Chemical identification of chlorophenylpiperazine in seized tablets

Designer drug is a term used to describe psychoactive drugs of abuse which are usually synthesized by modifying the molecular structures of existing drugs of abuse. The term gained widespread popularity when MDMA (ecstasy) experienced a popularity boom in the mid 1980´s. In Brazil, designer drugs seizures have increased in the last few years, and actually tablets with unknown psychoactive compounds began to be forwarded to the Forensic Laboratories. This work describes the analytical assays that were performed to identify the chlorophenylpiperazine, a psychoactive substance first time identified in seized tablets in Sao Paulo state

Chemical identification of chlorophenylpiperazine in seized tablets

sem informação333725729Designer drug is a term used to describe psychoactive drugs of abuse which are usually synthesized by modifying the molecular structures of existing drugs of abuse. The term gained widespread popularity when MDMA (ecstasy) experienced a popularity boom in the mid 1980´s. In Brazil, designer drugs seizures have increased in the last few years, and actually tablets with unknown psychoactive compounds began to be forwarded to the Forensic Laboratories. This work describes the analytical assays that were performed to identify the chlorophenylpiperazine, a psychoactive substance first time identified in seized tablets in Sao Paulo stat

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

A simple and fast capillary zone electrophoresis (CZE) method has been developed and validated for quantification of a non-nucleoside reverse transcriptase inhibitor (NNRTI) nevirapine, in pharmaceuticals. The analysis was optimized using 10 mmol L-1 sodium phosphate buffer pH 2.5, +25 kV applied voltage, hydrodynamic injection 0.5 psi for 5 s and direct UV detection at 200 µm. Diazepam (50.0 µg mL-1) was used as internal standard. Under these conditions, nevirapine was analyzed in approximately less than 2.5 min. The analytical curve presented a coefficient of correlation of 0.9994. Limits of detection and quantification were 1.4 µg mL-1 and 4.3 µg mL-1, respectively. Intra- and inter-day precision expressed as relative standard deviations were 1.4% and 1.3%, respectively and the mean recovery was 100.81%. The active pharmaceutical ingredient was subjected to hydrolysis (acid, basic and neutral) and oxidative stress conditions. No interference of degradation products and tablet excipients were observed. This method showed to be rapid, simple, precise, accurate and economical for determination of nevirapine in pharmaceuticals and it is suitable for routine quality control analysis since CE offers benefits in terms of quicker method development and significantly reduced operating costs.Um método simples e rápido por eletroforese capilar foi desenvolvido e validado para a quantificação do inibidor não nucleosídeo da transcritase reversa (NNRTI) nevirapina, em medicamentos. A análise foi otimizada utilizando tampão fosfato de sódio 10 mmol L-1, pH 2,5, voltagem aplicada de +25 kV, injeção hidrodinâmica a 0,5 psi por 5 s e detecção UV em 200 µm. Diazepam (50,0 µg mL-1) foi usado como padrão interno. Sob estas condições, nevirapina foi analisada em aproximadamente menos de 2,5 min. A curva analítica apresentou um coeficiente de correlação de 0,9994. Os limites de detecção e quantificação foram 1,4 µg mL-1 e 4,3 µg mL-1, respectivamente. Precisões intra e inter-dia expressas como desvio padrão relativo foram 1,4% e 1,3%, respectivamente e a recuperação média foi de 100,81%. O fármaco foi submetido a testes de hidrólises (ácida, básica e neutra) e a estresse oxidativo. Não foi observada interferência por parte dos produtos de degradação, nem dos excipients na análise da nevirapina. Este método mostrou ser rápido, simples, preciso, exato e econômico para a determinação de nevirapina em produtos farmacêuticos e é apropriado para o controle de qualidade em análise de rotina uma vez que a eletroforese capilar oferece benefícios em termos de desenvolvimento rápido dos métodos e custos muito reduzidos de operação.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Development of alternative methods for the quality control of antiretroviral drugs by capillary electrophoresis

