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Abstract(s)
O relatório de estágio apresentado é constituído por três capítulos. O primeiro é dedicado ao estágio curricular em Farmácia Comunitária realizado na Farmácia Holon Covilhã sob a orientação do Dr. Pedro Diamantino. O segundo capítulo descreve o estágio curricular em Farmácia Hospitalar orientado pela Dr.ª Olímpia Fonseca e realizado no Hospital Pêro da Covilhã, que faz parte do Centro Hospitalar da Cova da Beira. Em ambos os relatórios são abordados as atividades desenvolvidas, os conhecimentos adquiridos, as funções desempenhadas por cada colaborador e os aspetos legais relevantes de cada área.
O terceiro capítulo é referente à componente de investigação laboratorial, desenvolvida no Centro de Investigação em Ciências da Saúde da Universidade da Beira Interior (. O presente trabalho, intitulado como “Dried Saliva Spot: uma nova ferramenta na monitorização terapêutica de fármacos”, descreve o desenvolvimento e validação de um método analítico para a determinação de fenitoína, fenobarbital, carbamazepina e do seu metabolito ativo utilizando cromatografia líquida (HPLC) acoplada a um detetor diode array (DAD). Foi comprovada a existência de correlação entre o plasma e o fluido oral para estes compostos, tornando esta matriz um ótimo meio de monitorização não invasivo.
Para a preparação da amostra, adaptaram-se dos dispositivos utilizados em dried blood spot para fluido oral, resultando em dried saliva spot (DSS). O procedimento de extração, previamente otimizado, consistiu em aplicar 50 µL de fluido oral em papel de filtro WhatmanTM 903 protein saver, e secar por 1 hora. A extração foi feita com 1 mL de metanol acidificado (pH=5,5) e 5 minutos de agitação. Posteriormente, a amostra foi centrifugada durante 15 minutos a 3500 rpm. O sobrenadante foi evaporado e reconstituído com 80 µL de fase móvel. Para a separação dos compostos utilizou-se uma coluna Zorbax Rapid Resolution HT de 4,6x150mm;1,8 µm e uma fase móvel constituída por 35% acetonitrilo e 65% de água:metanol:trietilamina (75,6%:24,2%:0,3%) A metodologia descrita foi validada segundo normas de validação internacionais sendo os parâmetros estudados a seletividade, linearidade, limites de quantificação e deteção, precisão, exatidão, recuperação, estabilidade e fator de diluição.
O procedimento foi linear para o intervalo de concentrações de 0,1 (limite inferior de quantificação - LLOQ) até 10 µg/mL, com coeficientes de determinação (R2) superiores a 0,99 para todos os analitos. Precisões intra-e interdia foram tipicamente inferiores a 15% para todos os analitos a todas as concentrações estudadas, enquanto a exatidão se situou dentro do intervalo de ±15%. As recuperações variaram entre 40,8 % e 60,9%. O método validado mostrou ser aplicável à análise de amostras reais, sendo então uma ferramenta vantajosa no âmbito de análises de toxicologia clínica e monitorização terapêutica. Este é o primeiro método que utiliza DSS para determinação de fármacos anticonvulsivos, demonstrando ótimas perspetivas para o uso rotineiro em laboratório pela sua rápida e fácil aplicação, para além das vantagens inerentes ao uso de fluido oral enquanto amostra.
This report for the degree on master in pharmaceutical sciences is divided into three chapters. The first chapter reports the community pharmacy internship, developed at Farmácia Holon Covilhã and under the supevision of Dr. Pedro Diamantino. The second chapter describes the hospilar pharmacy internship, that was made under Dr. Olímpia Fonseca supervision at Hospital f Pêro da Covilhã, that is part of Centro Hospitalar da Cova da Beira. In both reports are mentioned the activities developed, the acquired knowledge, the responsibilities of each employee and the relevant legal aspect of each area. The third chapter approaches the laboratorial investigation component, developed at the Health Sciences Research Center from University of Beira Interior. This work, titled as “Dried Saliva Spot: a new tool in Therapeutic Drug Monitoring”, describes the development and validation of an analytic method for determination of phenytoin, phenobarbital, carbamazepine and its active metabolite using liquid chromatography (HPLC) coupled with a diode array detector (DAD). Correlation between plasma and oral fluid for these compounds has been previously reported, for this reason this matrix becomes an excellent specimen to drug monitoring. For the sample preparation, it was used the filter paper cards for blood (Dried blood spot - DBS). However, these were used with oral fluid samples, resulting in dried saliva spot (DSS). The extraction procedure, previously optimized, consisted in applying 50 µL of oral fluid in filter paper WhatmanTM 903 protein saver, dried for 1 hour. The extraction was made with 1 mL of acidified methanol (pH=5.5) and 5 minutes agitation. After that, the extract was centrifuged during 15 minutes at 3500 rpm. The supernatant was evaporated and reconstituted with 80 µL of mobile phase. For the compound separation it was used a Rapid Resolution HT column of 4.6x150mm;1.8 µm and a mobile phase constituted by 35% acetonitrile and 65% of water:methanol:triethylamine (75.6%:24.2%:0.3%) The described method was validated according to international criteria. The parameters evaluated were, selectivity, linearity, limits of quantitation and detection, precision, accuracy, recoveries, stability and dilution integrity The method shown to be linear between 0.1 (Lower limit of quantitation- LLOQ) and 10 µg/mL, with determination coefficients (R2) higher than 0.99 for all compounds. Intraday and interday precision were typically lower than 15% for all studied concentrations. The accuracy was with ± 15%. The recoveries ranged from 40.8 to 60.9 %. The validated methodology was successfully applied to authentic samples, proving to be a powerful tool for analysis in clinical toxicology and therapeutic drug monitoring. The described method is the first to use DSS for the determination of antiepileptic drugs, revealing itself as an excellent alternative to conventional methods for routine, due to the fast and user-friendly procedure with the use of a non-invasive sample.
This report for the degree on master in pharmaceutical sciences is divided into three chapters. The first chapter reports the community pharmacy internship, developed at Farmácia Holon Covilhã and under the supevision of Dr. Pedro Diamantino. The second chapter describes the hospilar pharmacy internship, that was made under Dr. Olímpia Fonseca supervision at Hospital f Pêro da Covilhã, that is part of Centro Hospitalar da Cova da Beira. In both reports are mentioned the activities developed, the acquired knowledge, the responsibilities of each employee and the relevant legal aspect of each area. The third chapter approaches the laboratorial investigation component, developed at the Health Sciences Research Center from University of Beira Interior. This work, titled as “Dried Saliva Spot: a new tool in Therapeutic Drug Monitoring”, describes the development and validation of an analytic method for determination of phenytoin, phenobarbital, carbamazepine and its active metabolite using liquid chromatography (HPLC) coupled with a diode array detector (DAD). Correlation between plasma and oral fluid for these compounds has been previously reported, for this reason this matrix becomes an excellent specimen to drug monitoring. For the sample preparation, it was used the filter paper cards for blood (Dried blood spot - DBS). However, these were used with oral fluid samples, resulting in dried saliva spot (DSS). The extraction procedure, previously optimized, consisted in applying 50 µL of oral fluid in filter paper WhatmanTM 903 protein saver, dried for 1 hour. The extraction was made with 1 mL of acidified methanol (pH=5.5) and 5 minutes agitation. After that, the extract was centrifuged during 15 minutes at 3500 rpm. The supernatant was evaporated and reconstituted with 80 µL of mobile phase. For the compound separation it was used a Rapid Resolution HT column of 4.6x150mm;1.8 µm and a mobile phase constituted by 35% acetonitrile and 65% of water:methanol:triethylamine (75.6%:24.2%:0.3%) The described method was validated according to international criteria. The parameters evaluated were, selectivity, linearity, limits of quantitation and detection, precision, accuracy, recoveries, stability and dilution integrity The method shown to be linear between 0.1 (Lower limit of quantitation- LLOQ) and 10 µg/mL, with determination coefficients (R2) higher than 0.99 for all compounds. Intraday and interday precision were typically lower than 15% for all studied concentrations. The accuracy was with ± 15%. The recoveries ranged from 40.8 to 60.9 %. The validated methodology was successfully applied to authentic samples, proving to be a powerful tool for analysis in clinical toxicology and therapeutic drug monitoring. The described method is the first to use DSS for the determination of antiepileptic drugs, revealing itself as an excellent alternative to conventional methods for routine, due to the fast and user-friendly procedure with the use of a non-invasive sample.
Description
Keywords
Antiepiléticos Dried Saliva Spots Farmácia Comunitária Farmácia Hospitalar Hplc-Dad