Aplicação da Toxicologia Analítica Verde no desenvolvimento de metodologias baseadas em LC-MS para detecção de Novas Substâncias Psicoativas em amostras biológicas / Application of Green Analytical Toxicology to the development of LC-MS-based methodologies for the detection of New Psychoactive Substances in biological samples

No início dos anos 2000, as Novas Substâncias Psicoativas (NPS) emergiram de forma sem precedentes causando uma drástica mudança no mercado de drogas sintéticas mundial. Estas substâncias são sintetizadas para fins ilícitos e mimetizam o efeito psicoativo das drogas tradicionais. Até o momento, mais de 1000 substâncias foram reportadas mundialmente, representando um grande problema de saúde pública principalmente associado ao desconhecimento das suas propriedades toxicológicas. Por este motivo, métodos analíticos para detectar e quantificar estas substâncias em materiais biológicos são importantes nos casos de toxicologia analítica e forense. Contudo, a tendência de reduzir o impacto ambiental destas metodologias tem ganhado popularidade com a Toxicologia Analítica Verde (GAT). Portanto, o objetivo do presente trabalho foi desenvolver novas técnicas analíticas para analisar as principais classes de NPS em amostras biológicas enquanto aplicando os princípios sustentáveis estabelecidos pela GAT. Os resultados obtidos no presente trabalho são apresentados como coletânea de artigos científicos publicados em revistas. Estes estão descritos nos capítulos 4 a 8. No capítulo 4, uma revisão sobre os desafios no desenvolvimento de técnicas de preparo de amostra para fins forenses é abordada com foco no uso das matrizes secas. No Capítulo 5, está descrito a aplicação da microextração líquido-líquido dispersiva para catinonas sintéticas em amostras de sangue total e urina. No capítulo 6, o artigo descreve o desenvolvimento da técnica microextração líquido-líquido homogênea com solventes de hidrofilicidade comutável para canabinoides sintéticos em amostras de plasma. No capítulo 7, a microextração em fase líquida em placas de 96 poços, cunhada extração paralela em membranas artificiais líquidas, foi desenvolvida para diferentes classes de drogas de abuso, incluindo NPS. O capítulo 8 mostra o desenvolvimento de uma extração por eletromembrana também no formato de placa de 96 poços para catinonas sintéticas em amostras de sangue total. Em todos os trabalhos, as técnicas de extração foram desenvolvidas, otimizadas e validadas. Os princípios da GAT foram aplicados de diferentes formas, como reduzindo o volume de amostra, simplificando os procedimentos, evitando o uso de solventes orgânicos, dentre outros. Assim, alternativas mais sustentáveis para a análise de drogas de abuso em amostras biológicas foram apresentadas e estas ajudam a consolidar e difundir o conceito do desenvolvimento de métodos analíticos com consciência ambiental além de fornecer ferramentas para auxiliar o controle das NPS no país

In the early 2000s, New Psychoactive Substances (NPS) emerged and unprecedentedly changed the illicit drug market. These substances are synthesized for illicit purposes and mimic the psychoactive effect of traditional drugs of abuse. To date, more than 1000 substances have been reported worldwide, representing a major public health problem mainly associated with their mostly unknown toxicological properties. In this context, analytical methods able to detect and quantitate these new drugs in biological specimens are important in cases of analytical and forensic toxicology. However, reducing the environmental impact of these methodologies has recently gained popularity with Green Analytical Toxicology (GAT). Therefore, the aim of this work was to develop new analytical techniques to analyze the main classes of NPS in biological samples while applying the environmentally friendly principles established by GAT. The results obtained throughout the development of the present work were split into four papers (chapters 4-8). In chapter 4, a review of common challenges faced during the development of new sample preparation techniques for forensic applications is described focusing on the use of dried matrices. In chapter 5, the application of dispersive liquid-liquid microextraction for synthetic cathinones in whole blood and urine samples is described. In chapter 6, the application of the somewhat recent switchable hydrophilicity solvent-based homogenous liquidliquid microextraction to synthetic cannabinoids in plasma samples is reported. In chapter 7, liquid-phase microextraction in the 96-well plate format, termed parallel artificial liquid membrane extraction, for different classes of drugs of abuse, including NPS, in plasma samples is presented. In chapter 8, an electromembrane extraction in the 96-well plate format for synthetic cathinones in whole blood samples was developed. In this work, sample preparation techniques were developed, optimized and validated. The principles of sustainable chemistry in method development were applied in different ways, such as reducing the sample volume, simplifying procedures, avoiding the use of organic solvents, among others. Thus, greener alternatives were presented for the analysis of drugs of abuse in biological samples and contribute to consolidate and spread this trend of environmental consciousness during method development. Additionally, valuable techniques that can be used in the combat against NPS were provided

Spongiform leukoencephalopathy unveiled in an autopsy of a drug abuser.

Toxic leukoencephalopathy (TLE) is a rare neurological debilitating and fatal condition. It has been previously associated with exposure to leukotoxic offenders such as chemotherapy, cranial radiation, certain drugs, and environmental factors. Currently, it is a commoner white matter syndrome resulting from increased substance abuse, classically by inhaled heroin and other opioids. Herein, we report a case of fatal TLE unveiled in an autopsy of a drug abuser. A 24-year-old male was found dead on the roadside. A day before, he was located in a state of delirium. In this case, the autopsy findings and histopathology characteristics of cerebral cortex involvement particularly directed to speculate the heroine as the principal offender.

Spongiform leukoencephalopathy unveiled in an autopsy of a drug abuser

ABSTRACT Toxic leukoencephalopathy (TLE) is a rare neurological debilitating and fatal condition. It has been previously associated with exposure to leukotoxic offenders such as chemotherapy, cranial radiation, certain drugs, and environmental factors. Currently, it is a commoner white matter syndrome resulting from increased substance abuse, classically by inhaled heroin and other opioids. Herein, we report a case of fatal TLE unveiled in an autopsy of a drug abuser. A 24-year-old male was found dead on the roadside. A day before, he was located in a state of delirium. In this case, the autopsy findings and histopathology characteristics of cerebral cortex involvement particularly directed to speculate the heroine as the principal offender.

The role of microorganisms in the biotransformation of psychoactive substances and its forensic relevance: a critical interdisciplinary review.

Microorganisms are widespread on the planet being able to adapt, persist, and grow in diverse environments, either rich in nutrient sources or under harsh conditions. The comprehension of the interaction between microorganisms and drugs is relevant for forensic toxicology and forensic chemistry, elucidating potential pathways of microbial metabolism and their implications. Considering the described scenario, this paper aims to provide a comprehensive and critical review of the state of the art of interactions amongst microorganisms and common drugs of abuse. Additionally, other drugs of forensic interest are briefly discussed. This paper outlines the importance of this area of investigation, covering the intersections between forensic microbiology, forensic chemistry, and forensic toxicology applied to drugs of abuse, and it also highlights research potentialities. Key points: Microorganisms are widespread on the planet and grow in a myriad of environments.Microorganisms can often be found in matrices of forensic interest.Drugs can be metabolized or produced (e.g. ethanol) by microorganisms.

Epidemio-toxicological profile of suicide cases: analysis from a forensic unit in Brazil.

The suicide phenomenon involves complex interactions between psychological, biological, cultural and socio-environmental factors. This study aimed to assess the epidemiological and toxicological profiles of officially confirmed suicide victims. A retrospective study was performed using autopsy reports, forensic anthropology examinations and police summaries of all suicide cases that underwent toxicological analysis at an Official Forensic Laboratory (Minas Gerais, Brazil) in 1 year. The sample set was composed of 351 suicide victims, predominantly men (70.32%), most of them were adults between 31 and 64 years old (62.11%), with mixed skin colour (48.89%) and low educational level (66.44%). The most common suicide method was hanging (57.79%), followed by intoxication (30.45%). Most victims presented positive toxicological results (56.41%), especially for the presence of medicines (37.6%), illicit drugs (36.3%) and pesticides (26.1%). Our study corroborated previous data that most suicide victims have low educational levels. The most common toxicological findings were medicines, especially prescription drugs, followed by illicit drugs and pesticides. We hope this study contributes to reflections and planning of preventive suicide programmes, considering the described profiles of victims.

Dispersive liquid-liquid microextraction of 11-nor-Δ9-tetrahydrocannabinol-carboxylic acid applied to urine testing.

Aim: THC-COOH is the major metabolite of Δ9-tetrahydrocannabinol commonly tested in urine to determine cannabis intake. In this study, a method based on dispersive liquid-liquid microextraction was developed for testing THC-COOH in urine. Materials & methods: Hydrolyzed urine specimens were extracted via dispersive liquid-liquid microextraction with acetonitrile (disperser solvent) and chloroform (extraction solvent). Derivatization was performed with N,O-Bis(trimethylsilyl)trifluoroacetamide with 1% trichloro(chloromethyl)silane. Analysis was performed by GC-MS/MS. Results: The method showed acceptable linearity (5-500 ng/ml), imprecision (<10.5%) and bias (<4.9%). Limits of detection and quantitation were 1 and 5 ng/ml, respectively. Twenty-four authentic samples were analyzed, with 22 samples being positive for THC-COOH. Conclusion: The proposed method is more environmentally friendly and provided good sensitivity, selectivity and reproducibility.

