Desenvolvimento de método analítico para análise de drogas de abuso utilizando a técnica de dried spot para fluido oral

A técnica de dried matrix spots (DMS) consiste em um procedimento de coleta de matrizes biológicas diretamente em papel filtro com especificações padronizadas para posterior análise das substâncias de interesse. Diante das vantagens descritas para esta técnica, novas metodologias vêm sendo desenvolvidas para aplicação em diversas áreas, dentre elas na toxicologia. Neste contexto, é de interesse o desenvolvimento de uma metodologia que utilize o fluido oral (FO) como matriz biológica, investigando a viabilidade da técnica de dried oral fluid spots (DOFS) na análise de drogas de abuso. O objetivo deste trabalho consistiu em validar método analítico quantitativo por cromatografia líquida acoplada à espectrometria de massas (CLAE-EM) para análise simultânea de cocaína, 3,4-metilenodioximetanfetamina (MDMA), anfetamina, benzoilecgonina (BZE) e cocaetileno utilizando DOFS. O FO foi aplicado em papel filtro Whatman® 903 e o tempo necessário para completa secagem foi estudado. Os solventes utilizados no processo de extração foram definidos através de abordagem quimiométrica, utilizando desenho simplex centroide para otimização. A curva de calibração foi definida cobrindo o intervalo linear de 40-500 ng/mL. Os parâmetros de validação avaliados foram: linearidade, precisão, exatidão, seletividade, efeito residual, efeitos de matriz e estabilidade. Além disso, foram investigadas a homogeneidade e a influência do volume de amostra aplicado no spot. O tempo de secagem ideal foi definido em 2,5 horas. O desenho quimiométrico permitiu a otimização da escolha dos solventes para a extração líquida, sendo definido o uso de acetonitrila:acetato de amônio:metanol (55:35:10 v/v) para a extração. A homocedasticidade da curva foi confirmada pelo teste de Cochran e o modelo linear proposto foi considerado adequado. Não foram observados interferentes na avaliação de seletividade e efeito residual. A avaliação da homogeneidade do spot apresentou menos de 15% de desvio do valor nominal de concentração e o volume de amostra aplicado de até 100 μL não prejudicou a exatidão, confirmando a homogênea difusão da amostra no papel. A realização deste trabalho abre precedentes ao demonstrar a viabilidade da aplicação de DOFS para a quantificação de drogas de abuso e oportuniza o surgimento de novos estudos que ampliem a utilização desta técnica em outras matrizes biológicas e novos contextos de aplicação.Dried matrix spots (DMS) consist of a sampling technique which comprises the collection of a small volume of a biological fluid onto a specific filter paper for further analysis. Due to the several advantages of the technique, DMS started to be employed for different purposes, with an increase of applications in toxicology in the last decade. In this context, it is of interest the development of a methodology using oral fluid (OF) to investigate the feasibility of dried oral fluid spots (DOFS) in drugs of abuse analysis. The aim of this work was to validate a liquid chromatography–mass spectrometry method using DOFS for the simultaneous quantification of cocaine, benzoylecgonine, cocaethylene, amphetamine, and 3,4-methylenedioxymethamphetamine. The oral fluid was applied to a Whatman® 903 grade paper and complete drying time was studied. The extraction procedure was optimized by chemometric approach using simplex centroid design. Calibration curves covered a linear concentration range of 40–500 ng/mL. Validation parameters of linearity, precision, accuracy, selectivity, carryover, matrix effects, and stability were evaluated. Besides, spot homogeneity and the influence of spot volume were also studied. The ideal drying time was set at 2.5 hours. The chemometric design allowed the optimization of liquid extraction, being defined a mixture of acetonitrile:ammonium acetate:methanol (55:35:10 v/v) as the solvent extraction. Homoscedasticity of the calibration curve was confirmed by Cochran’s test and the proposed linear model was considered adequate. No interferences were observed in the evaluation of carryover and selectivity. Spot homogeneity was considered satisfactory, with less than 15% of deviation from nominal concentration. Spot volume did not influence accuracy when less than 100 μL of the sample was applied onto the spot, confirming the homogeneous diffusion of the sample on the paper. The validation of the proposed method demonstrates the feasibility of DOFS application and set precedents for new studies that expand the use of this technique in other biological matrices and new application contexts

Gender differences in biochemical markers and oxidative stress of rats after 28 days oral exposure to a mixture used for weight loss containing p-synephrine, ephedrine, salicin, and caffeine

