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- Olanzapine: challenges in toxicological results interpretationPublication . Costa Lopes, Miguel; Abreu, Ana; Nascimento, João; Sardinha, Mário; Castañera, Antonio; Fonseca, Suzana; Franco, João; Nikolić, DobrilaFatal intoxications are a topic of great relevance in today's society and a massive challenge for pathologists all over the world. In post-mortem investigations it is often challenging to determine if and which drug(s) caused the death due to the post-mortem processes in a body. This study evaluates forensic fatal cases with positive blood levels for the antipsychotic Olanzapine. The aim of this study is to characterize the sample of such forensic autopsy cases and try to correlate the Olanzapine concentrations with the manner of death and cause of death attributed in the reports. We performed a retrospective, observational and descriptive study, analyzing the reports of forensic autopsies performed in the South Branch of the National Institute of Legal Medicine and Forensic Sciences, in Lisbon, Portugal, from 2013 until 2015. A total of 53 forensic autopsy cases characterized by the presence of Olanzapine were analyzed. Blood Olanzapine concentrations ranged from 10ng/ml to 4363ng/ml. The following variables were collected: sex, age, place of death, number of days between death and autopsy, clinical history, signals of putrefaction, cause of death, manner of death and ancillary tests performed, such as toxicological and histological exams. This study raises the need of awareness for the most common limitations that forensic pathologists face in the interpretation of toxicological results and indicate the need to create a reference table for each substance with our casuistic.
- 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.
- UHPLC-MS/MS methodology for analysis of new synthetic opioids and hallucinogens in whole bloodPublication . Pereira, Joana; Antunes, Mónica; Neng, N.R.; Mustra, Carla; Franco, João; Fonseca, SuzanaBackground & Aims: New Psychoactive Substances (NPS) are a real contemporaneous threat, due to their potency, dangerousness, and lack of control/monitoring. The NPS group that has grown the most is the synthetic opioids group, where fentanyl and its analogues stand out. However, the emerging concerning synthetic opioids are nitazenes. Due to their high potency, even minimal consumption doses can lead to severe health effects or even fatal overdoses, making them a public health issue. Notwithstanding, is it also important to remain vigilant towards more “traditional” psychoactive substances like hallucinogens once they have been associated with both intentional and unintentional poisonings/intoxications. This is particularly relevant now that they are also being used for clinical purposes. Therefore, it is essential to establish analytical methodologies for monitoring these compounds. As a result, the aim of this study was to develop, optimize and validate an easy to use, fast, simple, sensitive, robust, and routine flow method for the analysis of new synthetic opioids (fentanyls and nitazenes) and (classic) hallucinogens in whole blood. Methods: The present work describes a method that allows the screening, qualitative confirmation, and quantification of more than 10 psychoactive substances, including new synthetic opioids and hallucinogens. The sample preparation step consisted in 50 µL of whole blood protein precipitation with refrigerated acetonitrile containing formic acid and was optimized using a design of experiments (DoE) approach, namely Full Factorial Design, to achieve the best conditions for compounds extraction from matrix. Following centrifugation, the resulting supernatant extract can be directly injected into an ultra-high-performance liquid chromatograph coupled to a triple quadrupole linear ion trap mass spectrometer (Sciex UHPLC-QTRAP-MS® 6500+) and analyzed in a 5-minute run in Multiple Reaction Monitoring (MRM) mode with 2 transitions for each compound. The developed analytical methodology was fully validated according to the guiding principles of the ANSI/ASB Standard 036. To confirm its applicability in a real context, the proposed methodology was applied to the analysis of authentic forensic samples. Results & Discussion: In terms of validation, the methodology linearity was assessed between 1 and 20 ng/mL. The precision and accuracy were satisfactory, with values <15% and within ±15% (20% at the LOQ), respectively. The limits of detection were between 0.1 and 1 ng/mL, depending on the compound. Dilution ratios were also successfully evaluated. Selectivity was confirmed by analyzing spiked samples containing several therapeutic drugs and other drugs of abuse. Conclusion: The proposed methodology provides a valuable and powerful tool for toxicology laboratory, enabling the simultaneous identification, confirmation, and quantification of different families of psychoactive substances, including synthetic opioids and hallucinogens. Its speed, simplicity, effectiveness, and reliability make it particularly advantageous for routine analysis. These combined advantages make it a suitable alternative for routine implementation.
- 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.
- 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).
- 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.
- Munchausen by Proxy with Haloperidol: A case reportPublication . Fonseca, Suzana; Dias, MárioIntroduction: An 8-year-old male child with extrapyramidal symptoms was admitted at 3 pm at the Urgency of Pediatric Hospital. Extrapyramidal syndrome (EPS), sometimes referred to as neuroleptic malignant syndrome, is a neurological side effect of antipsychotic medication characterized by a set of symptoms, that include: hyperpyrexia (an early sign of this syndrome), generalized muscle rigidity, tremors, hypersalivation, altered mental status (including catatonic signs), and evidence of autonomic dysfunction (irregular pulse or blood pressure. This child had previous Hospital admissions with similar symptoms and the possibility of a Munchausen by Proxy Syndrome (MBPS) was considered for evaluation. This syndrome is a form of child abuse in which the carer (usually the mother) simulates, manipulates or produces symptoms of illness in the victim. In most cases the detrimental effect is caused by applying foreign substances, sometimes difficult to detect in the clinical laboratories. Serum and urine samples were collected and a toxicological screening in urine was performed at the hospital by immunoassay revealing positive results for barbiturates and amphetamines. A second urine sample, collected 2 hours later, tested negative to all groups screened. The analytical results didn´t matched with clinical disturbances of the patient and further diagnosis procedures yielded no pathological findings. The serum and urine samples were sent to the Department of Chemistry and Forensic Toxicology of National Institute of Legal Medicine and Forensic Sciences (INMLCF) for an extensive screening for drugs and medicines.
- 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.
- 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.
- Interactions between tramadol and selective serotonin reuptake inhibitors anti depressants in post mortem casesPublication . Fonseca, Suzana; Franco, JoãoBackground & Objectives Tramadol is very often prescribed for pain management in patients with depression in treatment with selective serotonin reuptake inhibitors (SSRI). Serotonergic antidepressants and its concomitant use with tramadol can lead to a drug–drug interaction, resulting in a decrease in the tramadol analgesic effect and a higher risk of serotonin syndrome caused by the accumulation of serotonin in the central nervous system. These interactions can also change the post-mortem concentrations of the therapeutic drugs and its metabolites, increasing the difficulty in the interpretation of forensic toxicology results, mainly when the concentrations are higher than the therapeutic level. The aim of this study was to review a five-year period of post-mortem cases, in order to examine the prevalence of concomitant use of tramadol and SSRI and the influence in the concentrations found.