Tweetable abstract Green analytical toxicology: Dispersive liquid­liquid microextraction applied to the analysis of THC-COOH in urine by GC­MS/MS.

Dried matrix spots in forensic toxicology.

Dried matrix spots (DMS) has gained the attention of different professionals in different fields, including toxicology. Investigations have been carried out in order to assess the potential of using DMS for the analysis of illicit substances, the main interest of forensic toxicologists. This technique uses minimal volumes of samples and solvents, resulting in simple and rapid extraction procedures. Furthermore, it has proved to increase analyte stability, improving storage and transportation. However, DMS presents some limitations: the hematocrit influencing accuracy and inconsistencies regarding the means of spotting samples and adding internal standard on paper. Thus, we provide an overview of analytical methodologies with forensic applications focusing on drugs of abuse and discussing the main particularities, limitations and achievements.

A systematic review of quantitative analysis of cannabinoids in oral fluid.

Cannabis sativa L. is a substance widely used around the world for recreational and medicinal purposes. Oral fluid has been investigated as an alternative biological matrix for demonstrating the illegal use of cannabis, particularly in situations where its recent use needs to be identified. In the last two decades, many methods have been developed to detect and quantify cannabinoids in oral fluid, especially for Δ9 -tetrahydrocannabinol, the primary psychoactive substance of cannabis. However, some aspects must be considered in the use of these techniques, such as cannabinoids recoveries or extraction efficiency from different oral fluid collection devices/containers. Pharmacokinetic studies have shown that the presence of minor cannabinoids and metabolites in the analysis of oral fluid may be valuable in interpreting tests, which indicates the need to improve the sensitivity of detecting low concentrations. The aim of this review is to summarize and to describe the methodologies for the quantitative analysis of cannabinoids in oral fluid that have previously been investigated. A systematic search for articles was performed of four different databases, using the descriptor "cannabinoids and oral fluid". Forty-seven studies that examined quantitative methods were identified. The analytical data described in these articles, including oral fluid collection, sample preparation, cannabinoids recovery and extraction efficiency, detection instruments, and quantification limits, were analyzed. The discussion of these particular features of cannabinoid analysis in oral fluid could help to improve or to develop methods for use in Forensic Toxicology.

[Toxicology of suicide cases in the state of Rio Grande do Sul, Brazil, 2017 to 2019Perfil toxicológico de los casos de suicidio en Rio Grande do Sul (Brasil), 2017-2019]. / Perfil toxicológico dos suicídios no Rio Grande do Sul, Brasil, 2017 a 2019.

OBJECTIVE: To describe the toxicology of suicide cases recorded in the state of Rio Grande do Sul, Brazil, from 2017 to 2019. METHOD: The present descriptive, cross-sectional study examined all the medico-legal reports and police records related to suicide deaths in the state. Multiple correspondence analyses were performed along with independent logistic regression models having ethanol, anxiolytic and antidepressant drugs, illicit drugs, and non-medical substances as dependent variables. RESULTS: Ethanol was investigated in 2 978 samples, with positive results in 28.5%. The odds of a positive ethanol finding were 0.5 time higher (95%CI: 1.1; 2.2) for suicides occurring at night, 1.0 (95%CI: 1.4; 2.9) time higher for suicides occurring on weekends, and 0.9 (95%CI: 1.3; 2.7) time higher in individuals with a prior criminal record. Investigation of psychotropic drugs (2 900 samples) was positive in 30.4% samples. Anxiolytics were the most common medication detected, with 1.5 (95%CI: 1.6; 4.1) time higher odds of occurrence in women and 0.8 time higher odds (95%CI: 1.2; 2.7) for suicides occurring in the fall-winter. The odds of detecting illicit drugs (n = 338) were 4.1 times higher (95%CI: 1.9; 14.4) in the regions of Pelotas (south of the state) vs. Passo Fundo (north), and 1.2 (95%CI: 1.3; 3.6) time higher in cases with positive ethanol results, without significant difference between adolescents and adults. CONCLUSIONS: Despite the lack of evidence on causality, the present results support a link between suicide and several psychoactive drugs. Medico-legal experts should be guided regarding the need to perform toxicological tests in all suicide cases.

OBJETIVO: Describir el perfil toxicológico de todas las víctimas de suicidio en Rio Grande do Sul desde el 2017 hasta el 2019. MÉTODOS: En este estudio descriptivo y transversal se consultaron todos los informes periciales y policiales sobre las muertes por suicidio en el estado. Se realizaron análisis de correspondencia múltiple y se crearon modelos independientes de regresión logística, con empleo de etanol, productos ansiolíticos y antidepresivos, sustancias ilícitas y agentes tóxicos no medicamentosos como variables dependientes. RESULTADOS: Se realizaron 2 978 exámenes de alcoholemia, con resultado positivo en un 28,5%. La probabilidad de obtener resultados positivos para alcoholemia aumentó 0,5 (IC95%: 1,1-2,2) en casos de suicidio durante la noche, 1,0 (IC95%: 1,4-2,9) en casos de suicidio en los fines de semana y 0,9 (IC95%: 1,3-2,7) cuando había antecedentes penales. En la investigación de productos psicotrópicos (2 900 muestras) se detectó algún medicamento en un 30,4%. Los ansiolíticos fueron la clase detectada con más frecuencia, con un aumento de la probabilidad de 1,5 (IC95%: 1,6-4,1) en las mujeres y de 0,8 (IC95%: 1,2-2,7) en casos de suicidio durante el otoño y el invierno. El aumento de la probabilidad de detección de sustancias ilícitas (n = 338) fue de 4,1 (IC95%: 1,9-14,4) en la macrorregión de Pelotas en comparación con la de Passo Fundo y de 1,2 (IC95%: 1,3-3,6) en personas con resultados positivos en la prueba de detección de etanol, sin que hubiera ninguna diferencia significativa entre adolescentes y adultos. CONCLUSIONES: Aun sin haberse comprobado la causalidad, los resultados muestran que existe un vínculo entre el suicidio y diversos productos psicoactivos. Es preciso orientar a los médicos legistas con respecto a la necesidad de realizar exámenes toxicológicos en todos los casos de suicidio.

Quantification of amphetamine and derivatives in oral fluid by dispersive liquid-liquid microextraction and liquid chromatography-tandem mass spectrometry.

The abuse of stimulants such as amphetamine, methamphetamine, ecstasy (MDMA), and their analogues (MDEA and MDA) has been increasing considerably worldwide since 2009. In this work, an analytical method using dispersive liquid-liquid microextraction (DLLME) to determine amphetamine and derivatives in oral fluid samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated. Linearity was achieved between 20 to 5000 ng/mL (r>0.992, 1/x² weighted linear regression), with a limit of quantification (LOQ) of 20 ng/mL. Imprecision (%relative standard deviation) and bias (%) were not higher than 9.1 and -12.3%, respectively. The matrix effect was lower than 14.6%, with no carryover observed up to 5000 ng/mL and no interference with 10 different oral fluid matrix sources and against 14 pharmaceuticals and other common drugs of abuse. MDMA, MDA, and MDEA in processed samples were stable up to 24 h at autosampler (10°C); and amphetamine and methamphetamine up to 18 h. The developed method was successfully applied to authentic oral fluid analyses (n = 140). The proposed method is an example of the Green Analytical Toxicology, since it reduces both the amount of solvent required in samples preparation and the quantity of solvents and reagents used in analytical-instrumental stage, as well as requires a minimal sample volume, being a cheaper, quicker and more ecological alternative to conventional methods. Obtained results showed that DLLME extraction combined with LC-MS/MS is a fast and simple method to quantify amphetamine derivatives in oral fluid samples.

Optimization of QuEChERS extraction for detection and quantification of 20 antidepressants in postmortem blood samples by LC-MS/MS.

In this study, a comprehensively optimization of QuEChERS (quick, easy, cheap, effective, rugged and safe) method using design of experiments (DOE) was conducted to evaluate the best conditions to obtain the most effective extraction. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis was performed to identify and quantify the antidepressants, with electrospray ionization acquired in positive mode. The method was validated for all analytes; the calibration curves were linear from 10-1000ng/mL, with R2>0.98, and with LOD and LOQ defined as 10ng/mL. Method imprecision and bias were less than 14.3% and 18.9%, respectively. Neither carryover nor interferences were observed. Overall, the optimized method was applied in postmortem real sample analysis to quantify the antidepressants. This study showed a viable method that can be applied for routine forensic analysis, with a quick and easy sample preparation and a rapid total run time of 8min for each analysis.

