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1.
PLoS Negl Trop Dis ; 13(2): e0007103, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30726203

RESUMO

Trypanosoma cruzi, the etiological agent of Chagas' disease, affects 8 million people predominantly living in socioeconomic underdeveloped areas. T. cruzi trypomastigotes (Ty), the classical infective stage, interact with the extracellular matrix (ECM), an obligatory step before invasion of almost all mammalian cells in different tissues. Here we have characterized the proteome and phosphoproteome of T. cruzi trypomastigotes upon interaction with ECM (MTy) and the data are available via ProteomeXchange with identifier PXD010970. Proteins involved with metabolic processes (such as the glycolytic pathway), kinases, flagellum and microtubule related proteins, transport-associated proteins and RNA/DNA binding elements are highly represented in the pool of proteins modified by phosphorylation. Further, important metabolic switches triggered by this interaction with ECM were indicated by decreases in the phosphorylation of hexokinase, phosphofructokinase, fructose-2,6-bisphosphatase, phosphoglucomutase, phosphoglycerate kinase in MTy. Concomitantly, a decrease in the pyruvate and lactate and an increase of glucose and succinate contents were detected by GC-MS. These observations led us to focus on the changes in the glycolytic pathway upon binding of the parasite to the ECM. Inhibition of hexokinase, pyruvate kinase and lactate dehydrogenase activities in MTy were observed and this correlated with the phosphorylation levels of the respective enzymes. Putative kinases involved in protein phosphorylation altered upon parasite incubation with ECM were suggested by in silico analysis. Taken together, our results show that in addition to cytoskeletal changes and protease activation, a reprogramming of the trypomastigote metabolism is triggered by the interaction of the parasite with the ECM prior to cell invasion and differentiation into amastigotes, the multiplicative intracellular stage of T. cruzi in the vertebrate host.


Assuntos
Matriz Extracelular/parasitologia , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Animais , Cromatografia Gasosa-Espectrometria de Massas , Regulação da Expressão Gênica/fisiologia , Interações Hospedeiro-Parasita , Humanos , Proteínas de Protozoários/genética
2.
Biochim Biophys Acta Gen Subj ; 1862(6): 1505-1515, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29526507

RESUMO

BACKGROUND: Impairment in mitochondrial biogenesis and function plays a key role in depression and anxiety, both of which being associated with changes in fatty acid and phospholipid metabolism. The antidepressant effects of (R,S)-ketamine have been linked to its conversion into (2S,6S;2R,6R)-hydroxynorketamine (HNK); however, the connection between structure and stereochemistry of ketamine and HNK in the mitochondrial homeostatic response has not yet been fully elucidated at a metabolic level. METHODS: We used a multi-platform, non-targeted metabolomics approach to study the change in mitochondrial metabolome of PC-12 cells treated with ketamine and HNK enantiomers. The identified metabolites were grouped into pathways in order to assess global responses. RESULTS: Treatment with (2R,6R)-HNK elicited the significant change in 49 metabolites and associated pathways implicated in fundamental mitochondrial functions such as TCA cycle, branched-chain amino acid biosynthetic pathway, glycoxylate metabolic pathway, and fatty acid ß-oxidation. The affected metabolites included glycerate, citrate, leucine, N,N-dimethylglycine, 3-hexenedioic acid, and carnitine and attenuated signals associated with 9 fatty acids and elaidic acid. Important metabolites involved in the purine and pyrimidine pathways were also affected by (2R-6R)-HNK. This global metabolic profile was not as strongly impacted by treatment with (2S,6S)-HNK, (R)- and (S)-ketamine and in some instances opposite effects were observed. CONCLUSIONS: The present data provide an overall view of the metabolic changes in mitochondrial function produced by (2R,6R)-HNK and related ketamine compounds and offer an insight into the source of the observed variance in antidepressant response elicited by the compounds.


