Simultaneous analysis of some club drugs in whole blood using solid phase extraction and gas chromatography–mass spectrometry

The use of psychoactive substances to improve social relations and increase body energy, in Rave Culture, has raised many legal and health public concerns, both for illicit trade and consumption. Therefore, forensic toxicology plays an important role in this area, mainly linked to the detection and quantitation of these substances, both in vivo and in post-mortem samples. In fact, at the moment, forensic sciences have been under public authorities’ scrutiny and critical look, due to the increasing attention of the media and public opinion, always applying for the use of scientific knowledge to help solving forensic cases. However, forensic toxicology results are only reliable to solve legal cases if all the analytical methodologies used are appropriately validated. In this work, a methodology for the extraction and analysis of 7-aminoflunitrazepam, buprenorphine, flunitrazepam, ketamine, methadone, phencyclidine (PCP) and d-propoxyphene was developed for whole blood samples, with Solid-Phase Extraction (SPE), using OASIS MCX SPE columns, and gas chromatography coupled to mass spectrometry. The procedure presented here proved to be reliable, specific, selective and sensitive, with good LODs and LOQs and good precision.The adoption of a SPE procedure with an automatic SPE extraction device, allowed an increased level of automation in sample treatment, being contemporarily less time-consuming, increasing productiveness, and allowing good recovery and appropriate selectivity being, also, simple and reproducible. The simultaneous detection and quantitation of all compounds by the same extraction and detection methodology is crucial and has a great potential for forensic toxicology and clinical analysis.info:eu-repo/semantics/acceptedVersio

Endogenous GHB concentrations in whole blood postmortem samples as a biomarker for post mortem interval estimation – A set of real cases analysis

Poster apresentado no 54th Annual Meeting of The International Association of Forensic Toxicologists, Brisbane (Austrália), 2016Gamma-hydroxybutyric acid (GHB) is an endogenous compound which has a story of clinical use and illicit abuse since the 1960’s. Its postmortem behaviour, namely regarding degradation and metabolism, has been increasingly studied to be used as a putative biomarker for post-mortem interval (PMI) estimation. Thus, whole blood post-mortem GHB levels were obtained in thirty two real cases with previous information on death and autopsy data. The samples were treated through sample methanolic precipitation followed by GC-MS/MS analysis (LLOQ=0.1 mg/L). No differences were encountered for the other parameters evaluated, including age [under 44 years-old - 7.87 2.06 mg GHB/L (n=7), 45 to 60 years-old - 6.80 3.67 mg GHB/L (n=13) and over 61 years-old - 5.72 2.39 mg GHB/L (n=12), p0.05], gender [men - 7.79 5.04 mg GHB/L (n=23), women - 6.72 2.60 mg GHB/L (n=9), p=0.273], cause of death [accident - 7.96 ± 2.26 mg GHB/L (n=8), suicide - 6.75 ± 3.22 mg GHB/L (n=7) and unknown/natural death - 5.14 ± 2.96 mg GHB/L (n=17), p0.05] and presence or absence of substances [absence - 6.37 2.61 mg GHB/L, presence - 6.96 3.38 mg GHB/L, p=0.405]. On the other hand, the results obtained suggest that the PMI (until 5 days between death and sampling) influences GHB whole blood concentration, noticed namely between 48 and 72 hours (24 - 48 hours (p=0.893), 48 - 72 hours (p<0.05); 72 - 96 hours (p=0.123). This study brings additional data regarding the usefulness of GHB levels in forensic toxicology, which might be further strengthened with larger, but comparable, studies from other laboratories and institutions in the forensic toxicology context.N/

A fast and reliable method for GHB quantitation in whole blood by GC–MS/MS (TQD) for forensic purposes

Gamma-Hydroxybutyric Acid (GHB) is an endogenous compound with a story of clinical use since the 1960’s. However, due to its secondary effects, it has become a controlled substance, entering the illicit market. A fully validated, sensitive and reproducible method for the quantification of GHB by methanolic precipitation and GC-MS/MS (TQD) in whole blood is presented. Using 100 µL of whole blood, obtained results included a LOD and LLOQ of 0.1 mg/L and a recovery of 86% in a working range between 0.1 and 100 mg/L. This method is sensitive and specific to detect the presence of GHB in small amounts of whole blood (both ante-mortem or post-mortem), and is, to the authors’ knowledge, the first GC-MS-MS TQD method that uses different precursor ions and product ions for the identification of GHB and GHB-D6 (internal standard). Hence, this method may be especially useful for the study of endogenous values in this biological sample.info:eu-repo/semantics/acceptedVersio