Atualmente cerca de 38,6 milhões de pessoas estão infectadas pelo vírus da síndrome da imunodeficiência adquirida (AIDS) em todo mundo. O único modo de tratamento para esta doença é através da utilização de medicamentos responsáveis por atuarem em diferentes pontos do ciclo replicativo do vírus. No Brasil esta doença é tratada como de calamidade pública, sendo seu tratamento feito através do programa nacional DST/AIDS, o qual distribui gratuitamente os medicamentos necessários para o tratamento. Tendo em vista que vários desses medicamentos são formulados pela indústria local, esta dissertação tem como objetivo o desenvolvimento de métodos analíticos passiveis de aplicação na rotina farmacêutica para a qualificação de matérias primas, bem como o controle de qualidade dos produtos acabados. As determinações nas formulações de nevirapina e lamivudina foram realizadas por CZE, em eletrólitos ácidos com pH < 2,5. Para a lamivudina a determinação foi realizada em um eletrólito de 0,5 % de TEA, 20 mmol.L-1 de TRIS, pH = 2,5, ajustado com ácido fosfórico, com um tempo de análise inferior a 4 minutos. O método desenvolvido para a nevirapina foi conduzido em um eletrólito de 10 mmol.L-1 de fosfato de sódio (pH =2,5), com um tempo de análise de 3 minutos. Um outro método foi desenvolvido permitindo a determinação de efavirenz, estavudina e ritonavir por MEKC, utilizando-se um planejamento fatorial 23 com ponto central, um tempo inferior a 9 minutos em um eletrólito consituído de 20 mmol.L-1 de tetraborato de sódio, 20 mmol.L-1 de SDS e 30 % de acetonitrila. Os métodos desenvolvidos foram validados de acordo com os protocolos oficiais, mostrando que estes métodos apresentam características adequadas para a análise de formulações farmacêuticas. Outra abordagem foi feita utilizando o acoplamento da eletroforese capilar à espectrometria de massas, onde amostras de urina fortificadas foram analisadas. As análise foram conduzidas utilizando 400 mmol.L-1 de ácido fórmico e líquido auxiliar constituído de 0,5 % de ácido fórmico diluído com uma solução metanol:água (1:1), permitindo a identificação inequívoca dos fármacos.There are approximately 38.6 million people infected by the immunodeficiency acquired virus (AIDS) over the world. The only way of treatment for this illness is administrating drugs that act in different points of the replicative cycle of the virus. In Brazil this illness is dealt as public calamity, being its treatment made through the national program DST/AIDS, which distributes free of charge necessary medicines for the treatment. Considering that many of these drugs are formulated by the local industries, this thesis has as objective the development of analytical methods to be applied in the pharmaceutical routine for the qualification of raw materials, as well as the quality control of the finished products. The analysis of drug formulations of nevirapine and lamivudine were carried by CZE, in acid electrolytes with pH < 2.5. For lamivudine the analysis was carried using an electrolyte composed of 0.5 % of TEA, 20 mmol.L-1 of TRIS, pH = 2.5, adjusted with phosphoric acid, with an analysis time less than 4 minutes. The method developed for the nevirapine, was lead in an electrolyte composed of 10 mmol.L-1 of phosphate buffer (pH = 2.5), with a time of analysis of 3 minutes. Another method was developed for efavirenz, estavudine and ritonavir by MEKC, using 23 a factorial design with central point, with an analysis time less then 9 minutes in an electrolyte of 20 mmol.L-1 of sodium tetraborate, 20 mmol.L-1 sodium dodecyl sulfate and 30 % acetonitrile. The developed methods were validated in accordance with official protocols, showing that these methods can be advantageously used in the analysis of pharmaceutical formulations. Another approach was to use the coupling of capillary electrophoresis with mass spectrometry, where fortified samples of urine had been analyzed. The analysis were performed using 400 mmol.L-1 formic acid and liquid sheath consisting of 0.5 % of formic acid diluted with a solution of (1:1) methanol:water, allowing the unequivocal identification detection of the drugs in the samples

Simple method for determination of cocaine and main metabolites in urine by CE coupled to MS

in this work, a simple method for the simultaneous determination of cocaine (COC) and five COC metabolites (benzoylecgonine, cocaethylene (CET), anhydroecgonine, anhydroecgonine methyl ester and ecgonine methyl ester) in human urine using CE coupled to MS via electrospray ionization (CE-ESI-MS) was developed and validated. Formic acid at 1 mol/L concentration was used as electrolyte whereas formic acid at 0.05 mol/L concentration in 1:1 methanol:water composed the coaxial sheath liquid at the ESI nozzle. The developed method presented good linearity in the dynamic range from 250 ng/mL to 5000 ng/mL (coefficient of determination greater than 0.98 for all compounds). LODs (signal-to-noise ratio of 3) were 100 ng/mL for COC and CET and 250 ng/mL for the other studied metabolites whereas LOQ`s (signal-to-noise ratio of 10) were 250 ng/mL for COC and CET and 500 ng/mL for all other compounds. Intra-day precision and recovery tests estimated at three different concentration levels (500, 1500 and 5000 ng/mL) provided RSD lower than 10% (except anhydroecgonine, 18% RSD) and recoveries from 83-109% for all analytes. The method was successfully applied to real cases. For the positive urine samples, the presence of COC and its` metabolites was further confirmed by MS/MS experiments

