Determination of levetiracetam in human plasma by dispersive liquid-liquid microextraction followed by gas chromatography-mass spectrometry

Levetiracetam (LEV) is an antiepileptic drug that is clinically effective in generalized and partial epilepsy syndromes. The use of this drug has been increasing in clinical practice and intra- or -interindividual variability has been exhibited for special population. For this reason, bioanalytical methods are required for drug monitoring in biological matrices. So this work presents a dispersive liquid-liquid microextraction method followed by gas chromatography-mass spectrometry (DLLME-GC-MS) for LEV quantification in human plasma. However, due to the matrix complexity a previous purification step is required. Unlike other pretreatment techniques presented in the literature, for the first time, a procedure employing ultrafiltration tubes Amicon (R) (10 kDa porous size) without organic solvent consumption was developed. GC-MS analyses were carried out using a linear temperature program, capillary fused silica column, and helium as the carrier gas. DLLME optimized parameters were type and volume of extraction and dispersing solvents, salt addition, and vortex agitation time. Under chosen parameters (extraction solvent: chloroform, 130 mu Ldispersing solvent: isopropyl alcohol, 400 mu Lno salt addition and no vortex agitation time), the method was completely validated and all parameters were in agreement with the literature recommendations. LEV was quantified in patient's plasma sample using less than 550 mu L of organic solvent.Sao Paulo Research Foundation (FAPESP) [2012/07210-8]Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, 14040-903 Ribeirão Preto, SP, BrazilDepartment of Exact and Earth Sciences, Institute of Environmental, Chemical and Pharmaceutical Sciences, Federal University of São Paulo, 09972-270 Diadema, SP, BrazilDepartament of Chemistry, Faculty of Philosophy, Sciences and Letters of Ribeirão Preto, University of São Paulo, 14040-901 Ribeirão Preto, SP, BrazilDepartment of Clinical Analysis, Toxicology and Food Science, Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, 14040-903 Ribeirão Preto, SP, BrazilDepartment of Exact and Earth Sciences, Institute of Environmental, Chemical and Pharmaceutical Sciences, Federal University of São Paulo, 09972-270 Diadema, SP, BrazilFAPESP: 2012/07210-8Web of Scienc

Avaliação da correlação dose / concentração plasmática da doxiciclina com evolução clínica, alterações hematológicas e plaquetárias em cães portadores de Erliquiose Canina / Evaluation of the dose / plasma correlation of doxycycline with clinical evolution, hematological and platelet changes in dogs with Canine Ehrlichiosis

A erliquiose é uma doença causada por bactérias do gênero Ehrlichia. A transmissão ocorre pelo vetor Rhipicephalus sanguineous. A doença manifesta-se na fase aguda, subaguda ou crônica e as manifestações clínicas e alterações hematológicas variam de acordo com a fase em que os animais se encontram. O presente estudo teve como objetivo a avaliação em parâmetros hematológicos e plaquetários e correlaciona-los com doses de 5, 7,5 e 10,0mg/kg de doxiciclina administrados em cães portadores de erliquiose. Ainda avaliou-se a concentração plasmática dos animais por UPLC-MS/MS. Com os resultados, observou-se que o método analítico demonstrou-se aplicável para a quantificação em questão. Dentre os parâmetros avaliados, somente as plaquetas demonstraram a elevação quando se comparou o valor do primeiro dia, com o do 28º dia. As concentrações plasmáticas demonstraram-se dentro de valores similares, porém, na dose de 10mg/kg uma diferença entre as concentrações dos animais foi constatada. Embora nas mesmas não tenha se observado uma proporcionalidade entre dose e concentração plasmática, sugere-se que a dose de 7,5mg/kg talvez seja a mais aplicável pelo fato da mesma não ter propiciado variações tão frequentes entre as concentrações detectadas nos animais. Ressalta-se ainda, a importância de estudos clínicos que visem correlacionar a dose administrada com concentração plasmática, o que induz a compreensão de que há um número significativo de variáveis que precisam ser consideradas, e que de forma frequente e acentuada interferem na terapêutica medicamentosa

Cyclam ''capa' POT.4' to ''capa' POT.3' denticity change upon mono-N-substitution with a carboxypropyl pendant arm in a ruthenium nitrosyl complex

The complex fac-[Ru(NO)Cl2(kappa(3)N(4),N(8),N(11)(1-carboxypropyl)cyclam)]Cl.H2O (1-carboxypropyl)cyclam=3-(1,4,8,11-tetraazacyclotetradecan-1-yl)propionic acid) was prepared in a one pot reaction by mixing equimolar amounts of RuNOCl 3 and (1-carboxypropyl)cyclam and was characterized by X-ray crystallography, electrospray ionization tandem mass spectrometry (ESI-MS/MS), elemental analysis, NMR, and electronic and vibrational (IR) spectroscopies. fac-[Ru(NO)Cl 2(kappa(3)N(4),N(8),N(11)(1-carboxypropyl)cyclam)]Cl.H2O crystallizes in the triclinic, space group P1, No. 2, with unit cell parameters of a=8.501(1) A, b=9.157(1) A, c=14.200(1) A, alpha=72.564(5) degrees , beta=82.512(5) degrees , gamma=80.308(5) degrees , and Z=2. The Ru-N interatomic distance and bond angle in the [Ru-NO] unit are 1.739(2) A and 167.7(2) degrees , respectively. ESI-MS/MS shows characteristic dissociation chemistry that initiates by HCl or NO loss. The IR spectrum displays a nu(NO) at 1881 cm(-1) indicating a nitrosonium character. The electronic spectrum shows absorptions bands at 264 nm (log epsilon=3.27), 404 nm (log epsilon=2.53), and 532 nm (log epsilon=1.88). (1)H and (13)C NMR are in agreement with the proposed molecular structure, which shows a very singular architecture where the cyclam ring N (with the carboxypropyl pendant arm) is not coordinated to the ruthenium resulting in a kappa(3) instead of the expected kappa(4) denticity.FAPESPCNPqCAPE

Padrão dose-resposta de extratos brutos produzidos por actinobactérias sobre a fermentação ruminal in vitro

As actinobactérias têm sido pesquisadas como fonte produtoras de extratos brutos que contêm compostos bioativos capazes de atuar como agentes antimicrobianos. O presente trabalho investigou o efeito dose-resposta de dois extratos brutos, AMC e Caat, na fermentação ruminal in vitro por: produção cumulativa de gás, digestibilidade in vitro da matéria seca (IVDMD) e matériaorgânica (IVOMD) e concentração de ácidos graxos de cadeia curta (SCFA). Três vacas leiteiras da raça Holandesa, multíparas e portadoras de fístula ruminal foram utilizadas como doadoras de inóculo ruminal e foram alimentadas com uma dieta basal composta por silagem de milho, farelo de soja, ureia, milho moído e suplemento mineral. As amostras de inóculo ruminal foram incubadas em garrafas de vidro com 1 g da dieta seca e moída, solução tampão e os extratos brutos avaliados em quatro doses (0,3, 0,6, 0,9 e 1,20 mg/10 mL de inóculo) em delineamento em blocos casualizados, sendo as doadoras consideradas os blocos como efeito aleatório. Além disso, foram utilizados controles negativos para a correção da produção de gás. Os resultados foram expressos como valores médios com base em análises triplicadas. A diminuição da produção cumulativa de gás foi observada de acordo com a dose em resposta linear às 24, 48 e 72 h de incubação com a adição de extrato de Caat. A IVOMD mostrou uma diminuição linear com 72 h de incubação com inclusão de Caat. Além disso, a inclusão do Caat reduziu linearmente as concentrações de ácido butírico e isovalérico, bem como a proporção de acetato/propionato. Diferentemente, a inclusão do extrato de AMC não afetou nenhuma das variáveis analisadas nas doses utilizadas. O extrato de Caat pode ser usado como um modulador da fermentação ruminal in vitro, uma vez que reduziu a proporção de acetato/propionato e a produção de gás acumulada.Actinobacteria have been researched as a source that produces crude extracts, which contain bioactive compounds able to act as antimicrobial agents. The present investigation evaluated the dose-response effect of two crude extracts, obtained at Caatinga rhizosphere (Caat) and Rhizophora mangle (AMC), on in vitro ruminal fermentation by: cumulative gas production, digestibility of dry (IVDMD) and organic matter (IVOMD), and short-chain fatty acids concentration (SCFA). Three multiparous Holstein dairy cows with ruminal fistula were used as the inoculum donors and fed a basal diet consisting of corn silage, soybean meal, urea, ground corn and mineral supplement. Ruminal fluid samples were incubated in glass bottles with 1 g of the dried and milled diet, a buffer solution, and the crude extracts evaluated in four doses (0.3, 0.6, 0.9 and 1.20 mg/10 mL inoculum) in a randomized block design, and the donators were considered as blocks with random effects. Additionally, negative controls were used. The results were expressed as average values based on triplicate analyses. Decreased cumulative gas production was observed according to linear dose response at 24, 48 and 72 h of incubation with the addition of Caat extract. The IVOMD showed a linear decrease at 72 h of incubation with dose Caat inclusion. Furthermore, the inclusion of Caat extract linearly reduced butyric and isovaleric acid concentrations, as well as acetate:propionate ratio. Finally, the Caat inclusion increased the propionic acid concentration in comparison to AMC extract. However, the inclusion of AMC extract did not affect any of the analyzed variables at the used doses. The Caat extract could be used as a modulator of in vitro ruminal fermentation, since it reduced acetate:propionate ratio and cumulative gas production