A gestão do risco na implementação e validação de métodos analíticos em Toxicologia Forense

Poster apresentado no 19 Congresso Nacional de Medicina Legal e Ciências Forenses, Coimbra, 2021Em 2017, a nova versão da norma ISO/IEC 17025 (versão portuguesa de 2018) incorporou a necessidade da Gestão de Riscos. A abordagem baseada no risco e a consciência do risco são acentuadas nesta nova versão da norma, sendo promovida uma abordagem ao “pensamento baseado no risco”. Segundo esta última revisão deste documento, o laboratorio é o responsável por decidir quais os riscos e as oportunidades que precisam de ser analisados e tratados. Mais ainda, a nova revisão da Norma, embora especifique que a organização deva planear ações para enfrentar os riscos, não contém requisitos para métodos formais de gestão de risco ou para um processo de gestão de risco documentado (à semelhança do que já vinha preconizado na ISO 9001:2015). Os laboratorios podem decidir desenvolver ou não uma metodologia mais extensa de gestão de risco do que o exigido na ISO 17025:2017, através da aplicação de outras orientações ou normas, como por exemplo, em conformidade com os requisitos da ISO 31000. O organismo de acreditação, contudo, avalia se o laboratorio estabeleceu ações apropriadas para lidar com os riscos e as oportunidades. Abraçar a questão da gestão dos riscos para prevenir possíveis falhas requer uma importante definição dos objetivos a nível global. Por outro lado, a abordagem do laboratorio como um conjunto de processos, permite definir objetivos para cada um deles, inclusivamente no da validação dos métodos, com uma avaliação profunda do que pode impedir a obtenção dos objetivos definidos. Para isso, torna-se necessário definir os riscos associados a cada fase de implementação de um método analítico. A aprovação da 3ª Edição do guia Eurachem/CITAC Guide to "Quality in Analytical Chemistry – An Aid to Accreditation" pelo "Eurachem Education and Training Working Group" irá naturalmente constituir uma excelente ferramenta na ajuda aos laboratorios no que a este novo tema diz respeito. Enquanto isso, neste trabalho, irá ser efetuada uma análise dos possíveis riscos associados ao desenvolvimento e à validação de métodos analíticos aplicados à Toxicologia Forense, de acordo com as causas possíveis e avaliação das respetivas consequências, em pontos chave como são a seleção dos métodos, a escolha dos parâmetros de validação, a definição dos recursos necessários, a análise dos resultados de validação e o controlo da qualidade implementado.info:eu-repo/semantics/publishedVersio

Comparison of endogenous GHB concentrations in blood and hair in death cases with emphasis on the post mortem interval

Gamma-hydroxybutyric acid (GHB) is an endogenous compound which has a story of clinical use and illicit abuse since the 1960’s. The possibility to use a multi-sample approach of GHB evaluation, including whole blood and hair, to better characterize a forensic toxicology case and evaluate a possible causal association with the death, is an exciting up-to-date issue. In addition, its post-mortem behaviour, namely regarding degradation and metabolism, has been increasingly investigated as a putative biomarker for post-mortem interval (PMI) estimation. Thus, In order to contribute to clarify this specific aspect, whole blood and hair post-mortem GHB levels were evaluated in thirty two real cases with previous information on death and autopsy data. The results obtained suggest that the PMI (until 5 days between death and sampling) influences GHB whole blood concentration, but not GHB levels in hair samples. No differences were encountered for the other parameters evaluated, including age, gender, cause of death and presence or absence of substances. This study brings new insights regarding the usefulness of GHB levels in forensic toxicology, which might be further strengthened with larger, but comparable, studies from other laboratories and institutions in the context of legal medicine.info:eu-repo/semantics/acceptedVersio

Aplicação do controlo de qualidade interno e avaliação de resultados em toxicologia forense