Acetyl Radical Production by the Methylglyoxal-Peroxynitrite System: A Possible Route for L-Lysine Acetylation

Methylglyoxal is an a-oxoaldehyde putatively produced in excess from triose phosphates, aminoacetone, and acetone in some disorders, particularly in diabetes. Here, we investigate the nucleophilic addition of ONOO(-), known as a potent oxidant and nucleophile, to methylglyoxal, yielding an acetyl radical intermediate and ultimately formate and acetate ions. The rate of ONOO(-) decay in the presence of methylglyoxal [k(2,app) = (1.0 +/- 0.1) x 10(3) M(-1) s(-1); k(2) approximate to 1.0 x 10(5) M(-1) s(-1)] at pH 7.2 and 25 degrees C was found to be faster than that reported with monocarbonyl substrates (k(2) < 10(3) M(-1) diacetyl (k(2) = 1.0 x 10(4) M(-1) s(-1)), or CO(2) (k(2) = 3-6 x 10(4) M(-1) s(-1)). The pH profile of the methylglyoxal peroxynitrite reaction describes an ascendant curve with an inflection around pH 7.2, which roughly coincides with the pK(a) values of both ONOOH and H(2)PO(4)(-) ion. Electron paramagnetic resonance spin trapping experiments with 2-methyl-2-nitrosopropane revealed concentration-dependent formation of an adduct that can be attributed to 2-methyl-2-nitrosopropane-CH(3)CO(center dot) (a(N) = 0.83 mT). Spin trapping with 3,5-dibromo-4-nitrosobenzene sulfonate gave a signal that could be assigned to a methyl radical adduct [a(N) = 1.41 mT; a(H) = 1.35 mT; a(H(m)) = 0.08 mT]. The 2-methyl-2-nitrosopropane-CH(3)CO(center dot) adduct could also be observed by replacement of ONOO(-) with H(2)O(2), although at much lower yields. Acetyl radicals could be also trapped by added L-lysine as indicated by the presence of W-acetyl-L-lysine in the spent reaction mixture. This raises the hypothesis that ONOO(-)/H(2)O(2) in the presence of methylglyoxal is endowed with the potential to acetylate proteins in post-translational processes.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Instituto Nacional de Ciencia e Tecnologia (INCT) RedoxomaInstituto Nacional de Ciencia e Tecnologia (INCT) Redoxom

cAMP signaling pathway controls glycogen metabolism in Neurospora crassa by regulating the glycogen synthase gene expression and phosphorylation

The cAMP-PKA signaling pathway plays an important role in many biological processes including glycogen metabolism. In this work we investigated its role in the Neurospora crassa glycogen metabolism control using mutant strains affected in components of the pathway, the cr-1 strain deficient in adenylyl cyclase activity therefore has the PKA pathway not active, and the mcb strain a temperature-sensitive mutant defective in the regulatory subunit of PKA therefore is a strain with constitutively active PKA. We analyzed the expression of the gene encoding glycogen synthase (gsn), the regulatory enzyme in glycogen synthesis as a potential target of the regulation. The cr-1 strain accumulated, during vegetative growth, glycogen levels much higher than the wild type strain indicating a role of the PKA pathway in the glycogen accumulation. The gsn transcript was not increased in this strain but the GSN protein was less phosphorylated "in vitro", and therefore more active, suggesting that the post-translational modification of GSN is likely the main mechanism controlling glycogen accumulation during vegetative growth. Heat shock down-regulates gsn gene transcription in the two mutant strains, as well as in the wild type strain, suggesting that the PKA pathway may not be the only pathway having a direct role in gsn transcription under heat shock. DNA-protein complexes were formed between the STRE motif in the gsn promoter and nuclear proteins from heat-shocked mycelium. However STRE was not able to induce transcription of a reporter gene in Saccharomyces cerevisiae, suggesting that the motif might be involved in a different way of regulation in the N. crassa gene expression under heat shock. The CRE-like DNA elements present in the gsn promoter were shown to be bound by different proteins from the PKA mutant strains. The DNA-protein complexes were observed with proteins from the strains grown under normal condition and under heat shock indicating the functionality of this DNA element. In this work we presented some evidences that the PKA signaling pathway regulates glycogen metabolism in N. crassa in a different way when compared to the well-characterized model of regulation existent in S. cerevisiae. (C) 2009 Elsevier B.V. All rights reserved.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq