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- Characterization of Antidepressant Consumption in a Portuguese Inland Population.Publication . Soares, Sofia; Rosado, Tiago; Santos, Vítor Hugo; Rei, Cristina; Amantegui, Patricia; Pissarra da Costa, António; Chaves, Telma; Valente, Rita; Duarte, Fábio; Pacheco, Susana; Martins, Marco; Dias, Kátia; Costa, Patricia; Costa, Rui; Castro, Sílvia; Sousa, Diana; Figueiredo, Diana; Soares, Isabel; Mouta, Salomé; Jesus, Bianca; Pires, Ana; Ribeiro, Cândida; Lobo, Sónia; Correia, Leonor; Malés, Sofia; Vale, Fátima; Moita, Carina; Moura, Carolina; Sousa, Joana; Afonso, Luís Rafael; Costa, Rita Santinho; Barroso, Mário; Gallardo, EugeniaMental disorders are a growing global concern, with depression being among the most prevalent. Portugal ranks second in antidepressant consumption within the OECD, following a threefold increase between 2000 and 2020. In inland regions such as Beira Interior, reduced healthcare services and distance from major hospitals further complicate access to care. This study analysed 142 patients from Beira Interior undergoing antidepressant therapy to characterise their demographic and clinical profile and to assess associations with adverse effects. A cross-sectional survey collected demographic data, clinical diagnoses, prescribed antidepressants, concomitant medications, and reported adverse effects. Both descriptive and inferential statistical analyses were performed. Most participants were female (81.0%), with a mean age of 57.8 years. Major depression was the most common diagnosis (76.1%). Selective serotonin reuptake inhibitors (47.4%) and trazodone (27.8%) were the most prescribed agents. Treatment had lasted one to five years in 59.9% of cases. Concomitant use of benzodiazepines (76.8%) and antipsychotics (48.6%) was frequent. Reported adverse effects included anticholinergic symptoms (38.7%) and confusion/agitation (26.8%). Women were more likely to use serotonin modulators, while patients >64 years had higher odds of using tetracyclic/unicyclic antidepressants, serotonin modulators, and multiple antidepressants. These classes were significantly associated with increased adverse effects. The findings reveal important risks related to polypragmasia and adverse reactions, underscoring the need for individualised prescribing, rigorous monitoring, and strict adherence to guidelines. Larger, stratified, and longitudinal studies are needed to clarify causality and optimise treatment outcomes.
- Chromatographic determination of antidepressants in plasma and saliva: Towards non-invasive therapeutic monitoringPublication . Soares, Sofia; Rosendo, Luana; Fonseca, Suzana; Gonçalves, Nuno; Franco, João; da Costa, António Pissarra; Rosado, Tiago; Barroso, Mário; Santos, Vítor Hugo; Rei, Cristina; Amantegui, Patricia; Chaves, Telma; Valente, Rita; Duarte, Fábio; Pacheco, Susana; Martins, Marco; Dias, Kátia; Costa, Patricia; Costa, Rui; Castro, Sílvia; Sousa, Diana; Figueiredo, Diana; Soares, Isabel; Mouta, Salomé; Jesus, Bianca; Pires, Ana; Ribeiro, Cândida; Lobo, Sónia; Correia, Leonor; Malés, Sofia; Vale, Fátima; Moita, Carina; Moura, Carolina; Sousa, Joana; Afonso, Luís Rafael; Costa, Rita Santinho; Gallardo, EugeniaDrug monitoring of antidepressants in plasma and oral fluid represents a valuable tool in clinical practice, enabling the optimisation of treatment efficacy and the reduction of adverse effects. Given the significant interindividual variability in antidepressant response-driven by factors such as metabolism, drug-drug interactions, and adherence to therapy-drug monitoring facilitates dose adjustment based on measured drug concentrations, ensuring levels remain within the therapeutic window. This study aimed at developing and validating a robust, rapid, and sensitive method for the simultaneous quantification of 21 selected antidepressants and their metabolites in only 100 μL of plasma and oral fluid. Sample preparation was performed using a simple protein precipitation protocol, followed by analysis via liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was validated in accordance with internationally accepted bioanalytical guidelines, demonstrating linearity over the concentration range of 0.98-1000 ng/mL. Limits of quantification were established at 0.98 ng/mL for all analytes across both matrices. The extraction procedure yielded high recovery rates, and the method showed excellent selectivity, sensitivity, precision, and accuracy, confirming its suitability for routine toxicological applications. The validated method was successfully applied to 142 paired authentic plasma and oral fluid specimens from patients undergoing antidepressant therapy. Antidepressant concentrations were determined in both matrices, and treatment adherence was considered high, being confirmed in 88.7 % of patients. Correlation analysis between plasma and oral fluid concentrations produced promising results for several of the compounds under investigation, reinforcing the potential utility of oral fluid as a non-invasive alternative matrix in drug monitoring.
