Percorrer por autor "Fonseca, Suzana"
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- 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 validação de um método analítico para a determinação de canabinóides em amostras de fluido oral por LC-MS/MSPublication . Antunes, Mónica; Simões, Susana; Fonseca, Suzana; Franco, João; Gallardo, Eugenia; Barroso, MárioA canábis é a droga de abuso mais consumida na Europa, pelo que várias metodologias analíticas têm sido desenvolvidas para a quantificação dos seus componentes em matrizes biológicas, especialmente sangue e urina. No entanto, cada vez mais se está a reconhecer a importância do estudo de matrizes biológicas não-convencionais, nomeadamente cabelo e fluido oral (ou saliva) – os seus procedimentos de recolha não são invasivos e estas amostras fornecem informações complementares sobre o uso de drogas, permitindo o controlo da exposição recente (saliva) e de médio a longo prazo (cabelo). Desta forma, foi desenvolvida uma metodologia analítica para a determinação e quantificação de tetrahidrocanabinol (THC), 11-hidroxi-tetrahidrocanabinol (THC-OH), 11-carboxitetrahidrocanabinol (THC-COOH), canabinol (CBN) e canabidiol (CBD) em amostras de saliva por cromatografia líquida acoplada à espectrometria de massa em tandem (LC-MS/MS). Segundo as Mandatory Guidelines for Federal Workplace Drug Testing Programs, o cut-off de THC em amostras de saliva é 2 ng/mL, exigindo um procedimento de extração otimizado e a utilização de uma técnica cromatográfica sensível. Resumidamente, a 200 μL de saliva foi adicionado o padrão interno, e as amostras foram submetidas a precipitação proteica com uma mistura de metanol/acetonitrilo (80:20, v/v) refrigerada. Depois de centrifugados, os extratos foram evaporados até à secura, reconstituídos em metanol, e 5 μL foram injetados no equipamento UPLC-QTRAP-MS 6500+ (SCIEX®), numa corrida de 14 minutos. A análise foi efetuada em modo MRM com duas transições para cada composto e uma transição para cada padrão interno. Os métodos foram validados de acordo com as diretrizes da norma ANSI/ASB 036, tendo sido estudados os parâmetros supressão iónica, interferentes, linearidade, precisão e exatidão, carryover, limites de deteção e quantificação, efeito de diluição e estabilidade das amostras extraídas. Todos os estudos destes parâmetros apresentaram resultados satisfatórios, e o método foi aplicado com sucesso a amostras reais. O método desenvolvido mostrou-se fácil, rápido, eficaz e robusto, estando apto para ser utilizado na rotina do laboratório, e sendo uma ferramenta complementar útil para o estudo do consumo recente de canabinóides.
- Detection and quantification of selected cannabinoids in hair samples by liquid-liquid extraction and LC-MS/MSPublication . Antunes, Mónica; Simões, Susana; Fonseca, Suzana; Franco, João; Barroso, Mário; Gallardo, EugeniaCannabis remains the most used illicit drug worldwide, with rising use linked to medical and recreational decriminalization. This has driven the development of analytical techniques to detect cannabinoids in biological matrices. Hair offers key advantages due to its non-invasive collection, extended detection window, stability, and easy storage. This study presents the development and validation of a robust method for extracting natural cannabinoids from hair, following ANSI/ASB 2019, FDA, and Society of Hair Testing guidelines. Hair samples were washed with methanol and cut into small pieces. Approximately 20 mg of hair was incubated with 1 M NaOH and methanol (30 min, 50 °C). The mixture was acidified with acetic acid and underwent liquid-liquid extraction using hexane/ethyl acetate (90/10, v/v). The organic phase was evaporated and reconstituted in 1-pentanol/methanol (50/50, v/v). Analysis was conducted by LC-MS/MS using multiple reaction monitoring (MRM) and triple-stage mass spectrometry (MS³). The method was selective, specific, precise, and linear, with working ranges of 5-2000 pg/mg for THC, CBN, and CBD; 50-2000 pg/mg for THC-OH; and 0.2-20 pg/mg for THC-COOH. Ion suppression was observed but did not affect sensitivity, with LLOQs and LODs from 0.2 to 50 pg/mg. Over 25 hair samples from university students tested positive for cannabis. THC ranged from 5.9 to 2430.7 pg/mg; one sample had THC-OH above LLOQ (61.4 pg/mg); THC-COOH ranged from 0.3 and 36.4 pg/mg; CBN from 5.7 to 461.0 pg/mg; and CBD from 5.7 to 850.2 pg/mg. Results aligned with self-reported use, confirming the method's forensic suitability.