Composto antifúngico produzido pelo endófito de mandioca Bacillus pumilus MAIIIM4a

Na busca de novos organismos e novos metabólitos secundários, um estudo foi conduzido visando avaliar a diversidade de bactérias endofíticas de etnovariedades de mandioca cultivadas por tribos indígenas da Amazônia brasileira e também para estudar metabólitos secundários produzidos por Bacillus pumilus. Sessenta e sete bactérias endofíticas de mandioca foram identificadas através do seqüenciamento do gene 16S rRNA e por meio da análise de ácidos graxos (FAME). Essas análises revelaram que 25% de todos os endofíticos pertenciam ao gênero Bacillus. O isolado Bacillus pumilus MAIIIM4a apresentou forte ação inibitória contra os fitopatógenos Rhizoctonia solani, Pythium aphanidermatum e Sclerotium rolfsii. Os metabólitos secundários deste isolado foram extraídos do sobrenadante usando-se hexano, diclorometano e acetato de etila. Esses extratos foram utilizados nas análises de bioautografia e LC-MS, as quais permitiram a identificação do composto pumilacidina, um antifúngico produzido por B. pumilus MAIIIM4a. A localização das bactérias endofíticas foi confirmada examinando-se o tecido celular da mandioca através de microscopia eletrônica.In the search for new organisms and new secondary metabolites, a study was conducted to evaluate the diversity of endophytic bacteria from ethnovarieties of cassava cultivated by Brazilian Amazon Indian tribes and also to study the secondary metabolites produced by a Bacillus pumilus strain. Sixty seven cassava endophytic bacteria were subjected to 16S rRNA sequencing and FAME analysis. The bacterial profile revealed that 25% of all endophytic isolates belonged to the genus Bacillus. The isolate B. pumilus MAIIIM4a showed a strong inhibitory activity against the fungi Rhizoctonia solani, Pythium aphanidermatum and Sclerotium rolfsii. Secondary metabolites of this strain were extracted using hexane, dichloromethane and ethyl acetate. Extracts were subjected to bioautography and LC/MS analysis, which allowed the identification of pumilacidin, an antifungal compound produced by B. pumilus MAIIIM4a. The bacterial endophytic localization was confirmed by cassava cell tissue examination using scanning electron microscopy

Determination of Levetiracetam in Human Plasma by Dispersive Liquid-Liquid Microextraction Followed by Gas Chromatography-Mass Spectrometry

Levetiracetam (LEV) is an antiepileptic drug that is clinically effective in generalized and partial epilepsy syndromes. The use of this drug has been increasing in clinical practice and intra- or -interindividual variability has been exhibited for special population. For this reason, bioanalytical methods are required for drug monitoring in biological matrices. So this work presents a dispersive liquid-liquid microextraction method followed by gas chromatography-mass spectrometry (DLLME-GC-MS) for LEV quantification in human plasma. However, due to the matrix complexity a previous purification step is required. Unlike other pretreatment techniques presented in the literature, for the first time, a procedure employing ultrafiltration tubes Amicon® (10 kDa porous size) without organic solvent consumption was developed. GC-MS analyses were carried out using a linear temperature program, capillary fused silica column, and helium as the carrier gas. DLLME optimized parameters were type and volume of extraction and dispersing solvents, salt addition, and vortex agitation time. Under chosen parameters (extraction solvent: chloroform, 130 μL; dispersing solvent: isopropyl alcohol, 400 μL; no salt addition and no vortex agitation time), the method was completely validated and all parameters were in agreement with the literature recommendations. LEV was quantified in patient’s plasma sample using less than 550 μL of organic solvent

Utilização do B-pineno na obtenção de sintons quirais para sintese de produtos naturais

Orientador: Anita J. MarsaioliDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de QuimicaMestrad

On the solvent and counter ion-free mechanism of ketalization reactions of gaseous activated carbonyls

CNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOKetalization of aldehydes and ketones is widely used in solution to protect their reactive carbonyl groups from undesired transformations. This reaction is known to be stereospecific and usually employs a diol [HOCH2CH2OH]. In acidic media, the reaction i421170177CNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOsem informaçãosem informaçãoThe authors are grateful to the Brazilian Research Foundations CNPq and FAPESP, as well as to Federal University of Parana (UFPR) for financial suppor