Perfil toxicológico dos suicídios no Rio Grande do Sul, Brasil, 2017 a 2019 / Perfil toxicológico de los casos de suicidio en Rio Grande do Sul (Brasil), 2017-2019 / Toxicology of suicide cases in the state of Rio Grande do Sul, Brazil, 2017 to 2019

RESUMO Objetivo. Descrever o perfil toxicológico de todas as vítimas de suicídio no Rio Grande do Sul, Brasil, de 2017 a 2019. Métodos. Neste estudo descritivo e transversal, foram consultados todos os laudos periciais e as ocorrências policiais relacionados aos óbitos por suicídio no estado. Foram realizadas análises de correspondência múltipla e construídos modelos independentes de regressão logística, tendo como variáveis dependentes o etanol, os ansiolíticos, os antidepressivos, as substâncias ilícitas e os agentes tóxicos não medicamentosos. Resultados. Foram realizados 2 978 exames de alcoolemia, com resultado positivo em 28,5%. A chance de resultados positivos para alcoolemia foi 0,5 (IC95%: 1,1 a 2,2) vez maior para suicídio durante a noite, 1,0 (IC95%: 1,4 a 2,9) vez maior para suicídio aos finais de semana e 0,9 (IC95%: 1,3 a 2,7) vez maior na presença de antecedentes criminais. A pesquisa de psicotrópicos (2 900 amostras) detectou algum medicamento em 30,4%. Os ansiolíticos foram a classe mais frequente, com chance 1,5 (IC95%: 1,6 a 4,1) vez maior em mulheres e 0,8 (IC95%: 1,2 a 2,7) vez maior para suicídios ocorridos no outono-inverno. As substâncias ilícitas (n = 338) tiveram chance 4,1 (IC95%: 1,9 a 14,4) vezes maior de detecção na macrorregião de Pelotas em relação à de Passo Fundo e 1,2 (IC95%: 1,3 a 3,6) vez maior em pessoas com resultados positivos para etanol. Não houve diferença significativa entre adolescentes e adultos. Conclusões. Embora sem evidência de causalidade, os resultados mostram um vínculo entre o suicídio e diversos psicoativos. Os médicos legistas devem ser orientados quanto à necessidade de realização de exames toxicológicos em todos os casos de suicídio.

ABSTRACT Objective. To describe the toxicology of suicide cases recorded in the state of Rio Grande do Sul, Brazil, from 2017 to 2019. Method. The present descriptive, cross-sectional study examined all the medico-legal reports and police records related to suicide deaths in the state. Multiple correspondence analyses were performed along with independent logistic regression models having ethanol, anxiolytic and antidepressant drugs, illicit drugs, and non-medical substances as dependent variables. Results. Ethanol was investigated in 2 978 samples, with positive results in 28.5%. The odds of a positive ethanol finding were 0.5 time higher (95%CI: 1.1; 2.2) for suicides occurring at night, 1.0 (95%CI: 1.4; 2.9) time higher for suicides occurring on weekends, and 0.9 (95%CI: 1.3; 2.7) time higher in individuals with a prior criminal record. Investigation of psychotropic drugs (2 900 samples) was positive in 30.4% samples. Anxiolytics were the most common medication detected, with 1.5 (95%CI: 1.6; 4.1) time higher odds of occurrence in women and 0.8 time higher odds (95%CI: 1.2; 2.7) for suicides occurring in the fall-winter. The odds of detecting illicit drugs (n = 338) were 4.1 times higher (95%CI: 1.9; 14.4) in the regions of Pelotas (south of the state) vs. Passo Fundo (north), and 1.2 (95%CI: 1.3; 3.6) time higher in cases with positive ethanol results, without significant difference between adolescents and adults. Conclusions. Despite the lack of evidence on causality, the present results support a link between suicide and several psychoactive drugs. Medico-legal experts should be guided regarding the need to perform toxicological tests in all suicide cases.

