco foi realizada mediante critérios da Environmental Protection Agency (EPA), índice de GUS e método de GOSS. Observou-se que vários princípios ativos sofrem risco de lixiviação, demonstrando a importância do monitoramento do rio para controle tanto da qualidade da água como da frequência e volume de herbicidas aplicado na região. A partir do resultado, foi realizado um monitoramento bimensal de julho de 2009 a julho de 2010, em dois pontos de amostragem. As amostras de água foram analisadas em laboratório, onde se, constatou a presença de Diuron e Ametrina. A qualidade da água na Sub-bacia do Rio Poxim está sendo influenciada pelo uso de herbicidas na região. Ocorreu um aumento nas concentrações dos herbicidas na água superficial, durante o período chuvoso, provocado possivelmente pelo escoamento superficial.

Analysis of carvedilol enantiomers in human plasma using chiral stationary phase column and liquid chromatography with tandem mass spectrometry

Carvedilol is an antihypertensive drug available as a racemic mixture. (-)-(S)-carvedilol is responsible for the nonselective beta-blocker activity but both enantiomers present similar activity on a1-adrenergic receptor. To our knowledge, this is the first study of carvedilol enantiomers in human plasma using a chiral stationary phase column and liquid chromatography with tandem mass spectrometry. The method involves plasma extraction with diisopropyl ether using metoprolol as internal standard and direct separation of the carvedilol enantiomers on a Chirobiotic T (R) (Teicoplanin) column. Protonated ions [M + H]+ and their respective ion products were monitored at transitions of 407 > 100 for the carvedilol enantiomers and 268 > 116 for the internal standard. The quantification limit was 0.2 ng ml-1 for both enantiomers in plasma. The method was applied to study enantioselectivity in the pharmacokinetics of carvedilol administered as a single dose of 25 mg to a hypertensive patient. The results showed a higher plasma concentration of (+)-(R)-carvedilol (AUC08 205.52 vs. 82.61 (ng h) ml-1), with an enantiomer ratio of 2.48. Chirality, 2012. (C) 2012 Wiley Periodicals, Inc.

A novel HS-SBSE system coupled with gas chromatography and mass spectrometry for the analysis of organochlorine pesticides in water samples

A methodology to analyze organochlorine pesticides (OCPs) in water samples has been accomplished by using headspace stir bar sorptive extraction (HS-SBSE). The bars were in house coated with a thick film of PDMS in order to properly work in the headspace mode. Sampling was done by a novel HS-SBSE system whereas the analysis was performed by capillary GC coupled mass spectrometric detection (HS-SBSE-GC-MS). The extraction optimization, using different experimental parameters has been established by a standard equilibrium time of 120 min at 85 degrees C. A mixture of ACN/toluene as back extraction solvent promoted a good performance to remove the OCPs sorbed in the bar. Reproducibility between 2.1 and 14.8% and linearity between 0.96 and 1.0 were obtained for pesticides spiked in a linear range between 5 and 17 ng/g in water samples during the bar evaluation.

Evaluation of comprehensive two-dimensional gas chromatography coupled to rapid scanning quadrupole mass spectrometry for quantitative analysis

Comprehensive two-dimensional gas chromatography (GC x GC) is a powerful technique that provides excellent separation and identification of analytes in highly complex samples with considerable increase in GC peak capacities. However, since second dimension analyses are very fast, detectors with a rapid acquisition rate are required. Over the last years, quite a number of studies have discussed the potential and limitations of the combination GC x GC with a variety of quadrupole mass spectrometers. The present research focuses on the evaluation of qMS effectiveness at a 10,000-amu/s scan speed and 20-Hz scan frequency for the identification (full scan mode acquisition-TIC) and quantification (extracted ion chromatogram) of target pesticide residues in tomato samples. The following MS parameters have been evaluated: number of data points per peak, mass spectrum quality, peak skewing, and sensitivity. The validated proposed GC x GC/qMS method presented satisfactory results in terms of repeatability (coefficient of variation lower than 15%), accuracy (84-117%), and linearity (ranging from 25 to 500 ng/g), while significant enhancement in sensitivity was observed (a factor of around 10) under scan conditions. (C) 2012 Elsevier B.V. All rights reserved.

