Browsing by Author "Machado, Francisca"
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- Desenvolvimento e validação de uma metodologia analítica para a determinação de α-PHP em sangue por GC-MS-EIPublication . Margalho, Cláudia; Machado, Francisca; Franco, João; Vieira, Duarte NunoIntrodução: As Novas Substâncias Psicoativas (NSP) são alvo de grande preocupação a nível global, apesar dos contínuos esforços empregues pelos sistemas de controlo nacionais e internacionais para limitar a sua disseminação. A alfa-pirrolidinohexanofenona (α-PHP) é uma NSP recente, pertencente ao grupo das catinonas, com um reduzido número de estudos pulicados. Embora exista uma baixa incidência de consumo de NSP em Portugal, houve um notório aumento recente de doses apreendidas de α-PHP e um consequente aumento de casos detetados da substância em matrizes biológicas durante a realização das análises toxicológicas forenses de rotina de drogas de abuso realizadas no Serviço de Química e Toxicologia Forenses da Delegação do Centro do Instituto Nacional de Medicina Legal e Ciências Forenses, I.P. Justificou-se, assim, desenvolver e validar uma metodologia analítica para a determinação e quantificação de α-PHP em sangue. Métodos: O procedimento analítico foi devidamente validado segundo a norma publicada pela American Academy of Forensic Sciences (AAFS) Standard Practices for Method Validation in Forensic Toxicology (ANSI/ASB Standard 036, 2019). Foram usados volumes de 500 μL de amostra de sangue às quais se adicionou o padrão interno, cocaína-d3. Seguidamente foi empregue a extração em fase sólida (SPE) com colunas de troca catiónica de modo misto (Oasis MCX®). Após secagem, os extratos foram reconstituídos em 60 µL de metanol e posteriormente analisados por GC-MS-EI com monitorização dos iões (77, 105 e 140) previamente selecionados em modo full-scan. Resultados: O método apresentou linearidade entre 10 e 1000 ng/mL, com coeficientes de determinação (r2) superiores a 0.999, exatidão de cada calibrador dentro do intervalo de aceitação de ± 20% e uma distribuição aleatória dos valores dos residuais em torno da linha zero. Foi alcançado um limite de deteção de 5 ng/mL e um limite de quantificação de 10 ng/mL, tendo a eficiência da extração variado entre 98.5% e 103.3%. Foram cumpridos os critérios de aceitação para a precisão intra-dia e intermédia (< 20%) e para o bias (± 20): CV < 17.7% e bias < 11.6%. O α-PHP apresentou estabilidade no sangue por um período de 6h, quando as amostras foram mantidas na bancada à temperatura do laboratório; até 48h, quando os extratos resultantes das amostras processadas foram mantidos no amostrador automático à temperatura da sala dos equipamentos de GC-MS; e por 21 dias após 5 ciclos de congelamento/descongelamento. Não foi observado o fenómeno de carryover. Discussão: A metodologia validada demonstrou ser suficientemente sensível para a determinação de α-PHP em amostras de sangue. Conclusão: A técnica analítica GC-MS-EI revelou-se suficientemente sensível para a determinação do analito estudado em sangue, apresentando uma grande utilidade para ser usada em qualquer laboratório de toxicologia forense, com ou sem tecnologia mais sofisticada. Este é o primeiro procedimento analítico que combina SPE e GC-MS-EI para a determinação de α-PHP em sangue.