RESUMEN Objetivo. Describir el perfil toxicológico de todas las víctimas de suicidio en Rio Grande do Sul desde el 2017 hasta el 2019. Métodos. En este estudio descriptivo y transversal se consultaron todos los informes periciales y policiales sobre las muertes por suicidio en el estado. Se realizaron análisis de correspondencia múltiple y se crearon modelos independientes de regresión logística, con empleo de etanol, productos ansiolíticos y antidepresivos, sustancias ilícitas y agentes tóxicos no medicamentosos como variables dependientes. Resultados. Se realizaron 2 978 exámenes de alcoholemia, con resultado positivo en un 28,5%. La probabilidad de obtener resultados positivos para alcoholemia aumentó 0,5 (IC95%: 1,1-2,2) en casos de suicidio durante la noche, 1,0 (IC95%: 1,4-2,9) en casos de suicidio en los fines de semana y 0,9 (IC95%: 1,3-2,7) cuando había antecedentes penales. En la investigación de productos psicotrópicos (2 900 muestras) se detectó algún medicamento en un 30,4%. Los ansiolíticos fueron la clase detectada con más frecuencia, con un aumento de la probabilidad de 1,5 (IC95%: 1,6-4,1) en las mujeres y de 0,8 (IC95%: 1,2-2,7) en casos de suicidio durante el otoño y el invierno. El aumento de la probabilidad de detección de sustancias ilícitas (n = 338) fue de 4,1 (IC95%: 1,9-14,4) en la macrorregión de Pelotas en comparación con la de Passo Fundo y de 1,2 (IC95%: 1,3-3,6) en personas con resultados positivos en la prueba de detección de etanol, sin que hubiera ninguna diferencia significativa entre adolescentes y adultos. Conclusiones. Aun sin haberse comprobado la causalidad, los resultados muestran que existe un vínculo entre el suicidio y diversos productos psicoactivos. Es preciso orientar a los médicos legistas con respecto a la necesidad de realizar exámenes toxicológicos en todos los casos de suicidio.

Use of bone marrow for detection of toxic chemicals for the elucidation of poisoning in forensic veterinary medicine / Uso de medula óssea para detecção de produtos químicos tóxicos para a elucidação de intoxicação em patologia veterinária forense

In forensic toxicology, the detection of toxic chemicals from human bone marrow is often used in cases with an extended post mortem interval; however, in veterinary medicine, this practice is not used. Therefore, this study was performed to investigate the suitability of bone marrow for toxicological analysis in dogs and cats. Six animals with suspected poisoning were selected; the carcasses were sent for necropsy, and the organs were collected and preserved in buffered formalin and processed routinely for histological examination. In addition, bone marrow samples from the femur, humerus, and tibia were collected for toxicological analysis by liquid chromatography coupled to mass spectrometry detection (LC-MS). This analysis confirmed the presence of aldicarb, aldicarb sulfone, asulam, carbendazim, chlorpyrifos, dichlorvos, thifensulfuron methyl and trifloxysulfuron-sodium and associated with clinical symptoms and anatomo-histopathological alterations it was recognized the poisonings. It is expected that this study will promote the toxicological investigation of bone marrow and open avenues for the use of this tissue as an option for the detection of toxic chemicals in cases of forensic pathology.(AU)

Na toxicologia forense, a detecção de substâncias químicas tóxicas provenientes da medula óssea humana é frequentemente usada em casos com intervalo post mortem prolongado; no entanto, na medicina veterinária, essa prática não é utilizada. Portanto, este estudo foi realizado para investigar a utilização da medula óssea nas análises toxicológicas em cães e gatos. Seis animais com suspeita de intoxicação foram selecionados; as carcaças foram enviadas para necropsia e os órgãos foram coletados e preservados em formalina tamponada e processados rotineiramente para exame histológico. Amostras de medula óssea de fêmur, úmero e tíbia foram coletadas para análise toxicológica por cromatografia líquida acoplada à espectrometria de massa-massa (LC-MS). A análise por LC-MS confirmou a presença dos agrotóxicos aldicarbe, aldicarbe sulfona, asulam, carbendazim, clorpirifós, diclorvós, tifensulfuron metil e trifloxisulfuron-sódico, e em associação com sinais clínicos e achados anatomo-histopatológicos comprovou-se as intoxicações. Espera-se que este estudo promova a utilização da medula óssea como uma opção na investigação toxicológica para a detecção de produtos químicos tóxicos em casos de patologia forense.(AU)

Use of bone marrow for detection of toxic chemicals for the elucidation of poisoning in forensic veterinary medicine / Uso de medula óssea para detecção de produtos químicos tóxicos para a elucidação de intoxicação em patologia veterinária forense

In forensic toxicology, the detection of toxic chemicals from human bone marrow is often used in cases with an extended post mortem interval; however, in veterinary medicine, this practice is not used. Therefore, this study was performed to investigate the suitability of bone marrow for toxicological analysis in dogs and cats. Six animals with suspected poisoning were selected; the carcasses were sent for necropsy, and the organs were collected and preserved in buffered formalin and processed routinely for histological examination. In addition, bone marrow samples from the femur, humerus, and tibia were collected for toxicological analysis by liquid chromatography coupled to mass spectrometry detection (LC-MS). This analysis confirmed the presence of aldicarb, aldicarb sulfone, asulam, carbendazim, chlorpyrifos, dichlorvos, thifensulfuron methyl and trifloxysulfuron-sodium and associated with clinical symptoms and anatomo-histopathological alterations it was recognized the poisonings. It is expected that this study will promote the toxicological investigation of bone marrow and open avenues for the use of this tissue as an option for the detection of toxic chemicals in cases of forensic pathology.(AU)