- Detection and quantification of selected cannabinoids in oral fluid samples by protein precipitation and LC-MS/MSPublication . Antunes, Mónica; Simões, Susana; Fonseca, Suzana; Franco, João; Gallardo, Eugenia; Barroso, MárioCannabis is the most widely consumed illicit drug worldwide. As consumption rates increase, partially due to the decriminalization of its use for medicinal and recreational purposes, analytical methods for monitoring different cannabinoids in several biological matrices have been developed. Herein, a simple and fast extraction procedure to extract natural cannabinoids from oral fluid (OF) samples was developed and fully validated according to the ANSI/ASB 2019 Standard Practices for Method Validation in Forensic Toxicology. Using only 0.2 mL of neat OF, the analytes [Δ9-tetrahidrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (THC-OH), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH), cannabinol (CBN) and cannabidiol (CBD)] were extracted by protein precipitation with a mixture of methanol:acetonitrile (80:20, v/v); the extracts were centrifuged, evaporated to dryness and reconstituted in 100 µL of methanol. Analysis was performed by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). The developed methodology produced linear results for all compounds, with working ranges of 0.1–50 ng/mL for THC, 0.5–50 ng/mL for THC-OH, CBN and CBD, and 0.05–1 ng/mL for THC-COOH. Ion suppression was observed for THC, CBN and CBD, which did not impair sensitivity considering the low limits of quantification (LOQs) and limits of detection (LODs) obtained (which varied between 0.05 and 0.5 ng/mL). The extraction procedure produced great recoveries, and the compounds were stable. No interferences were found, and the method proved to be extremely fast, selective, precise, and accurate for use in routine analysis. The method was successfully applied to authentic samples.
- Determination of biomarkers of cannabis consumption in hair samples: preliminary resultsPublication . Antunes, Mónica; Fonseca, Suzana; Simões, Susana; Franco, João; Gallardo, Eugenia; Barroso, MárioOver 96 million European adults are estimated to have consumed illicit drugs at some point in their lives, and cannabis is still the most consumed drug. The Portuguese data is obtained using surveys, but this type of study has several disadvantages, such as under or overestimation of consumption rates, which may lead to biased conclusions.Consequently, it is desirable that these studies are accompanied by drug monitoring in biological samples to circumvent the associated drawbacks.
- Development and validation of a multi-substance method for routine analysis of pesticides in post-mortem samples by UPLC-MS/MSPublication . Matos, Francisco; Barroso, Mário; Antunes, Mónica; Franco, João; Fonseca, SuzanaPesticides play an important role in forensic toxicology and are usually classified as a single class of chemicals, but in fact there are several different types of compounds, including organophosphates, carbamates, pyrethroids or organochlorines, among others, with different toxicities. Pesticide analysis in post-mortem samples can be difficult due to the complexity of the samples and to the high toxicity of these compounds. The aim of this study was to develop and validate an easy to use, sensitive, and robust method, using ultra-performance liquid chromatography-tandem mass spectrometry to be incorporated in the routine flow for more than fifty pesticide analysis in post-mortem blood. The developed analytical method was fully validated according to the guiding principles of the ANSI/ASB Standard 036 and applied to routine post-mortem samples, being already detected more than 15 positive cases. This UPLC-MS/MS method is useful and a powerful tool in a toxicology lab because it is fast, simple, effective, and trustworthy. This method is able to detect and analyse pesticides in post-mortem samples. It is set to use for routine analysis and successfully applied to intoxication cases.