Assuntos
Ketamina/análogos & derivados , Ketamina/farmacologia , Redes e Vias Metabólicas/efeitos dos fármacos , Metaboloma , Metabolômica/métodos , Mitocôndrias/metabolismo , Animais , Mitocôndrias/efeitos dos fármacos , Células PC12 , Ratos , Estereoisomerismo
3.
Trials ; 18(1): 601, 2017 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-29258572

RESUMO

BACKGROUND: Early reperfusion of the occluded coronary artery during acute myocardial infarction is considered crucial for reduction of infarcted mass and recovery of ventricular function. Effective microcirculation and the balance between protective and harmful lymphocytes may have roles in reperfusion injury and may affect final ventricular remodeling. METHODS/DESIGN: BATTLE-AMI is an open-label, randomized trial comparing the effects of four therapeutic strategies (rosuvastatin/ticagrelor, rosuvastatin/clopidogrel, simvastatin plus ezetimibe/ticagrelor, or simvastatin plus ezetimibe/clopidogrel) on infarcted mass and left ventricular ejection fraction (LVEF) (blinded endpoints) in patients with ST-segment elevation myocardial infarction submitted to fibrinolytic therapy before coronary angiogram (pharmacoinvasive strategy). All patients (n = 300, 75 per arm) will be followed up for six months. The effects of treatment on subsets of B and T lymphocytes will be determined by flow-cytometry/ELISPOT and will be correlated with the infarcted mass, LVEF, and microcirculation perfusion obtained by cardiac magnetic resonance imaging. The primary hypothesis is that the combined rosuvastatin/ticagrelor therapy will be superior to other therapies (particularly for the comparison with simvastatin plus ezetimibe/clopidogrel) for the achievement of better LVEF at 30 days (primary endpoint) and smaller infarcted mass (secondary endpoint) at 30 days and six months. The trial will also evaluate the improvement in the immune/inflammatory responses mediated by B and T lymphocytes. Omics field (metabolomics and proteomics) will help to understand these responses by molecular events. DISCUSSION: BATTLE-AMI is aimed to (1) evaluate the role of subsets of lymphocytes on microcirculation improvement and (2) show how the choice of statin/antiplatelet therapy may affect cardiac remodeling after acute myocardial infarction with ST elevation. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02428374 . Registered on 28 September 2014.


Assuntos
Anti-Inflamatórios/administração & dosagem , Linfócitos B/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Mediadores da Inflamação/sangue , Inibidores da Agregação de Plaquetas/administração & dosagem , Infarto do Miocárdio com Supradesnível do Segmento ST/tratamento farmacológico , Linfócitos T/efeitos dos fármacos , Terapia Trombolítica , Adenosina/administração & dosagem , Adenosina/análogos & derivados , Anti-Inflamatórios/efeitos adversos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Biomarcadores/sangue , Brasil , Protocolos Clínicos , Clopidogrel , Angiografia Coronária , Quimioterapia Combinada , ELISPOT , Ezetimiba/administração & dosagem , Feminino , Citometria de Fluxo , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/efeitos adversos , Imagem por Ressonância Magnética , Masculino , Metabolômica , Inibidores da Agregação de Plaquetas/efeitos adversos , Proteômica , Projetos de Pesquisa , Rosuvastatina Cálcica/administração & dosagem , Infarto do Miocárdio com Supradesnível do Segmento ST/sangue , Infarto do Miocárdio com Supradesnível do Segmento ST/diagnóstico por imagem , Infarto do Miocárdio com Supradesnível do Segmento ST/imunologia , Sinvastatina/administração & dosagem , Volume Sistólico/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Terapia Trombolítica/efeitos adversos , Ticagrelor , Ticlopidina/administração & dosagem , Ticlopidina/análogos & derivados , Fatores de Tempo , Resultado do Tratamento , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos
4.
Toxicology ; 376: 94-101, 2017 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-27234314

RESUMO

The prevalence of marijuana use among pregnant women is high. However, the effects on gestation and fetal development are not well known. Epidemiological and experimental studies present conflicting results because of the route of administration, dose, time of exposure, species used, and how Cannabis toxicity is tested (prepared extracts, specific components, or by pyrolysis). In this study, we experimentally investigated the effects of maternal inhalation of Cannabis sativa smoke representing as nearly as possible real world conditions of human marijuana use. Pregnant mice (n=20) were exposed (nose-only) daily for 5min to marijuana smoke (0.2g of Cannabis) from gestational day (GD) 5.5 to GD17.5 or filtered air. Food intake and maternal weight gain were recorded. Ultrasound biomicroscopy was performed on 10.5 and 16.5dpc.On GD18.5, half of the dams were euthanized for the evaluation of term fetus, placenta, and resorptions. Gestation length, parturition, and neonatal outcomes were evaluated in the other half. Five minutes of daily (low dose) exposure during pregnancy resulted in reduced birthweight, and litter size was not altered; however, the number of male pups per litter was higher. Besides, placental wet weight was increased and fetal to placental weight ratio was decreased in male fetuses, showing a sex-specific effect. At the end of gestation, females from the Cannabis group presented reduced maternal net body weight gain, despite a slight increase in their daily food intake compared to the control group. In conclusion, our results indicate that smoking marijuana during pregnancy even at low doses can be embryotoxic and fetotoxic.