Poster apresentado no XII Encontro Nacional de Cromatografia, Aveiro, dezembro de 2022A garantia da qualidade dos resultados do Serviço de Química e Toxicologia Forense (SQTF) do INMLCF,IP é assegurada pelo cumprimento do programa de controlo de qualidade interno (e, sempre que possível, pela participação em ensaios interlaboratoriais). O Controlo de Qualidade em laboratórios forenses constitui uma ferramenta essencial para garantir a confiança dos seus clientes. Paralelamente, enquanto laboratório forense de caráter essencialmente analítico, o seu produto corresponde à execução de ensaios laboratoriais e à correspondente emissão dos resultados obtidos. Assim, para um produto deste tipo, as ações de Controlo da Qualidade dividem-se em ações de âmbito interno (orientado para o controlo da precisão e cujos critérios dependem do laboratório) e externo (voltado para o controlo da exatidão, normalmente dependente duma intervenção externa). Este trabalho pretende abordar as ações de âmbito interno, como são os conceitos de controlo de qualidade interno e de avaliação dos resultados aplicadas a ensaios realizados por cromatografia, bem como a sua correspondente aplicação efetiva no Laboratório. O Controlo de Qualidade Interno deve definir um conjunto de mecanismos capazes de garantir a verificação da conformidade de parâmetros predefinidos para aceitação dos resultados dos ensaios e a sua metodologia deve ser adaptada aos diferentes tipos de procedimentos de ensaio. A avaliação e o correspondente cálculo dos resultados revelam-se tão importantes quanto analisar amostras representativas e executar adequadamente a técnica de análise selecionada. Avaliar e calcular corretamente os resultados e expressá-los de forma adequada tem uma influência crítica no valor probatório do resultado apresentado em cada relatório emitido. As principais medidas adotadas pelo SQTF para garantir a qualidade dos seus resultados, baseadas em referências internacionais, preveem a inclusão de controlos internos da qualidade. Paralelamente, todos os métodos analíticos possuem critérios de aceitação/rejeição de resultados, os métodos quantitativos utilizam padrões internos e os controlos são representativos da matriz das amostras a analisar. Com esta política, o SQTF pretende a manutenção da qualidade dos serviços prestados, reconhecendo o impacto que têm para a sociedade em geral e para a Administração da Justiça em particular.info:eu-repo/semantics/publishedVersio

GC-MS – Still standing for clinical and forensic analysis: validation of a multidrug method to detect and quantify illicit drugs

An SPE-GC-MS analytical method using whole blood samples has been developed and validated to detect and quantify nineteen compounds belonging to the Drugs of Abuse (DA) groups of cocaine and metabolites, opiates and new psychoactive substances (NPS). The method detailed here is necessary because the recreational consumption of these DA has increased considerably in recent years and poly-drug-consumption is now very common. The method developed was both specific and selective. Three different working ranges have been defined due to the differences between therapeutic, toxic and lethal concentrations of DA. Linearity was confirmed for the defined working ranges of all DA, except pseudoephedrine, ephedrine, norephedrine, and TFMPP. Since the remaining nineteen substances showed heteroscedasticity, six ponderation factors were studied to find the best fit for each compound. Limits of Detection and Lower Limits of Quantification have been studied and defined. No carryover was noted, with acceptable extraction recoveries. The achievement of all validation criteria according to international guidelines allows the application of the proposed method in routine forensic analysis. Using this method can also significantly reduce response times and GC-MS analysis costs.info:eu-repo/semantics/acceptedVersio

Analysis of organophosphorus pesticides in whole blood by GC-MS-μECD with forensic purposes

In the present work, two multi-residue methods for the determination of ten organophosphorus pesticides (OPs), namely chlorfenvinphos, chlorpyrifos, diazinon, dimethoate, fenthion, malathion, parathion, phosalone, pirimiphos-methyl and quinalphos, in post-mortem whole blood samples are presented. The adopted procedure uses GC-MS for screening and quantitation, and GC-µECD (electron capture detector) for compound confirmation. Three different Solid Phase Extraction (SPE) procedures for OPs with Oasis® hydrophilic lipophilic balanced (HLB) and Sep-Pak® C18 cartridges were tested, and followed by GC-µECD and GC-MS analysis. The Sep-Pak® C18 cartridges extraction procedure was selected since it generated analytical signals 5 times higher than those obtained with the two different Oasis® HLB cartridges extraction procedures. The method has shown to be selective for the isolation of selected OPs as well as to the chosen internal standard (ethion) in postmortem blood samples. Calibration curves between 50 and 5000 ng/mL were prepared using weighted linear regression models (1/x2). It was not possible to establish a working range for fenthion by GC-µECD due to the lower sensitivity of the detector to this compound, whereas for pirimiphos-methyl it was set between 500 and 5000 ng/mL. The limit of quantitation was established at 50 ng/mL for all analytes, except for pirimiphos-methyl by GC-µECD analysis (500 ng/mL). The average extraction efficiency ranged from 72 to 102%. The developed methods were considered robust and fit for the purpose, and had already been adopted in the laboratory routine analysis.info:eu-repo/semantics/acceptedVersio