- Development and validation of a simple and fast method for routine analysis of new synthetic opioids and hallucinogens in whole blood using protein precipitation and UHPLC-MS/MSPublication . Pereira, Joana R.P.; Antunes, Mónica; Neng, N.R.; Mustra, Carla; Franco, João; Fonseca, SuzanaIn forensic toxicology, the rapid and reliable detection of emerging synthetic opioids and hallucinogens is crucial for case investigations and public health monitoring. This work describes the development, optimization and validation of a simple, fast and sensitive methodology for the simultaneous analysis of 6 new synthetic opioids (carfentanil, fentanyl, isotonitazene, metonitazene, norfentanyl, and sufentanil) and 2 hallucinogens (lysergide [LSD] and mescaline), together with the main LSD metabolite 2-oxo-3-hydroxy-lysergide [LSD-OH], in whole blood samples by liquid chromatography coupled to tandem mass spectrometry. Under optimized experimental conditions, linearity was verified between 0.1 and 20 ng/mL for all analytes except mescaline (2.5-500 ng/mL), with r2 > 0.99 for 1/x weighting, and no significant carryover or matrix effects were observed. Good precision (% RSD < 13 %) and trueness (% Bias within ± 20 %) values were achieved. The estimated limit of quantification (LOQ) was 0.1 ng/mL for all compounds except mescaline (2.5 ng/mL). Authentic forensic samples were also analyzed, and positive samples for fentanyl, norfentanyl, and sufentanil were identified. The proposed methodology allows the simultaneous analysis of compounds from different families of psychoactive substances, in both postmortem and in vivo samples, using only 50 µL of whole blood. The demonstrated speed, simplicity, and effectiveness make it particularly advantageous for routine implementation in forensic toxicology laboratories.
- Development and validation of a simple and fast method for routine analysis of new synthetic opioids and hallucinogens in whole blood using protein precipitation and UHPLC-MS/MSPublication . Pereira, Joana R. P.; Antunes, Mónica; Neng, Nuno R.; Mustra, Carla; Franco, João; Fonseca, SuzanaIn forensic toxicology, the rapid and reliable detection of emerging synthetic opioids and hallucinogens is crucial for case investigations and public health monitoring. This work describes the development, optimization and validation of a simple, fast and sensitive methodology for the simultaneous analysis of 6 new synthetic opioids (carfentanil, fentanyl, isotonitazene, metonitazene, norfentanyl, and sufentanil) and 2 hallucinogens (lysergide [LSD] and mescaline), together with the main LSD metabolite 2-oxo-3-hydroxy-lysergide [LSD-OH], in whole blood samples by liquid chromatography coupled to tandem mass spectrometry. Under optimized experimental conditions, linearity was verified between 0.1 and 20 ng/mL for all analytes except mescaline (2.5–500 ng/mL), with r2 > 0.99 for 1/x weighting, and no significant carryover or matrix effects were observed. Good precision (% RSD < 13 %) and trueness (% Bias within ± 20 %) values were achieved. The estimated limit of quantification (LOQ) was 0.1 ng/mL for all compounds except mescaline (2.5 ng/mL). Authentic forensic samples were also analyzed, and positive samples for fentanyl, norfentanyl, and sufentanil were identified. The proposed methodology allows the simultaneous analysis of compounds from different families of psychoactive substances, in both postmortem and in vivo samples, using only 50 µL of whole blood. The demonstrated speed, simplicity, and effectiveness make it particularly advantageous for routine implementation in forensic toxicology laboratories.