Assuntos
Cannabis/toxicidade , Desenvolvimento Fetal/efeitos dos fármacos , Fumar Maconha/efeitos adversos , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal/diagnóstico por imagem , Drogas Ilícitas/toxicidade , Animais , Ingestão de Alimentos/efeitos dos fármacos , Ingestão de Alimentos/fisiologia , Feminino , Desenvolvimento Fetal/fisiologia , Exposição por Inalação/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Gravidez , Ultrassonografia Pré-Natal
5.
Trials ; 18(1): 601-601, 2017.
Artigo em Inglês | Sec. Est. Saúde SP, SESSP-IDPCPROD, Sec. Est. Saúde SP | ID: ses-36830

RESUMO

BACKGROUND: Early reperfusion of the occluded coronary artery during acute myocardial infarction is considered crucial for reduction of infarcted mass and recovery of ventricular function. Effective microcirculation and the balance between protective and harmful lymphocytes may have roles in reperfusion injury and may affect final ventricular remodeling. METHODS/DESIGN: BATTLE-AMI is an open-label, randomized trial comparing the effects of four therapeutic strategies (rosuvastatin/ticagrelor, rosuvastatin/clopidogrel, simvastatin plus ezetimibe/ticagrelor, or simvastatin plus ezetimibe/clopidogrel) on infarcted mass and left ventricular ejection fraction (LVEF) (blinded endpoints) in patients with ST-segment elevation myocardial infarction submitted to fibrinolytic therapy before coronary angiogram (pharmacoinvasive strategy). All patients (n = 300, 75 per arm) will be followed up for six months. The effects of treatment on subsets of B and T lymphocytes will be determined by flow-cytometry/ELISPOT and will be correlated with the infarcted mass, LVEF, and microcirculation perfusion obtained by cardiac magnetic resonance imaging. The primary hypothesis is that the combined rosuvastatin/ticagrelor therapy will be superior to other therapies (particularly for the comparison with simvastatin plus ezetimibe/clopidogrel) for the achievement of better LVEF at 30 days (primary endpoint) and smaller infarcted mass (secondary endpoint) at 30 days and six months...(AU)


Assuntos
Infarto do Miocárdio , Linfócitos B , Espectroscopia de Ressonância Magnética , Metabolômica , Proteômica
6.
Electrophoresis ; 37(7-8): 1039-47, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26826549

RESUMO

A CE-ESI-MS method was developed and validated for the separation and quantitative analysis of amino acids (AA) in urine. Experimental parameters related to the CE-MS interface, BGE, and mass spectrometer (MS) settings were optimized providing a good separation of 27 AA, including the isomers L-leucine, L-isoleucine, and L-alloisoleucine, in less than 30 min. The sheath liquid was composed by 0.50% formic acid in 60% (v,v) methanol-water delivered at a flow rate of 5 µL/min. The BGE consisted of 0.80 mol/L formic acid at pH 1.96 and 15% methanol. A pH stacking procedure was implemented to enhance sensitivity (a 12.5% NH4 OH solution was injected at 0.5 psi/9 s prior to samples injected at 0.6 psi/20 s). The proposed method was validated according to FDA and ICH protocols exhibiting acceptable parameters. Analytical curves presented coefficients of determination from 0.996 to 0.9997 (with large F statistics and low p-values). LODs and quantification ranged from 0.63 to 29 µmol/L and from 1.9 to 86 µmol/L, respectively. Practical repeatability was obtained for all AA with coefficients of variation better than 0.55% CV (migration time) and 1.7% CV (peak area ratios; methionine sulfone as internal standard). Recoveries of AA in spiked urine ranged from 92.0 to 123% with few exceptions. Moreover, a successful quantification of AA in pooled control and test urine samples, which compose a vesicoureteral reflux cohort, was achieved showing the potential applicability of the proposed method for targeted metabolomics studies using CE-ESI-MS with an Ion Trap as mass analyzer.