- Desenvolvimento e otimização de uma nova metodologia para determinação de antidepressivos em plasma com extração por MEPS e análise por GC-MS/MSPublication . Soares, Sofia; Rosado, Tiago; Barroso, Mário; Gallardo, EugeniaO consumo de antidepressivos constitui uma problemática mundial e Portugal apresenta uma das taxas de doenças mentais mais elevadas da Europa1. A monitorização terapêutica está estabelecida para um pequeno número de medicamentos para os quais se verifica uma relação direta entre concentração e efeito farmacológico. Este trabalho tem como objetivo desenvolver uma metodologia para deteção de antidepressivos (fluoxetina, venlafaxina, citalopram, sertralina, paroxetina e metabolitos) em 250 μL de plasma por microextração em seringa empacotada (MEPS) e cromatografia gasosa acoplada à espectrometria de massa em tandem (GC-MS/MS). A técnica de MEPS foi otimizada utilizando a ferramenta estatística Design of Experiments e o número de strokes foi o único fator significativo. O procedimento final de extração foi: adicionar 250 μL de amostra com 500 μL de acetonitrilo, centrifugar, decantar e evaporar; dissolver o resíduo com 1 mL de tampão fosfato 25 mM (pH 5), adicionar o padrão interno e homogeneizar; acondicionamento com 250 μL de metanol e 250 μL de 0,1% de ácido fórmico em H2O; loading de 12x150 μL de amostra; lavagem de 4x50 μL com 1% de ácido fórmico em H2O; secagem por aspiração de 4x50 μL de ar; eluição de 6x100 μL com 1% de hidróxido de amónia em metanol; evaporar até a secura, derivatizar durante 2 minutos com microondas a 800 W e injetar 2 μL no sistema cromatográfico. Foram obtidas recuperações entre 12-93% e limites de quantificação entre 10-100 ng/mL. Este é o primeiro trabalho a utilizar a MEPS e GC-MS/MS na determinação de antidepressivos e metabolitos em amostras de plasma.
- Development and optimization of a new method to determine antidepressants in oral fluid by microextraction by packed sorbent and analysis by GC-MS/MSPublication . Soares, Sofia; Rosado, Tiago; Barroso, Mário; Gallardo, EugeniaBetween 2013 and 2016, the consumption of antidepressants doubled in Portugal, with around 30 million packages of medication for depression, anxiety and other mental health problems dispensed annually. This phenomenon has even given rise to several alerts, which was highlighted in the 2017 report of the National Program for Mental Health, as Portugal has one of the highest rates of mental illness in Europe1. Therapeutic drug monitoring is well established for a small number of drugs, namely for those where a direct relationship between concentration and pharmacological effect at the site of action exists, which in turn is predictably reflected in the response. Drug concentrations in the various biological fluids are used in conjunction with other clinical observation measures to assess the patient's condition, and further support the individualization of therapy. Within this group of drugs targeted for monitoring are antidepressants. In recent years, oral fluid has gained more and more importance in the field of therapeutic drug monitoring, as a non-invasive and painless alternative to traditionally sampled specimens (e.g. blood, plasma). MEPS is a miniaturized solid-phase extraction (SPE) and therefore has a great advantage in the reduced operating volumes and consequently lower amounts of sample and organic solvents are required. As a result, MEPS decreases the time required for extraction, clean-up and concentration of analytes. This is the first work that uses MEPS as sample preparation technique for the determination of antidepressants and metabolites in oral fluid samples (250 µL). The simple extraction procedure proved to be efficient, requiring only 250 µL of biological sample, making it an excellent alternative for the determination of these compounds in routine clinical and forensic toxicology laboratories and for therapeutic monitoring purposes.