Antidepressants detection and quantification in whole blood samples by GC–MS/MS, for forensic purposes

Depression is among the most prevalent psychiatric disorders of our society, leading to an increase in antidepressant drug consumption that needs to be accurately determined in whole blood samples in Forensic Toxicology Laboratories. For this purpose, this work presents a new gas chromatography tandem mass spectrometry (GC–MS/MS) method targeting the simultaneous and rapid determination of 14 common Antidepressants in whole blood: 13 Antidepressants (amitriptyline, citalopram, clomipramine, dothiepin, fluoxetine, imipramine, mianserin, mirtazapine, nortryptiline, paroxetine, sertraline, trimipramine and venlafaxine) and 1 Metabolite (N-desmethylclomipramine). Solid-phase extraction was used prior to chromatographic separation. Chromatographic and MS/MS parameters were selected to improve sensitivity, peak resolution and unequivocal identification of the eluted analyte. The detection was performed on a triple quadrupole tandem MS in selected ion monitoring (SIM) mode in tandem, using electronic impact ionization. Clomipramine-D3 and trimipramine-D3 were used as deutered internal standards. The validation parameters included linearity, limits of detection, lower limit of quantification, selectivity/ specificity, extraction efficiency, carry-over, precision and robustness, and followed internationally accepted guidelines. Limits of quantification and detection were lower than therapeutic and subtherapeutic concentration ranges. Overall, the method offered good selectivity, robustness and quick response (<16 min) for typical concentration ranges, both for therapeutic and lethal levels.info:eu-repo/semantics/acceptedVersio

MDMA Intoxication in potential donor with cardiac arrest – A case report

Poster apresentado no 57th Annual Meeting of The International Association of Forensic Toxicologists, Birmingham (Reino Unido), 2019Amphetamines consumption is still an important public health issue, namely in terms of compounds variability and disposition to consumers. However, some of them, still classic substances, keep standing in the illicit market, with remarkable and continuous success. That is the case of MDMA. Nevertheless, there is always new information and data on MDMA intoxication, both in vivo as in post-mortem context. The authors report an intoxication case with MDMA in an 18 years old male, who was considered a potential organ donor, after a cardiac arrest. Whole blood samples were collected in vivo, at the Emergency Room (ER), in different moments, and post-mortem, at the Forensic Pathology Service of the National Institute of Legal Medicine and Forensic Science. After a general screening procedure, samples were extracted by an SPE procedure (OASIS® MCX) followed by a GC-MS single quad analysis. The post-mortem whole blood sample was positive for Lidocaine (< 500 ng/mL), compatible with ER intervention, and positive for MDMA (2278 ng/mL) and MDA (49 ng/mL), while the whole blood samples collected in vivo (during the maintenance of the individual under advanced life support), were positive for MDMA (between 504 ng/mL and 1918 ng/mL) and MDA (between 20 ng/mL and 89 ng/mL). The samples were negative for other substances, namely ethanol, other drugs of abuse and medicines. Interpretation of these results is pivotal to understand the behaviour of the substance. So, in this case, MDMA post-mortem behaviour should be carefully evaluated, considering as possible influencers, in the specific context of the case, the time lapse between the death verification, the maintenance of the advanced life support and the body manipulation for organ collection purposes. Also referred and discussed is the ante-mortem/post-mortem ratio of MDMA obtained values, compared with literature references. No doubt that death was due to MDMA intoxication, but information from the in vivo samples analysis suggests that this type of sample should also be considered, in a complementary role, whenever possible.N/