Assuntos
Aminoácidos/urina , Eletroforese Capilar/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Criança , Humanos , Limite de Detecção , Modelos Lineares , Metabolômica/métodos , Reprodutibilidade dos Testes
7.
Food Chem ; 192: 566-74, 2016 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-26304385

RESUMO

A RPLC-DAD method for the analysis of eight anthocyanins was developed, validated and applied to strawberry extracts. The chromatographic method was conducted under gradient elution in acidulated water-methanol mobile phase and octadecyl-silica columns. An ultrasound extraction procedure was optimized by a 3(2) factorial design (%HCl in methanol, temperature, and time) and response surface methodology. Method validation was performed according to the following parameters: linearity (R(2)>0.99, p-value<10(-4), F>725), LOD (3-7 µmol L(-1)) and LOQ (9-22 µmol L(-1)), selectivity/specificity (baseline separation of all analytes and peak purity), instrumental precision (<6.4%CV), repeatability (<6.3%CV) and intermediate precision (<9.9%CV), recovery (83-99%), robustness (mobile phase pH, column temperature and flow rate) and stability (high temperatures and storage; 1st order kinetics). The antioxidant power of anthocyanins was measured on-line (ABTS(+) reaction; Trolox as reference). Ten strawberry extracts were quantified (average values: 24.2 µg/g for cyanidin-3-glucoside and 49.1 µg/g for pelargonidin-3-glucoside).


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Fragaria/química , Antioxidantes/análise , Cinética
8.
PLoS One ; 10(7): e0130675, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26161866

RESUMO

There is a rising resistance against antimony drugs, the gold-standard for treatment until some years ago. That is a serious problem due to the paucity of drugs in current clinical use. In a research to reveal how these drugs affect the parasite during treatment and to unravel the underlying basis for their resistance, we have employed metabolomics to study treatment in Leishmania infantum promastigotes. This was accomplished first through the untargeted analysis of metabolic snapshots of treated and untreated parasites both resistant and responders, utilizing a multiplatform approach to give the widest as possible coverage of the metabolome, and additionally through novel monitoring of the origin of the detected alterations through a 13C traceability experiment. Our data stress a multi-target metabolic alteration with treatment, affecting in particular the cell redox system that is essential to cope with detoxification and biosynthetic processes. Additionally, relevant changes were noted in amino acid metabolism. Our results are in agreement with other authors studying other Leishmania species.


Assuntos
Antimônio/farmacologia , Antiprotozoários/farmacologia , Resistência a Medicamentos , Leishmania infantum/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Metaboloma/efeitos dos fármacos , Cromatografia Líquida/métodos , Humanos , Leishmania infantum/metabolismo , Leishmaniose Visceral/parasitologia , Metabolômica/métodos , Espectrometria de Massas em Tandem/métodos
9.
Electrophoresis ; 36(18): 2336-2347, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26095472

RESUMO

This review article compiles in a critical manner literature publications regarding seven neglected diseases (ND) prioritized in Brazil (Chagas disease, dengue, leishmaniasis, leprosy, malaria, schistosomiasis, and tuberculosis) under the perspective of metabolomics. Both strategies, targeted and untargeted metabolomics, were considered in the compilation. The majority of studies focused on biomarker discovery for diagnostic purposes, and on the search of novel or alternative therapies against the ND under consideration, although temporal progression of the infection at metabolic level was also addressed. Tuberculosis, followed by schistosomiasis, malaria and leishmaniasis are the diseases that received larger attention in terms of number of publications. Dengue and leprosy were the least studied and Chagas disease received intermediate attention. NMR and HPLC-MS technologies continue to predominate among the analytical platforms of choice in the metabolomic studies of ND. A plethora of metabolites were identified in the compiled studies, with expressive predominancy of amino acids, organic acids, carbohydrates, nucleosides, lipids, fatty acids, and derivatives.