A associação de p-sinefrina, efedrina, salicina, e cafeína em suplementos alimentares e produtos para perda de peso é muito utilizada em todo o mundo, embora a efedrina tenha sido proibida em muitos países. O objetivo deste estudo foi avaliar o perfil de toxicidade à exposição oral de 28 dias à associação de p-sinefrina, efedrina, salicina e cafeína (na proporção de 10:4:6:80 m/m respectivamente) em ratos Wistar machos e fêmeas. Diariamente, os animais foram observados quanto ao peso corporal, sinais de toxicidade, morbidade e mortalidade. Após 28 dias, os animais foram sacrificados e o sangue coletado para avaliações hematológicas, bioquímicas e de estresse oxidativo. Não se observaram sinais clínicos de toxicidade, tampouco perda significativa de peso, mortes, ou quaisquer alterações significativas nos parâmetros hematológicos. Biomarcadores do estresse oxidativo e bioquímicos mostraram peroxidação lipídica, danos renais e hepáticos (p < 0,05; ANOVA/Bonferroni) em ratos machos (100 e 150 mg/kg) e a redução (p < 0,05; ANOVA/Bonferroni) nos níveis de glutationa reduzida (GSH) em todos os grupos de machos tratados. Nas fêmeas, não houve indícios de estresse oxidativo, nem alterações bioquímicas. O diferente perfil de toxicidade entre os gêneros sugere influência hormonal nos efeitos de mistura administrada. A associação testada pode alterar o estado oxidativo e promover danos renais e hepáticos.The association of p-synephrine, ephedrine, salicin, and caffeine in dietary supplements and weight loss products is very common worldwide, even though ephedrine has been prohibited in many countries. The aim of this study was to evaluate a 28-day oral exposure toxicity profile of p-synephrine, ephedrine, salicin, and caffeine mixture (10:4:6:80 w/w respectively) in male and female Wistar rats. Body weight and signs of toxicity, morbidity, and mortality were observed daily. After 28 days, animals were euthanized and blood collected for hematological, biochemical, and oxidative stress evaluation. No clinical signs of toxicity, significant weight loss or deaths occurred, nor were there any significant alterations in hematological parameters. Biochemical and oxidative stress biomarkers showed lipid peroxidation, and hepatic and renal damage (p < 0.05; ANOVA/Bonferroni) in male rats (100 and 150 mg/kg) and a reduction (p < 0.05; ANOVA/Bonferroni) in glutathione (GSH) levels in all male groups. Female groups displayed no indications of oxidative stress or biochemical alterations. The different toxicity profile displayed by male and female rats suggests a hormonal influence on mixture effects. Results demonstrated that the tested mixture can alter oxidative status and promote renal and hepatic damages

Multianalytical Method Validation for Qualitative and Quantitative Analysis of Solvents of Abuse in Oral Fluid by HS-GC/MS

The use of oral fluid as a biological matrix to monitor the use of drugs of abuse is a global trend because it presents several advantages and good correlation to the blood level. Thus, the present work aimed to develop and validate an analytical method for quantification and detection of solvents used as inhalants of abuse in oral fluid (OF), using Quantisal™ as collector device by headspace and gas chromatography coupled with a mass detector (HS-GC/MS). Chromatographic separation was performed with a ZB-BAC1 column and the total time of analysis was 11.8 min. The method showed good linearity (correlation coefficient higher than 0.99 for all solvents). The limits of detection ranged from 0.05 to 5 mg/L, while the lower limits of quantification ranged from 2.5 to 12.5 mg/L. Accuracy, precision, matrix effect, and residual effect presented satisfactory results, meeting the criteria accepted for the validation of bioanalytical methods. The method showed good selectivity considering that, for solvents coeluting at the same retention time, resolution was performed by the mass detector. The method developed proved to be adequate when applied in OF samples from users of drugs and may be used to monitor the abuse of inhalants in routine forensic analyses

Estudo do conteúdo de sinefrina em frutos de espécies de Citrus

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Estudo do conteúdo de sinefrina em frutos de espécies de Citrus

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Optimization and validation of a method for simultaneous analysis of organic solvents in oral fluid collected by Quantisal™

The development of analytical methods is required for the biological monitoring of occupational exposure. Considering that oral fluid (OF) is a promising matrix, the aim of this study was to optimize the headspace extraction by using the central composite design and to validate a methodology for the simultaneous analysis of ethyl ether, dichloromethane and ethyl acetate in OF by headspace-gas chromatography with mass spectrometry detector. The best condition chosen for the headspace analysis was 85°C for 5 minutes. The method was satisfactorily validated, making it adequate for use in toxicological analysis of occupational exposure to organic solvents