Desenvolvimento e aplicação de método bioanalítico para detecção de biomarcadores do consumo de cocaína e crack em amostras de colostro e mecônio através de LC-MS

O abuso de drogas durante a gestação e/ou amamentação é uma preocupação constante das equipes de saúde, pois pode causar inúmeros efeitos adversos aos recém-nascidos. O desenvolvimento de métodos analíticos para detectar drogas de abuso em amostras de colostro (primeiro estágio do leite materno) e mecônio (primeiras fezes do recém-nascido) tem grande relevância, pois permite o monitoramento e o correto tratamento dos bebês expostos e também das usuárias. Método de cromatografia líquida acoplado ao espectrômetro de massas (LC-MS) foi desenvolvido e validado para a determinação de biomarcadores do uso de cloridrato de cocaína e de sua forma fumada “crack” (cocaína base) em amostras de colostro e mecônio. A cocaína (COC) e seu metabólito benzoilecgonina (BZE), os produtos de pirólise éster metilanidroecgonina (AEME) e anidroecgonina (AEC) foram analisados após um simples procedimento de precipitação de proteínas e centrifugação utilizando atropina (ATP) como padrão interno (PI). Aplicando estudo de quimiometria, todos os picos foram separados em condição isocrática por 12 minutos através de uma coluna para compostos polares Kinetex HILIC a 30 °C. Um íon foi utilizado para a quantificação e três íons para a confirmação de cada analito. O método foi linear para todos os analitos no intervalo de concentração de 5 – 300 ng/mL com coeficientes de correlação (r) entre 0,9983 – 0,9996 para as amostras de colostro e no intervalo de 15 – 500 ng/mg com r entre 0,9971 – 0,9986 para as amostras de mecônio. O limite inferior de quantificação (LIQ) foi de 5 ng/mL para o colostro e 15 ng/mg para o mecônio, com parâmetros de validação dentro do preconizado. O efeito matriz foi avaliado e apresentou resultados adequados, demonstrando que ambos os procedimentos de limpeza das amostras são rápidos e confiáveis, exigindo pequenas quantidades de solvente orgânico. O método por LC- MS é mais rápido e barato quando comparado com outros disponíveis na literatura e também foi aplicado com sucesso para avaliar amostras reais de colostro e mecônio.Development and application of bioanalytical method for detection of cocaine and crack use biomarkers in colostrum and meconium samples by LC-MS. Drug abuse during pregnancy and/or breastfeeding is an ongoing concern of health professionals, because it can cause several adverse effects to newborns. The development of analytical methods to detect drugs of abuse in colostrum (first stage of breast milk) and meconium (first stool of the new born) has great relevance, because it allows the monitoring and the appropriate treatment of the exposed newborns and mothers. A liquid chromatography coupled to mass spectrometry (LC- MS) method was developed and validated for the determination of cocaine hydrochloride and its smoked form “crack” (cocaine base) biomarkers in samples of colostrum and meconium. Cocaine (COC) and its metabolite benzoylecgonine (BZE), its pyrolytic products anhydroecgonine methyl ester (AEME) and anhydroecgonine (AEC), were analyzed after a simple protein precipitation and centrifugation procedure using atropine (ATP) as internal standard (IS). Applying a chemometric approach study, all the peaks were separated at isocratic conditions in an 12 minutes run using a column for polar compounds Kinetex HILIC at 30 °C. One ion was used for quantification and three ions for confirmation of each analyte. The method was linear for all analytes within the concentration range of 5 – 300 ng/mL with correlation coefficients (r) between 0.9983 – 0.9996 for colostrum samples and within 15 – 500 ng/mg with r between 0.9971 – 0.9986 for meconium samples. Lower limit of quantification (LLOQ) was 5 ng/mL for colostrum and 15 ng/mg for meconium, with validation parameters within recommended values. Matrix effect was evaluated and showed adequate results, demonstrating that both sample cleaning procedures are fast and reliable, requiring small amounts of organic solvent. The LC-MS method is faster and cheaper compared to others available in the literature and it has also been successfully applied to assess real samples of colostrum and meconium