10.
J Forensic Sci ; 60 Suppl 1: S241-7, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25413634

RESUMO

In this work, two methods were developed to determine herbicides paraquat, glyphosate, and aminomethylphosphonic acid (AMPA) in marijuana samples by capillary electrophoresis. For paraquat analysis, sample was extracted with aqueous acetic acid solution and analyzed by capillary zone electrophoresis with direct UV detection. The running electrolyte was 50 mmol/L phosphate buffer (pH 2.50). For glyphosate and AMPA, indirect UV/VIS detection was used, as these substances do not present chromophoric groups. Samples were extracted with 5 mmol/L hydrochloric acid. The running electrolyte was 10 mmol/L gallic acid, 6 mmol/L TRIS, and 0.1 mmol/L CTAB (pH = 4.7). The methods presented good linearity, precision, accuracy, and recovery. Paraquat was detected in 12 samples (n = 130), ranging from 0.01 to 25.1 mg/g. Three samples were positive for glyphosate (0.15-0.75 mg/g), and one sample presented AMPA as well. Experimental studies are suggested to evaluate the risks of these concentrations to marijuana user.


Assuntos
Cannabis/química , Glicina/análogos & derivados , Herbicidas/análise , Organofosfonatos/análise , Paraquat/análise , Contaminação de Medicamentos , Eletroforese Capilar , Glicina/análise , Isoxazóis , Tetrazóis
11.
Anal Bioanal Chem ; 406(14): 3459-76, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24722876

RESUMO

Miltefosine (MT) (hexadecylphosphocholine) was implemented to cope with resistance against antimonials, the classical treatment in Leishmaniasis. Given the scarcity of anti- Leishmania (L) drugs and the increasing appearance of resistance, there is an obvious need for understanding the mechanism of action and development of such resistance. Metabolomics is an increasingly popular tool in the life sciences due to it being a relatively fast and accurate technique that can be applied either with a particular focus or in a global manner to reveal new knowledge about biological systems. Three analytical platforms, gas chromatography (GC), liquid chromatography (LC) and capillary electrophoresis (CE) have been coupled to mass spectrometry (MS) to obtain a broad picture of metabolic changes in the parasite. Impairment of the polyamine metabolism from arginine (Arg) to trypanothione in susceptible parasites treated with MT was in some way expected, considering the reactive oxygen species (ROS) production described for MT. Importantly, in resistant parasites an increase in the levels of amino acids was the most outstanding feature, probably related to the adaptation of the resistant strain for its survival inside the parasitophorous vacuole.


Assuntos
Resistência a Medicamentos , Leishmania donovani/metabolismo , Metabolômica , Fosforilcolina/análogos & derivados , Arginina/química , Carbono/química , Cromatografia Gasosa , Cromatografia Líquida , Eletroforese Capilar , Cromatografia Gasosa-Espectrometria de Massas , Glutationa/análogos & derivados , Glutationa/química , Hidrodinâmica , Espectrometria de Massas , Fosforilcolina/análise , Fosforilcolina/química , Controle de Qualidade , Espécies Reativas de Oxigênio , Espermidina/análogos & derivados , Espermidina/química
12.
J Pharm Biomed Anal ; 84: 140-7, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23831489

RESUMO

A simple capillary electrophoretic method with spectrophotometric UV detection at 236 nm has been developed for the selective separation and determination of 1-(2-chlorophenyl)piperazine (oCPP), 1-(3-chlorophenyl)piperazine (mCPP) and 1-(4-chlorophenyl)piperazine (pCPP) in confiscated pills. Several cyclodextrin derivatives were tested to compose the background electrolyte (BGE). The optimized BGE contained 20 mmol/L phosphoric acid adjusted to pH 2.5 with triethylamine and 10 mmol/L α-cyclodextrin, which provided acceptable resolution of analytes and candidate interferents in less than 15 min. The analyses were performed at constant voltage of 25 kV in 60 cm (effective length 50 cm; 50 µm i.d.) uncoated fused-silica capillary maintained at 25°C with sample injection at 4,826 Pa for 8s. Procaine at a concentration of 0.1mg/mL was used as internal standard (IS). Possible interference from other drugs such as amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxy-N-ethylamphetamine, 1-(3-trifluoromethylphenyl)piperazine and cocaine was also examined. The analytical curves were linear (R(2)=0.9994-0.9995) in the range of 10-200 µg/mL (for oCPP and mCPP) and 20-200 µg/mL for pCPP. Limits of detection (LODs) were 2.0 µg/mL (oCPP), 2.5µg/mL (mCPP) and 3.5 µg/mL (pCPP). Intraday precision at three concentration levels and six replicates of each level (10, 100, 200 µg/mL of each analyte; n=18) was evaluated for the corrected peak area ratio of analyte to IS and the migration times giving RSDs ≤ 4.9%. The accuracy was estimated for mCPP by a recovery test at the same three concentration levels and recoveries varied from 101.0 to 101.6%. The method has been successively applied to the analysis of 17 confiscated pills based mostly on mCPP.


