A validated dilute-and-shoot LC-MS-MS urine screening for the analysis of 95 illicit drugs and medicines: Insights from clinical and forensic Brazilian cases.

Urine toxicological analysis is a relevant tool in both clinical and forensic scenarios, enabling the diagnosis of acute poisonings, elucidation of deaths, verification of substance use in the workplace and identification of drug-facilitated crimes. For these analyses, the dilute-and-shoot technique associated with liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS) is a promising alternative since it has demonstrated satisfactory results and broad applicability. This study developed and validated a comprehensive LC-MS-MS screening method to analyze 95 illicit drugs and medicines in urine samples and application to clinical and forensic Brazilian cases. The dilute-and-shoot protocol was defined through multivariate optimization studies and was set using 100 µL of sample and 300 µL of solvent. The total chromatographic run time was 7.5 min. The method was validated following the recommendations of the ANSI/ASB Standard 036 Guideline. The lower limits of quantification varied from 20 to 100 ng/mL. Within-run and between-run precision coefficient of variations% were <20%, and bias was within ± 20%. Only 4 of the 95 analytes presented significant ionization suppression or enhancement (>25%). As proof of applicability, 839 urine samples from in vivo and postmortem cases were analyzed. In total, 90.9% of the analyzed samples were positive for at least one substance, and 78 of the 95 analytes were detected. The most prevalent substances were lidocaine (40.2%), acetaminophen (38.0%) and benzoylecgonine (31.5%). The developed method proved to be an efficient and simplified alternative for analyzing 95 therapeutic and illicit drugs in urine samples. Additionally, the results obtained from sample analysis are essential for understanding the profile of Brazilian substance use, serving as a valuable database for the promotion of health and safety public policies.

Suicide and associated factors across life span / Suicídio e fatores associados ao longo da vida

ABSTRACT Objective To assess the epidemiological and toxicological profile of all suicide victims in 2017 in Rio Grande do Sul, Brazil. Methods The victims were classified by gender, age, parental absence, city, suicide form, death context, and toxicological results, using the police occurrences and the reports issued by the Instituto-Geral de Perícias do RS. Multiple correspondence analysis and the Cochran-Mantel-Haenszel X 2 test were used to evaluate associations between the parameters studied. Results There were 1,284 suicides (11.3 cases/100,000 inhabitants) in RS in 2017, 80% of which were men and 46% were young and old. Porto Alegre had the highest number of victims and the region of the Vale do Rio Pardo, the highest rate (20.8 cases/100,000 inhabitants). The hanging was the most used medium and the depression, the most mentioned context in the occurrences. The presence of ethanol was observed in 30% of the samples analyzed, with an adult male profile associated with the presence of other psychotropic substances, whose class was most frequently detected with anxiolytics. The nitrite was the most detected poison among the samples sent for this purpose. There was an association between parental absence and young people, between suicidal intoxication method and women and among young people and the presence of illicit compounds. Conclusion Mortality due to suicide continues to increase in RS, which, historically, has the highest Brazilian index. The information obtained in this study supports new research, promoting awareness raising, guidance to health services and the elaboration of more preventive public policies.

RESUMO Objetivo Determinar o perfil epidemiológico e toxicológico de todas as vítimas de suicídio no Rio Grande do Sul em 2017. Métodos As vítimas foram classificadas por gênero, idade, ausência parental, cidade, forma de suicídio, contexto do óbito e resultados toxicológicos, utilizando as ocorrências policiais e os laudos emitidos pelo Instituto-Geral de Perícias do RS. A análise de correspondência múltipla e o teste Cochran-Mantel-Haenszel X 2 foram utilizados para avaliar associações entre os parâmetros estudados. Resultados Foram registrados 1.284 suicídios (11,3 casos/100 mil habitantes) no RS em 2017, sendo 80% homens e 46% jovens e idosos. Porto Alegre apresentou o maior número de vítimas e a região do Vale do Rio Pardo, o maior índice (20,8 casos/100 mil habitantes). O enforcamento foi o meio mais empregado e a depressão, o contexto mais citado nas ocorrências. A presença de etanol foi observada em 30% das amostras analisadas, com perfil masculino adulto associado à presença de outros psicotrópicos, cuja classe mais frequentemente detectada foi a dos ansiolíticos. O nitrito foi o veneno mais detectado entre as amostras encaminhadas para essa finalidade. Houve associação entre a ausência parental e os jovens, entre o método suicida intoxicação e as mulheres e entre os jovens e a presença de compostos ilícitos. Conclusão A mortalidade por suicídio segue aumentando no RS, que, historicamente, apresenta o maior índice brasileiro. As informações obtidas neste trabalho servem de apoio a novas pesquisas, promovendo ações de conscientização, orientação aos serviços de saúde e na elaboração de mais políticas públicas preventivas.

Acute administration of 5-oxoproline induces oxidative damage to lipids and proteins and impairs antioxidant defenses in cerebral cortex and cerebellum of young rats.

5-Oxoproline accumulates in glutathione synthetase deficiency, an autossomic recessive inherited disorder clinically characterized by hemolytic anemia, metabolic acidosis, and severe neurological symptoms whose mechanisms are poorly known. In the present study we investigated the effects of acute subcutaneous administration of 5-oxoproline to verify whether oxidative stress is elicited by this metabolite in vivo in cerebral cortex and cerebellum of 14-day-old rats. Our results showed that the acute administration of 5-oxoproline is able to promote both lipid and protein oxidation, to impair brain antioxidant defenses, to alter SH/SS ratio and to enhance hydrogen peroxide content, thus promoting oxidative stress in vivo, a mechanism that may be involved in the neuropathology of gluthatione synthetase deficiency.

N-acetylaspartic acid impairs enzymatic antioxidant defenses and enhances hydrogen peroxide concentration in rat brain.

N-Acetylaspartic acid accumulates in Canavan Disease, a severe inherited neurometabolic disease clinically characterized by severe mental retardation, hypotonia, macrocephaly and generalized tonic and clonic type seizures. Considering that the mechanisms of brain damage in this disease remain poorly understood, in the present study we investigated the in vitro and in vivo effects of N-acetylaspartic acid on the activities of catalase, superoxide dismutase and glutathione peroxidase, as well as on hydrogen peroxide concentration in cerebral cortex of 14-day-old rats. Catalase and glutathione peroxidase activities were significantly inhibited, while hydrogen peroxide concentration was significantly enhanced by N-acetylaspartic acid both in vitro and in vivo. In contrast, superoxide dismutase activity was not altered by N-acetylaspartic acid. Our results clearly show that N-acetylaspartic acid impairs the enzymatic antioxidant defenses in rat brain. This could be involved in the pathophysiological mechanisms responsible for the brain damage observed in patients affected by Canavan Disease.

Glutaric acid induces oxidative stress in brain of young rats.

This study investigated the effects of glutaric acid, which predominantly accumulates in glutaric acidemia type I, on some in vitro parameters of oxidative stress in brain of young rats. We evaluated chemiluminescence, total radical-antioxidant potential (TRAP) and the activities of the antioxidant enzymes catalase, glutathione peroxidase and superoxide dismutase in brain tissue homogenates in the presence of glutaric acid at concentrations ranging from 0.05 to 2.0 mM. The acid significantly increased chemiluminescence (up to 65%) and reduced total radical-antioxidant potential (up to 28%) and glutathione peroxidase activity (up to 46%), without affecting catalase and superoxide dismutase activities. The results provide evidence that glutaric acid induces oxidative stress in vitro in rat brain. If these findings also occur in humans, it is possible that they may contribute to the neuropathology of patients affected by glutaric acidemia type I.