Na toxicologia forense, a detecção de substâncias químicas tóxicas provenientes da medula óssea humana é frequentemente usada em casos com intervalo post mortem prolongado; no entanto, na medicina veterinária, essa prática não é utilizada. Portanto, este estudo foi realizado para investigar a utilização da medula óssea nas análises toxicológicas em cães e gatos. Seis animais com suspeita de intoxicação foram selecionados; as carcaças foram enviadas para necropsia e os órgãos foram coletados e preservados em formalina tamponada e processados rotineiramente para exame histológico. Amostras de medula óssea de fêmur, úmero e tíbia foram coletadas para análise toxicológica por cromatografia líquida acoplada à espectrometria de massa-massa (LC-MS). A análise por LC-MS confirmou a presença dos agrotóxicos aldicarbe, aldicarbe sulfona, asulam, carbendazim, clorpirifós, diclorvós, tifensulfuron metil e trifloxisulfuron-sódico, e em associação com sinais clínicos e achados anatomo-histopatológicos comprovou-se as intoxicações. Espera-se que este estudo promova a utilização da medula óssea como uma opção na investigação toxicológica para a detecção de produtos químicos tóxicos em casos de patologia forense.(AU)

SNPs from BCHE and DRD3 genes associated to cocaine abuse amongst violent individuals from Sao Paulo, Brazil.

Violence and drug abuse are highly destructive phenomena found world-wide, especially in Brazil. They seem to rise proportionally to one another and possibly related. Additionally, genetics may also play a role in drug abuse. This study has focused on identifying the use of cocaine within postmortem cases arriving at the Institute of Legal Medicine of Sao Paulo as well as the presence of certain single nucleotide polymorphisms (SNPs) to better understand one's susceptibility to abuse the drug. Both hair and blood samples have been extracted through a simple methanol overnight incubation or a rapid dilute-and-shoot method, respectively. The samples were then analyzed using an UPLC-ESI-MS/MS and genotyped through RT-PCR. Statistical analyses were performed via SPSS software. From 105 postmortem cases, 53% and 51% of the cases shown to be positive for cocaine in hair and blood, respectively. Genetic wise, a significant difference has been observed for SNP rs4263329 from the BCHE gene with higher frequencies of the genotypes A/G and G/G seen in cocaine users (OR=8.91; 95%CI=1.58-50.21; p=0.01). Likewise, also SNP rs6280 from the DRD3 gene presented a significant association, with both genotypes T/C and C/C being more frequent in users (OR=4.96; 95% CI=1.07-23.02; p=0.04). To conclude, a rather high proportion of cocaine has been found, which may suggest a connotation between the use of the drug and risky/violent behaviors. Additionally, significant associations were also found within two SNPs related to cocaine use, however, due to several inherent limitations, these must be confirmed.

Psychoactive substances in human breast milk: a review of analytical strategies for their investigation.

WHO recommends breastfeeding for the first 6 months of newborn's life. Due to its physicochemical properties, breast milk may contain undesirable components originated from mother's feeding, medication and illicit drugs consumption. Some of these substances transferred from bloodstream to milk and delivered to the infant can cause harmful effects. For the last decades, analytical advances enabled the analysis of several substances in milk using different techniques. Thereby, it is possible to evaluate infant's level of exposure to these substances. This review presents the information published in the main scientific dissemination media about psychoactive drugs investigation in human breast milk, involving the sample preparation techniques and chromatographic validated methods developed in the past 10 years.

Analysis of biofluids by paper spray-MS in forensic toxicology.

Direct ambient ionization techniques have been developed with the aim to reduce the complexity of mass spectrometry analysis by minimizing sample preparation and chromatographic separation. In this context, paper spray-MS (PS-MS) is an innovative approach that provides faster and cheaper analysis of biofluids by the addition of the sample directly to a paper. In forensic toxicology, the analytical workflow for the detection and quantification of drugs of abuse is onerous, including sample treatment, extraction and clean up, especially regarding complex biological matrices. PS-MS allows the detection of analytes of toxicological interest in blood, plasma and urine using low sample volume. This review aims to discuss the potential use, advances and challenges of PS-MS in forensic toxicology.