Assuntos
Ciclodextrinas/química , Eletroforese Capilar/métodos , Piperazinas/química , Isomerismo , Procaína/química , Padrões de Referência , Comprimidos/química
13.
Molecules ; 18(4): 3859-71, 2013 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-23538900

RESUMO

"Chimarrão" and "tererê" are maté (dried, toasted and milled Ilex paraguariensis leaves and stemlets) beverages widely consumed in South America. This paper describes the application of HPLC-DAD-ESI/MS method for the identification and quantification of caffeoylquinic acids (CQA), flavonol glycosides and purine alkaloids in these beverages. The beverage samples were prepared from commercial lots of maté from Southern Brazil. The caffeoylquinic acids, 4,5-diCQA, 3-CQA, 5-CQA, and 4-CQA were the major compounds, having 238-289, 153-242, 183-263, and 123-188 µg/mL, respectively, for chimarrão and 206-265, 122-218, 164-209, 103-169 µg/mL, respectively, for tererê. Caffeine also had high amounts while glycosides of quercetin and kaempferol were found at much lower levels. The individual antioxidant activity was also determined by an on-line system that measured their ABTS•+ radical scavenging activity, showing that the antioxidant capacity was not proportional to the concentrations of the phenolic compounds. 3-CQA, quercetina-3-O-ramnosylglucoside, and quercetina-3-O-glucoside were the major contributors to the antioxidant capacity, although the quercetin glycosides had concentrations less than 10 times that of 3-CQA.


Assuntos
Antioxidantes/análise , Bebidas/análise , Cromatografia Líquida de Alta Pressão/métodos , Ilex paraguariensis/química , Espectrometria de Massas/métodos , Fenóis/análise , Benzotiazóis/análise , Benzotiazóis/química , Brasil , Cafeína , Ácido Clorogênico/análise , Flavonóis/análise , Quempferóis/análise , Fenóis/química , Extratos Vegetais/análise , Folhas de Planta/química , Quercetina/análise , Ácido Quínico/análogos & derivados , Ácido Quínico/análise , Ácidos Sulfônicos/análise , Ácidos Sulfônicos/química
14.
Anal Chem ; 84(16): 6963-72, 2012 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-22799605

RESUMO

Combining data from multiple analytical platforms is essential for comprehensive study of the molecular phenotype (metabotype) of a given biological sample. The metabolite profiles generated are intrinsically dependent on the analytical platforms, each requiring optimization of instrumental parameters, separation conditions, and sample extraction to deliver maximal biological information. An in-depth evaluation of extraction protocols for characterizing the metabolome of the hepatobiliary fluke Fasciola hepatica , using ultra performance liquid chromatography and capillary electrophoresis coupled with mass spectroscopy is presented. The spectrometric methods were characterized by performance, and metrics of merit were established, including precision, mass accuracy, selectivity, sensitivity, and platform stability. Although a core group of molecules was common to all methods, each platform contributed a unique set, whereby 142 metabolites out of 14,724 features were identified. A mixture design revealed that the chloroform:methanol:water proportion of 15:59:26 was globally the best composition for metabolite extraction across UPLC-MS and CE-MS platforms accommodating different columns and ionization modes. Despite the general assumption of the necessity of platform-adapted protocols for achieving effective metabotype characterization, we show that an appropriately designed single extraction procedure is able to fit the requirements of all technologies. This may constitute a paradigm shift in developing efficient protocols for high-throughput metabolite profiling with more-general analytical applicability.