- Evaluation of Antipsychotic Drugs’ Stability in Oral Fluid SamplesPublication . Gameiro, Carina; Gonçalves, Joana; Soares, Sofia; Rosado, Tiago; Araujo, André R. T. S.; Passarinha, Luís A.; Barroso, Mário; Gallardo, EugeniaAntipsychotics have narrow therapeutic windows, and their monitoring in biological fluids is therefore important; consequently, stability in those fluids must be investigated during method development and validation. This work evaluates the stability of chlorpromazine, levomepromazine, cyamemazine, clozapine, haloperidol, and quetiapine in oral fluid (OF) samples, using the dried saliva spots (DSS) sampling approach and gas chromatography coupled to tandem mass spectrometry. Since many parameters can influence the stability of the target analytes, design of experiments was adopted to check the crucial factors that affect that stability in a multivariate fashion. The studied parameters were the presence of preservatives at different concentrations, temperature, light, and time. It was possible to observe that antipsychotic stability improved when OF samples in DSS were stored at 4 °C, with a low ascorbic acid concentration, and in the absence of light. With these conditions, chlorpromazine and quetiapine were stable for 14 days, clozapine and haloperidol were stable for 28 days, levomepromazine remained stable for 44 days, and cyamemazine was stable for the entire monitored period (146 days). This is the first study that evaluates the stability of these antipsychotics in OF samples after application to DSS cards.
- O fluido oral na monitorização terapêutica de fármacos antidepressivosPublication . Soares, Sofia; Rosado, Tiago; Barroso, Mário; Gallardo, EugeniaIntrodução: Entre 2013 e 2016, o consumo de fármacos antidepressivos duplicou em Portugal, com cerca de 30 milhões de embalagens de medicamentos dispensadas anualmente, apresentando uma das taxas de doenças mentais mais elevadas da Europa (Soares et al., 2021). A monitorização terapêutica é uma prática instituída para um pequeno número de fármacos, como os antidepressivos, para os quais há uma relação direta entre a sua concentração e o efeito farmacológico no local de ação. As concentrações dos fármacos nos fluidos biológicos são utilizadas conjuntamente com outras medidas de observação clínica para avaliar o estado do doente, sendo o suporte para a individualização da terapêutica ao permitir a caracterização de alterações farmacocinéticas observadas durante o curso do tratamento, a deteção de alterações no estado fisiopatológico do doente, ou a modificação da farmacocinética base do fármaco, mas também para a avaliação da adesão à terapêutica. Este trabalho descreve o desenvolvimento de um método analítico com potencial na aplicação clínica para a determinação de antidepressivos (fluoxetina, venlafaxina, citalopram, sertralina, paroxetina e metabolitos) em apenas 0,1 mL de fluido oral com amostragem por dried saliva spots (DSS) e análise por cromatografia gasosa-espectrometria de massa em tandem (GC-MS/MS). Métodos: Foi utilizado um GC-MS/MS modelo 7000B da Agilent Technologies. Todas as condições relativas ao equipamento foram previamente otimizadas. Relativamente à otimização dos DSS, foram avaliados os tempos de secagem e extração, solvente de extração e volume de solvente de extração com recurso à ferramenta