Main degradation products of dabigatran etexilate evaluated by LC-UV and LC-ESI-MS, degradation kinetics and in vitro cytotoxicity studies

The present study reports the stability profile of an antithrombotic drug: dabigatran etexilate (DAB). The drug was subjected to thermal degradation at 60 °C and products formed were investigated by liquid chromatography-UV (LC-UV) and liquid chromatography-mass spectrometry (LC-ESI-MS). Chromatographic separation of the degradation products was performed on a GL Sciences Inc. Inertsil ODS-2 column (250 mm × 4.6 mm i.d., with a particle size of 5 µm and pore size of 110 Å) with mobile phase consisting of acetonitrile and ammonium acetate buffer (pH 5.5; 10 mmol L−1 ) (65:35, v/v) pumped at 1.0 mL min−1 flow rate. Column temperature was set at 30 °C and detection at 225 nm using a UV detector. LC-UV method previously validated was extended to LC-ESI-MS for the characterization of the degradation products (DP-01 and DP-02) formed, without complicated isolation or purification processes, based on retention times and confirmation of molecular weight. Degradation kinetics of DAB was also evaluated and could be described as a first-order process (R2 = 0.9900). Furthermore, no evidence of cytotoxicity in human mononuclear cells was observed for DAB degraded samples

Determination of phenobarbital in human plasma by a specific liquid chromatography method: application to a bioequivalence study

A liquid chromatography method was developed and validated for the determination of phenobarbital in human plasma using phenytoin as internal standard. The drugs were extracted from plasma by liquid-liquid extraction and separated isocratically on a C12 analytical column, maintained at 35 ºC, with water:acetonitrile:methanol (58.8:15.2:26, v/v/v) as mobile phase, run at a flow rate of 1.2 mL/min with detection at 205 nm. The method was linear in the range of 0.1-4 μg/mL (r²=0.9999) and demonstrated acceptable results for the precision, accuracy and stability studies. The method was successfully applied for the bioequivalence study of two tablet formulations (test and reference) of phenobarbital 100 mg after single oral dose administration to healthy human volunteers

Avaliação de pirogênios em produtos de uso veterinário pelos testes da hipertermia em coelhos e do lisado de amebócitos do Limulus Pyrogens in veterinary products by the rabbit pyrogen test and the Limulus amoebocyte lysate test

Realizou-se a avaliação de pirogênios em produtos veterinários de uso parenteral, pelo método da hipertermia em coelhos, calculando-se, para o teste das amostras, doses com concentrações de três a sete vezes superiores à terapêutica. Preconizou-se como resposta positiva o aumento de temperatura de 0,6°C. Utilizou-se também o ensaio do lisado de amebócitos do Limulus (LAL) por geleificação, semiquantitativo, executando o teste de interferentes, validando o procedimento e estabelecendo a máxima diluição válida para a análise de cada produto. Paralelamente, efetuou-se avaliação comparativa de amostras com o método do LAL cromogênico, quantitativo, demonstrando correlação e reprodutibilidade dos resultados. Avaliaram-se vinte e oito produtos de diferentes classes farmacológicas, observando-se que dois não cumpriram as especificações, sendo reprovados. Sugere-se que as especificações estudadas sejam adotadas, contribuindo para aprimorar o controle de contaminantes, garantindo a qualidade e a segurança dos produtos veterinários.The rabbit pyrogen test was used to evaluate veterinary products, suggesting the temperature rise of 0.6°C as the ending point for the positive results. The test doses were calculated based on the therapeutic dose increased from three to seven times. The semi-quantitative Limulus amoebocyte lysate (LAL) gel clot test was performed and compared to the LAL spectrophotometric chromogenic, quantitative assay. The comparative evaluation of the samples showed correlation and reproducibility of the results. The interference test was carried out, the procedure validated and the maximum valid dilution established for the analysis of the products without Pharmacopoeial specifications. Two of the twenty-eight products of different pharmacological groups evaluated didn't meet the requirements and were reproved. The specifications investigated are suggested to be used for the purity evaluation of the veterinary parenteral products, as a contribution to assure the quality and safety of the products