Assuntos
Fracionamento Químico/métodos , Fasciola hepatica/metabolismo , Metabolômica/métodos , Animais , Cromatografia Líquida de Alta Pressão
15.
Electrophoresis ; 33(4): 705-12, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22451064

RESUMO

A fast and sensitive method for the simultaneous determination of Sudan dyes (I, II, III, and IV) in food samples was developed for the first time using partial filling micellar electrokinectic chromatography-mass spectrometry (MEKC-MS). The use of MEKC was essential to achieve the separation of these neutral analytes, while the partial filling technique was necessary to avoid the contamination of the ion source with non-volatile micelles. MEKC separation and MS detection conditions were optimized in order to achieve a fast, efficient, and sensitive separation of the four dyes. Filling 25% of the capillary with an MEKC solution containing 40 mM ammonium bicarbonate, 25 mM SDS, and 32.5% (v/v) acetonitrile, a baseline separation of the four azo-dyes was obtained in 10 min. Tandem MS was investigated in order to improve the sensitivity and selectivity of the analysis. Limits of detection (LOD) values 5, 8, 15, and 29 times better were obtained for Sudan III, I, II, and IV, respectively, using partial filling MEKC-MS/MS instead of partial filling MEKC-MS. Under optimized conditions, LOD from 0.05 to 0.2 µg/mL were obtained. The suitability of the developed method was demonstrated through the fast and sensitive determination of Sudan I, II, III, and IV in spiked chilli powder samples. This determination could not be achieved by MEKC-UV due to the existence of several interfering compounds from the matrix.


Assuntos
Compostos Azo/análise , Capsicum/química , Cromatografia Capilar Eletrocinética Micelar/métodos , Corantes/análise , Especiarias/análise , Naftóis/análise , Espectrometria de Massas em Tandem/métodos
16.
J Agric Food Chem ; 59(22): 11903-9, 2011 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-21985218

RESUMO

A new method based on partial filling micellar electrokinetic chromatography (MEKC) for the quantitative determination of Sudan dyes (I, II, III, and IV) in chilli sauces is presented. The separation is achieved filling 25% of the capillary with a MEKC buffer composed of 40 mM NH(4)HCO(3), 25 mM sodium dodecyl sulfate, and 32.5% (v/v) acetonitrile (ACN). The rest of the capillary is filled using a capillary zone electrophoresis (CZE) buffer composed of 40 mM NH(4)HCO(3) and 32.5% (v/v) ACN. Under optimized conditions, the azo dyes are baseline separated in less than 8 min with limits of detection ranging from 0.57 to 0.71 µg mL(-1) (S/N > 3). Using an internal standard, the repeatability of the quantitative determination is improved almost four times. The applicability of the method for rapid screening and determination of Sudan dyes is corroborated by analyzing spiked chilli sauce samples with recoveries from 85 to 99%. The reported conditions are demonstrated to be compatible with mass spectrometry detection.


Assuntos
Compostos Azo/análise , Cromatografia Capilar Eletrocinética Micelar/métodos , Corantes/análise , Contaminação de Alimentos/análise , Lycopersicon esculentum/química
17.
J Anal Toxicol ; 35(5): 274-9, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21619722

RESUMO

The aim of this work was to develop a simple and fast method for the detection of paraquat in oral fluid, plasma, and urine by capillary electrophoresis with diode-array detection (CE-DAD), to diagnose of acute poisoning related to this herbicide. The use of oral fluid in analytical toxicology has been established for drugs of abuse, but not for diagnosis of pesticides poisoning. Oral fluid was collected without stimulation using absorbent cotton swabs. Sample preparation included diluting, vortex mixing, and centrifuging of biological fluid, using ethyl paraquat as internal standard. CE-DAD analyses were performed in a fused-silica capillary, and separation was performed under constant voltage condition of 21 kV, with detection at 195 nm. The electrolyte was a 40 mmol/L phosphate buffer at pH 2.50. The proposed method provided a fast and simple assay for the determination of paraquat in human oral fluid, plasma, and urine. To our knowledge, this is the first mention of use of oral fluid as a biological fluid diagnosis of quaternary ammonium herbicide poisoning. After validation, the method was applied to two cases of acute poisoning by this herbicide.