estatística Design of Experiments. Resultados: Após otimização, as condições finais do processo de extração foram: 1 hora de secagem, 1 mL de metanol e 5 minutos de extração. O método foi validado apresentando linearidade dentro da gama estudada (dentro do intervalo terapêutico), com limites de deteção e quantificação entre 10 e 100 ng/mL. Todos os parâmetros de validação estudados estiveram de acordo com o preconizado por agências internacionais como a EMA e a FDA. Conclusões: Este é o primeiro trabalho que utiliza DSS como técnica de extração de antidepressivos e metabolitos em amostras de fluido oral, mostrando-se uma alternativa sensível, simples e rápida às técnicas convencionais, podendo ser aplicado rotineiramente na monitorização terapêutica, avaliação da adesão à terapêutica, e ainda no âmbito de toxicologia clínica e forense. Agradecimentos: Este trabalho tem o apoio dos fundos FEDER através de POCI-COMPETE 2020-Operational Programme Competitiveness and Internationalisation in Axis I-Strengthening research, technological development and innovation (Project POCI-01-0145-FEDER-007491) e da FCT-Fundação para a Ciência e a Tecnologia (Projecto UID/Multi/00709/2020). Sofia Soares gostaria de agradecer à FCT a bolsa com a referência SFRH/BD/148753/2019.
- Microextraction action by packed sorbent in forensic drug analysisPublication . Rosado, Tiago; Pelixo, Rodrigo; Pires, Bruno; Catarro, João Miguel; Rosendo, Luana M.; Brinca, Ana T.; Antunes, Mónica; Soares, Sofia; Simão, Ana Y.; Barroso, Mário; Gallardo, EugeniaMicroextraction by packed sorbent (MEPS) efficiently combines extraction, pre-concentration, and cleanup in a single device comprising two parts: the MEPS syringe and the packed sorbent bed1. MEPS has been used for several bioanalytical applications, including extraction of endogenous metabolites, biomarkers, and drugs from biological samples. It is particularly useful in metabolomics and pharmacokinetic studies2,3. Regarding MEPS applicability to forensic toxicology, urine is the most used specimen, followed by oral fluid (despite of its relatively high viscosity). Protein precipitation followed by centrifugation and with, or without dilution of the supernatant is the most commonly reported approach. The most detected compounds in forensic settings using MEPS are drugs of abuse [opiates and opioids (26%), cocaine (13%) cathinones (11%), dissociative hallucinogens (11%), cannabinoids and amphetamines (9% each) and other drugs (10%)] and medicinal drugs [antidepressants (9%), benzodiazepines/Z drugs (4%)]. MEPS was also applied to a beverage for forensic purposes e.g. to evaluate its composition in drug-facilitated crimes. An important feature in MEPS is the miniaturization of the sorbent. A careful selection of the sorbent will allow working with complex matrices, separating the target analytes from interferences and improve recoveries. The most widely selected sorbent was the silica based C18 that is a popular reversed-phase material (41%). Starting in the 2000s, new modifications of sorbents appeared. Overall, what the future holds for MEPS applications in forensic toxicology is promising, and ongoing research and technological advancements are likely to enhance the capabilities of MEPS approaches, making this technique an increasingly valuable tool for toxicological investigations.