- Documenting human exposure to cannabinoids using oral fluidPublication . Antunes, Mónica; Simões, Susana; Fonseca, Suzana; Franco, João Miguel; Gallardo, Eugenia; Barroso, MárioThe importance of studying non-conventional biological matrices such as oral fluid (OF) is increasingly being recognized. This sample presents several advantages, mainly related to its collection procedure: it is non-invasive, easy to perform by non-medical personnel, can be performed under supervision to prevent adulteration, and provides low biohazard risk. OF samples are more likely to contain parent drugs, reflecting recent drug use – a major advantage of this matrix1. A fast and robust analytical methodology was developed in OF samples for the determination of tetrahydrocannabinol (THC), 11-hydroxy-tetrahydrocannabinol (THC-OH), 11-carboxytetrahydrocannabinol (THC-COOH), cannabinol (CBN) and cannabidiol (CBD) by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), aiming at documenting cannabis consumption. Briefly, 200-μL aliquots of OF were subjected to protein precipitation with a refrigerated methanol/acetonitrile mixture (80:20, v/v). After centrifugation, the extracts were evaporated to dryness, reconstituted in methanol, and 5-μL aliquots were injected into the UPLC-QTRAP-MS 6500+ (SCIEX®) system (in a 14-minute run). The analysis was carried out in MRM mode with two transitions for each compound and one transition for each internal standard. The method was validated according to the guidelines of ANSI/ASB 0362. Parameters such as ion suppression/enhancement, interferents, linearity, precision and accuracy, limits of detection and quantification, dilution integrity and stability were studied and showcased satisfactory results. The 2 ng/mL cut-off for THC3 was achieved, and the method was successfully applied to real samples (57.95- 898.28 ng/mL for THC; 0.17-4.09 ng/mL for THC-COOH; 1.26-44.57 ng/mL for CBN; 0.42-1007.86 ng/mL).
- Estudo de estabilidade de controlo analítico em método multi-substâncias por LC/MS-MS do SQTF.Publication . Mustra, Carla; Fonseca, Suzana; Monsanto, Paula; Franco, João MiguelINTRODUÇÃO/OBJETIVOS O desenvolvimento e validação de métodos analíticos é uma tarefa fundamental e frequente do Serviço de Química e Toxicologia Forenses (SQTF) que envolve o estudo de vários parâmetros como modelo de calibração, seletividade, sensibilidade, exatidão, precisão, estabilidade, entre outros. O grau de complexidade de uma validação aumenta a par com o número de substâncias abrangidas pelo método, bem como com a especificidade de cada substância. Tão ou mais importante que a validação do método é o seu controlo analítico ao longo do tempo, o que designamos por controlo de qualidade interno, o qual permite avaliar o desempenho do método e do equipamento. O SQTF tem vindo a ampliar o leque de substâncias pesquisadas nos seus laboratórios e a melhorar os níveis de sensibilidade dos métodos, com vista a dar resposta às solicitações. Este crescimento e melhoria dos métodos só foi possível devido à aquisição de novos equipamentos, o que implicou desafios acrescidos relativamente a vários aspetos, em particular quanto à metodologia usada para assegurar um controlo de qualidade interno eficaz. O objetivo deste trabalho é demonstrar como um estudo preliminar da estabilidade de um controlo analítico pode contribuir para a adoção de critérios que permitam avaliar o desempenho do método/equipamento ou mesmo conduzir a novas propostas, como por exemplo a escolha de padrões internos. MATERIAL E MÉTODOS Durante 2 meses foi injetado juntamente com as sequências analíticas de confirmação/quantificação de substâncias medicamentosas de amostras de rotina, um controlo analítico preparado a partir de uma amostra branca (sangue onde previamente se confirmou a ausência das substâncias em estudo) fortificada com cerca de 180 substâncias a uma concentração de 50 ng/mL. Foram avaliadas 177 substâncias medicamentosas relativamente à sua resposta ao longo do tempo (razão entre a área do analito e área de padrão interno: clomipramina d3) num total de 13 injeções. O tratamento estatístico dos dados foi efetuado com recurso à aplicação Microsoft Office Excel®. RESULTADOS E DISCUSSÃO Verificou-se que a maioria das substâncias, cerca de 80%, apresentaram um comportamento estável ao longo do período estudado. Identificaram-se, no entanto, algumas substâncias com variações assinaláveis ao longo do tempo, nomeadamente algumas estatinas e benzodiazepinas cujo coeficiente de variação da razão das áreas é superior a 60%. Nestes casos será importante avaliar as causas destas variações cuja correção poderá, eventualmente, ser implementada através da seleção de um padrão interno mais específico. CONCLUSÕES O estudo de estabilidade de um controlo analítico revelou-se uma ferramenta importante para definir critérios de monitorização do desempenho do método e do próprio equipamento. A maioria das substâncias parece ter estabilidade nas condições testadas, no entanto identificaram-se alguns analitos menos estáveis para os quais será adequado selecionar marcadores de estabilidade ou redefinir padrões internos que sejam mais específicos.