Assuntos
Herbicidas/metabolismo , Paraquat/metabolismo , Envenenamento/diagnóstico , Adulto , Eletroforese Capilar , Herbicidas/envenenamento , Humanos , Limite de Detecção , Masculino , Paraquat/envenenamento , Saliva/metabolismo
18.
Food Chem ; 127(2): 651-5, 2011 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-23140714

RESUMO

A sulfated-ß-cyclodextrin (s-ß-CD) modified reduced flow micellar electrokinetic chromatography (RF-MEKC) method was developed and validated for the determination of catechins in green tea. The optimal electrolyte consisted of 0.2% triethylamine, 50mmol/L SDS and 0.8% s-ß-CD (pH=2.9), allowing baseline separation of five catechins in 4min. The samples and standards were injected at 0.6psi for 5s under constant voltage of -30kV. Sample preparation simply involved extraction of 2g of tea with 200mL water at 95°C under constant stirring for 5min. The method demonstrated excellent performance, with limits of detection (LOD) and quantification (LOQ) of 0.02-0.1 and 0.1-0.5µg/mL, respectively, and recovery percentages of 94-101%. The method was applied to six samples of Brazilian green tea infusions. Epigallocatechin gallate (23.4-112.4µg/mL) was the major component, followed by epigallocatechin (18.4-78.9µg/mL), epicatechin gallate (5.6-29.6µg/mL), epicatechin (4.6-14.5µg/mL) and catechin (3.2-8.2µg/mL).

19.
Phytochem Anal ; 21(5): 428-33, 2010 Sep-Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20310075

RESUMO

INTRODUCTION: A large number of natural and synthetic compounds having butenolides as a core unit have been described and many of them display a wide range of biological activities. Butenolides from P. malacophyllum have presented potential antifungal activities but no specific, fast, and precise method has been developed for their determination. OBJECTIVE: To develop a methodology based on micellar electrokinetic chromatography to determine butenolides in Piper species. METHODOLOGY: The extracts were analysed in an uncoated fused-silica capillaries and for the micellar system 20 mmol/L SDS, 20% (v/v) acetonitrile (ACN) and 10 mmol/L STB aqueous buffer at pH 9.2 were used. The method was validated for precision, linearity, limit of detection (LOD) and limit of quantitation (LOQ) and the standard deviations were determined from the standard errors estimated by the regression line. RESULTS: A micellar electrokinetic chromatography (MEKC) method for determination of butenolides in extracts gave full resolution for 1 and 2. The analytical curve in the range 10.0-50.0 µg/mL (r(2) = 0.999) provided LOD and LOQ for 1 and 2 of 2.1/6.3 and 1.1/3.5 µg/mL, respectively. The RSD for migration times were 0.12 and 1.0% for peak area ratios with 100.0 ± 1.4% of recovery. CONCLUSIONS: A novel high-performance MEKC method developed for the analysis of butenolides 1 and 2 in leaf extracts of P. malacophyllum allowed their quantitative determined within an analysis time shorter than 5 min and the results indicated CE to be a feasible analytical technique for the quantitative determination of butenolides in Piper extracts.


Assuntos
4-Butirolactona/análogos & derivados , Piper/química , 4-Butirolactona/análise , Cromatografia Líquida de Alta Pressão , Cromatografia Capilar Eletrocinética Micelar , Indicadores e Reagentes , Folhas de Planta/química , Padrões de Referência , Reprodutibilidade dos Testes , Soluções
20.
J Pharm Biomed Anal ; 51(2): 430-8, 2010 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-19545963

RESUMO

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 3(2) 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 microg mL(-1), R(2)>0.999; LOD and LOQ from 1.32 to 3.80 microg mL(-1) and from 4.01 to 11.5 microg 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 microg g(-1) were found, with smaller amounts occurring as free phenolic acids.


Assuntos
Eletroforese Capilar/métodos , Frutas/química , Hidroxibenzoatos/análise , Boratos/análise , Brasil , Tampões (Química) , Eletrólitos/análise , Eletroforese Capilar/instrumentação , Concentração de Íons de Hidrogênio , Hidroxibenzoatos/química , Metanol/análise , Estrutura Molecular , Extratos Vegetais/análise , Extratos Vegetais/química , Padrões de Referência , Reprodutibilidade dos Testes , Solventes/análise , Fatores de Tempo , Água/química
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