Methane emission from soil under long-term no-till cropping systems

Methane (CH4) emission from agricultural soils increases dramatically as a result of deleterious effect of soil disturbance and nitrogen fertilization on methanotrophic organisms; however, few studies have attempted to evaluate the potential of long-term conservation management systems to mitigate CH4 emissions in tropical and subtropical soils. This study aimed to evaluate the long-term effect (>19 years) of no-till grass- and legume-based cropping systems on annual soil CH4 fluxes in a formerly degraded Acrisol in Southern Brazil. Air sampling was carried out using static chambers and CH4 analysis by gas chromatography. Analysis of historical data set of the experiment evidenced a remarkable effect of high C- and N-input cropping systems on the improvement of biological, chemical, and physical characteristics of this no-tilled soil. Soil CH4 fluxes, which represent a net balance between consumption (-) and production (+) of CH4 in soil, varied from -40 +/- 2 to +62 +/- 78 mu g C m(-2) h(-1). Mean weighted contents of ammonium (NH4+-N) and dissolved organic carbon (DOC) in soil had a positive relationship with accumulated soil CH4 fluxes in the post-management period (r(2) = 0.95, p = 0.05), suggesting an additive effect of these nutrients in suppressing CH4 oxidation and stimulating methanogenesis, respectively, in legume-based cropping systems with high biomass input. Annual CH4 fluxes ranged from -50 +/- 610 to +994 +/- 105 g C ha(-1), which were inversely related to annual biomass-C input (r(2) = 0.99, p = 0.003), with the exception of the cropping system containing pigeon pea, a summer legume that had the highest biologically fixed N input (>300 kg ha(-1) yr(-1)). Our results evidenced a small effect of conservation management systems on decreasing CH4 emissions from soil, despite their significant effect restoring soil quality. We hypothesized that soil CH4 uptake strength has been off-set by an injurious effect of biologically fixed N in legume-based cropping systems on soil methanotrophic microbiota, and by the methanogenesis increase as a result of the O-2 depletion in niches of high biological activity in the surface layer of the no-tillage soil. (C) 2012 Elsevier B.V. All rights reserved.

DEVELOPMENT AND VALIDATION OF AN ANALYTICAL METHOD FOR QUANTITATION OF THE DRUG BEVACIZUMAB BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

DEVELOPMENT AND VALIDATION OF AN ANALYTICAL METHOD FOR QUANTITATION OF THE DRUG BEVACIZUMAB BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY. In this study, an analytical method was developed and validated for quantitation of the drug bevacizumab (Avastin (R)) by high performance liquid chromatography (HPLC). The HPLC column was a BioSuite 250 (R) HR SEC, 300 x 7.8 mm x 5 mu m (Waters, USA). The mobile phase consisted of phosphate buffered saline (PBS). The results revealed that the method was specific, precise. accurate, robust and linear (r(2) = 0.998) from 5 to 75 mu g mL(-1). Therefore, this method can be used in drug release studies or in quality control ampoules of the drug.

QUANTIFICATION OF TRYPTOPHAN IN PLASMA BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

A simple, rapid and selective method using high-performance liquid chromatography with ultraviolet detection (267 nm) was applied for the determination of tryptophan in plasma. Separation was carried out on a C18 column (150 x 4.6 mm internal diameter) in 6 min. The mobile phase consisted of 5 mM the sodium acetate and acetonitrile (92:8, v/v). The method was shown to be precise and accurate, and good recovery of analyte was achieved, characterizing the method as efficient and reliable for use in laboratory analysis.

Nutrients and Nonessential Elements in Soil after 11 Years of Wastewater Irrigation

Irrigation of citrus (Citrus aurantium L. x Citrus paradise Macf.) with urban reclaimed wastewater (RWW) can be economical and conserve fresh water. However, concerns remain regarding its deleterious effects on soil quality. We investigated the ionic speciation (ISP) of RWW and potential impacts of 11 yr of irrigation with RWW on soil quality, compared with well-water (WW) irrigation. Most of nutrients (similar to 53-99%) in RWW are free ionic species and readily available for plant uptake, such as: NH4+, NO3-, K+, Ca2+, Mg2+, SO42-, H3BO3, Cl-, Fe2+, Mn2+, Zn2+, Co2+, and Ni2+, whereas more than about 80% of Cu, Cr, Pb, and Al are complexed with CO3-, OH-, and/or organic matter. The RWW irrigation increased the availability and total concentrations of nutrients and nonessential elements, and soil salinity and sodicity by two to three times compared with WW-irrigated soils. Although RWW irrigation changed many soil parameters, no difference in citrus yield was observed. The risk of negative impacts from RWW irrigation on soil quality appears to be minimal because of: (i) adequate quality of RWW, according to USEPA limits; (ii) low concentrations of metals in soil after 11 yr of irrigation with RWW; and (iii) rapid leaching of salts in RWW-irrigated soil during the rainy season.

Determination of anticonvulsants in human plasma using SPME in a heated interface coupled online to liquid chromatography (SPME-LC)

A simple and sensitive method using solid phase microextraction (SPME) and liquid chromatography (LC) with heated online desorption (SPME-LC) was developed and validated to analyze anticonvulsants (AEDs) in human plasma samples. A heated lab-made interface chamber was used in the desorption procedure, which allowed the transference of the whole extracted sample. The SPME conditions were optimized by applying an experimental design. Important factors are discussed such as fiber coating types, pH, extraction time and desorption conditions. The drugs were analyzed by LC, using a C18 column (150 mm x 4.6 mm x 5 mm); and 50 mmol L-1, pH 5.50 ammonium acetate buffer : acetonitrile : methanol (55 : 22 : 23 v/v) as the mobile phase with a flow rate of 0.8 mL min(-1). The suggested method presented precision (intra-assay and inter-assay), linearity and limit of quantification (LOQ) all adequate for the therapeutic drug monitoring (TDM) of AEDs in plasma.