- Development and Validation of a GC–MS-EI Method to Determine α-PHP in Blood: Application to Samples Collected during Medico-Legal AutopsiesPublication . Machado, Francisca; Franco, João Miguel; Vieira, Duarte Nuno; Margalho, CláudiaNew psychoactive substances (NPSs) still represent an issue of great concern worldwide despite efforts made by national and international control systems to limit the spread of these substances. Alpha-pyrrolidinohexanophenone (α-PHP) is a fairly recent synthetic cathinone (the second largest group of monitored substances in Europe) with only a few published studies on the substance. Though there is a low incidence of NPS consumption in Portugal, a recent increase in apprehensions and detections in biological matrices of the substance was verified. An analytical methodology was developed and validated for determining and quantitating α-PHP in blood. Solid-phase extraction was employed for sample preparation (500 μL), which was further analyzed by gas chromatography-mass spectrometry-electron ionization in single-ion monitoring mode with cocaine-d3 as the internal standard. Method validation followed the guidelines of the American National Standards Institute/AAFS Standards Board (ANSI/ASB Standard 036). The procedure was linear between 10 and 1,000 ng/mL, with determination coefficients (r2) higher than 0.999. Carryover was not observed. A limit of detection of 5 ng/mL and a limit of quantitation of 10 ng/mL were achieved. Intraday and intermediate precision and bias assessment showed satisfactory results (coefficient of variation <17.7%; bias <11.6%), and extraction efficiency ranged from 98.5% to 103.3%. The stability of the substance was considered acceptable for at least 6 h at room temperature, 48 h in the autosampler and 21 days after five freeze/thaw cycles. The developed methodology was applied to 15 real samples from the Laboratory of Chemistry and Forensic Toxicology, Centre Branch of the National Institute of Legal Medicine and Forensic Sciences, Portugal, with drug concentrations ranging from 15 to 227 ng/mL. Available information for each case is also detailed in the present article.
- Α-Pyrrolidinoisohexanophenone (Α-PIHP) in three fatalPublication . Dinis, Pedro; Machado, Francisca; Franco, João; Margalho, CláudiaBackground & Aims The New Psychoactive Substances (NPSs) are widely spread worldwide through the illicit markets and are increasingly the cause of intoxication deaths. Alpha-pyrrolidinoisohexanophenone (α-PiHP) is a positional isomer of α-pyrrolidinohexanophenone (α-PHP) and has been reported for the first time in 2016 at seized materials in China. Both substances are pyrovalerone derivatives from the synthetic cathinones group, and mainly exert cardiovascular, psychological, and neurologic effects. In addition, as published in literature and regardless of their isomerism, both cathinones can appear separately or together in a mixture. This work presents three forensic fatal cases associated with the consumption of α-PiHP and/or α-PHP: case 1 involves a 41-year-old man, drug user, mostly of “bloom”, who committed suicide by hanging; case 2 reports to a 32-year-old man, addicted to synthetic drugs, associated with a sample of “weed” collected next to the corpse; case 3 concerns to a 58-year-old male drug addict, who died after being admitted to the emergency department in a state of coma. Methods The toxicological analyses were carried out in peripheral blood in the three cases and, in case 2, the sample of “weed” was also analysed. Samples (500µL) were prepared with 0.1 M phosphate buffer, extracted by solid-phase extraction and analysed through gas chromatography coupled with mass spectrometry (GC-MS) in full-Scan monitoring mode to search for unknown substances. Subsequently, the samples were analysed in single-ion monitoring mode to confirm the substances detected in full-Scan. The ions 140, 98 and 77 were monitored for α-PiHP; 140, 105 and 77 for α-PHP; and 185 for cocaine-d3 (internal standard). Results & Discussion In case 1 was detected and confirmed α-PiHP. Other substances were also detected and confirmed such as tramadol, fluoxetine, diazepam, nordiazepam, cyamemazine, olanzapine, paliperidone and risperidone. Regarding case 2, in the “weed” we were able to detect several cannabinoids (cannabicyclol, cannabichromene, cannabidiol, cannabidivarol, cannabigerol, cannabinol, delta-9-tetrahydrocannabinol, delta-8-tetrahydrocannabinol and tetrahydrocannabivarin), nicotine and α-PiHP. In peripheral blood was confirmed α-PiHP, cocaine and its metabolites (benzoylecgonine and ecgonine methyl ester), delta-9-tetrahydrocannabinol and 11-nor-9-carboxy-delta-9-tetrahydrocannabinol, phenacetine (a cocaine cutting agent), morphine, paracetamol, alprazolam, nordiazepam, sertraline and mirtazapine. In case 3, both α-PHP and α-PiHP were detected and confirmed in peripheral blood, such as paracetamol. Therefore, the toxicological results of the three cases are relevant to complement the case histories since the presence of α-PiHP in the peripheral blood can be related with the death of individuals. Conclusion With the three cases presented in this work, we can conclude that the search for NPS in biological and non-biological specimens plays an important role, especially in cases of drug-related deaths. The concomitant use of traditional drugs of abuse, prescription medication, α-PHP and α-PiHP, either separately or in combination, increases the possibility of a fatal intoxication since both substances have a high toxic potential.