- Monitorização de antidepressivos: comparação de dois métodos de extração em amostras de fluido oralPublication . Soares, Sofia; Rosado, Tiago; Barroso, Mário; Gallardo, EugeniaO consumo de antidepressivos representa uma problemática mundial, apresentando Portugal uma das taxas de prevalência de doenças mentais mais elevadas da Europa1. A monitorização terapêutica é instituída para um pequeno número de fármacos para os quais existe uma relação direta entre a concentração e o efeito farmacológico. Este trabalho compara duas metodologias desenvolvidas para a determinação de antidepressivos (fluoxetina, venlafaxina, sertralina, citalopram, paroxetina e metabolitos) em amostras de fluido oral com recurso à cromatografia gasosa acoplada à espectrometria de massa em tandem. O primeiro método utiliza amostragem por dried saliva spots (DSS) como técnica de extração. O volume de amostra foi de 100 μL e o procedimento foi otimizado utilizando a ferramenta estatística Design of Experiments (DoE) para avaliação dos tempos de secagem e extração e do solvente e volume do solvente de extração. As recuperações variaram entre 13 e 46%2. A segunda metodologia implementa a microextração em seringa empacotada (MEPS), utilizando 250 μL de amostra. Esta técnica foi otimizada utilizando a ferramenta de DoE para avaliação do número de strokes no load da amostra, número de lavagens e número de eluições. As recuperações variaram entre 12 e 93%. Ambos os métodos foram validados seguindo diretrizes internacionais. Os limites de quantificação foram estabelecidos entre 10-100 ng/mL e o intervalo de linearidade variou entre 10-500 ng/mL para ambas as metodologias (dentro do intervalo terapêutico). Este trabalho é o primeiro a utilizar DSS e MEPS para determinação destes antidepressivos em fluido oral.
- New Method for the Monitoring of Antidepressants in Oral Fluid Using Dried Spot SamplingPublication . Soares, Sofia; Rosado, Tiago; Barroso, Mário; Gallardo, EugeniaThe increase in the consumption of antidepressants is a public health problem worldwide, as these are a class of compounds widely used in the treatment of several illnesses, such as depression and anxiety. This work aimed to develop and optimize a method for the quantification of a number of antidepressants and their metabolites (fluoxetine, venlafaxine, O-desmethylvenlafaxine, citalopram, sertraline, and paroxetine) in 100 μL of oral fluid using the dried saliva spots (DSS) sampling approach and gas chromatography coupled with tandem mass spectrometry (GC–MS/MS). The method was validated, presenting linearity within the studied range, with detection and quantification limits ranging between 10 and 100 ng/mL, and coefficients of determination (R2) of at least 0.99 for all analytes. Recoveries were between approximately 13 and 46%. The analysis of precision and accuracy presented acceptable coefficients of variation and relative errors, considering the criteria usually accepted in the validation of bioanalytical procedures. The method herein described is the first to be reported using DSS for the extraction of antidepressants, proving to be a sensitive, simple, and fast alternative to conventional techniques, and capable of being routinely applied in clinical and forensic toxicology scenarios
- Novel synthetic opioids – toxicological aspects and analysisPublication . Tabarra, Inês; Soares, Sofia; Rosado, Tiago; Gonçalves, Joana; Luís, Ângelo; Malaca, Sara; Barroso, Mário; Keller, Thomas; Restolho, José; Gallardo, EugeniaOver the past few years, there has been an emerging number of new psychoactive drugs. These drugs are frequently mentioned as “legal highs”, “herbal highs”, “bath salts” and “research chemicals”. They are mostly sold and advertised on online forums and on the dark web. The emerging new psychoactive substances are designed to mimic the effects of psy- choactive groups, which are often abused drugs. Novel synthetic opioids are a new trend in this context and represent an alarming threat to public health. Given the wide number of fatalities related to these compounds reported within the last few years, it is an important task to accurately identify these compounds in biologic matrices in order to administer an effective treatment and reverse the respiratory depression caused by opioid related substan- ces. Clinicians dealing with fentanyl intoxication cases should consider that it could, in fact, be a fentanyl analogue. For this reason, it is a helpful recommendation to include synthetic opioids in the routine toxicological screening procedures, including analysis in alternative matrices, if available, to investigate poly-drug use and possible tolerance to opioids. To address this public health problem, better international collaboration, effective legislation, effective investigation, control of suspicious “research chemicals” online forums and continu- ous community alertness are required. This article aims to review diverse reported fatalities associated with new synthetic opioids describing them in terms of pharmacology, metabol- ism, posology, available forms, as well as their toxic effects, highlighting the sample proce- dures and analytical techniques available for their detection and quantification in biological matrices
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