Fatal metaldehyde poisoning in a dog confirmed by gas chromatography.

BACKGROUND: Metaldehyde is a toxic pesticide used mainly as a molluscicide, responsible for intoxication and deaths in both humans and animals. Accidental exposure to metaldehyde in dogs is considered rare, but severe. Data concerning clinical and veterinary forensic toxicology are largely incomplete, especially regarding case reports in dogs. The present work reports a complete and detailed description of a case from the history, clinical evolution, pathological exams and toxicological diagnosis in an accidental case of metaldehyde poisoning in dog. CASE PRESENTATION: An eleven-month-old, 3.0 kg, male German Spitz was presented for emergency care with acute vomiting and seizures 3 hours after suspected accidental ingestion of commercial molluscicide containing 3% metaldehyde (Lesmax®). The animal was in lateral recumbency and showed stuporous mentation, salivation, tonic-clonic status epilepticus, systemic tremors, bilateral miosis, absent palpebral, corneal, oculovestibular and gag reflexes, severely depressed spinal reflexes, dyspnea and tachycardia. Despite treatment, the patient progressed to comatose mentation and died. Necropsy examination revealed discrete lesions in the liver and central nervous system, while stomach examination revealed content of feed, activated charcoal and blue-green granules, compatible to the commercial formula of metaldehyde. Histology examination revealed extensive hemorrhage and severe centrolobular necrosis of the liver and tumefaction of Kupfer cells. Brain samples showed discrete hemorrhage and hyperemia. In order to confirm the diagnosis, samples from feces, stomach content, spleen, liver, heart, kidneys and brain were submitted gas chromatography analysis. Results confirmed the presence of metaldehyde in all samples. We describe clinicopathological abnormalities of a fatal case of metaldehyde poisoning in a dog, as well as postmortem diagnosis using gas chromatography. CONCLUSION: Metaldehyde poisoning is rarely reported, since the diagnosis is often difficult and the notifications scarce. To our knowledge, this is the first report describing clinical signs, pathological findings and chromatographic diagnosis. This report aims to contribute to the understanding of the pathogenesis of metaldehyde intoxication, to further explore veterinary forensic toxicology diagnosis.

Simultaneous Determination of Cocaine and Metabolites in Human Plasma Using Solid Phase Micro-Extraction Fiber Tips C18 and UPLC-MS/MS.

The determination of cocaine (COC) and its metabolites ecgonine methyl ester (EME), benzoylecgonine (BZE), norcocaine (NCOC) and cocaethylene (CE) in human plasma is relevant in clinical and forensic toxicology. An efficient extraction and clean-up of plasma specimens for the simultaneous determination of BZE along with COC and basic metabolites is challenging due to their widely different polarities and ionization characteristics. Recently, biocompatible SPME LC tips C18 became commercially available. We applied SPME LC tips C18 to the simultaneous extraction of COC, BZE, EME, NCOC, and CE by direct immersion of the fiber in plasma diluted with a buffer at pH 8.0. Analytes were desorbed from the fiber to methanol containing formic acid and injected into a UPLC-MS/MS system. The assay was linear from 5 to 500 ng mL-1. Precision assays presented CV% in the range of 2.22 to 10.54%, and accuracy was in the range of 93.4-108.1%. The assay requires minimal quantities of plasma and organic solvents, allowing multiple extractions in parallel. Biocompatible SPME is a promising alternative for preparing biological samples prior to drug measurement by UPLC-MS/MS.

Seizures of Clandestinely Produced Tablets in Santa Catarina, Brazil: The Increase in NPS from 2011 to 2017.

Ecstasy is the name given to a large group of substances known as "club drugs" traded in the form of tablets, powder and liquid that present a varied composition, including mainly 3,4-methylenedioxymethamphetamine (MDMA) and analogous substances, in addition to the growing presence of new psychoactive substances. Based on this, clandestinely produced tablets ("ecstasy tablets") seized by police in Santa Catarina in the period from 2011 to 2017 were analyzed. During the study period, 3472 seizures occurred, which resulted in total 498,443 tablets seized. Seventy nine substances were identified. In the year 2011, about 90% of the tablets contained MDMA. This number decreased to 66.6% in 2017. On the other hand, there was an increase in the number of tablets that mainly contained synthetic cathinones, as well as tryptamine and piperazine derivatives. Police seizures in the metropolitan region of Florianópolis and on the coast prevailed over the rest of the state.