Metodologias de detecção e de confirmação de canabinoides em amostras brutas de cannabis sativa L. e em amostras biológicas de fluido oral

A Cannabis sativa L. (Cannabaceae) é uma das drogas mais consumidas no mundo. Os efeitos psicoativos dessa planta podem ser associados majoritariamente à presença de Δ9- tetrahidrocanabinol (Δ9- THC), motivo pelo qual esse é considerado o marcador de escolha para análises toxicológicas conforme a legislação de diversos países. Considerando o atual cenário do Brasil, em que a legalização e/ou descriminalização da Cannabis sativa encontram-se em discussão, torna-se evidente a necessidade de metodologias simples, de baixo custo e que sejam confiáveis, permitindo a detecção, a identificação e a quantificação dos canabinoides presentes tanto em amostras de apreensão quanto em amostras biológicas. Assim, esta tese tem como objetivo desenvolver e validar uma metodologia analítica confirmatória para a quantificação indireta do Δ9-THC empregando o método de número de carbonos efetivos, bem como desenvolver uma metodologia de triagem para uma possível estimativa da idade da amostra, utilizando análise multivariada de imagens; ambas em amostras brutas de Cannabis sativa. Adicionalmente, desenvolver uma revisão sistemática a respeito de métodos imunocromatográficos de triagem para investigação da presença de canabinoides (Δ9-THC, canabidiol e canabinol) em fluido oral. No capítulo I, são apresentados os resultados da metodologia analítica proposta para a quantificação indireta de Δ9-THC por CBN empregando cromatografia em fase gasosa acoplada a detector de ionização de chama (CG/DIC), a qual mostrou-se viável tanto para quantificação indireta quanto para a direta de Δ9-THC, proporcionando resultados semelhantes em ambas as abordagens. Assim, o método pode ser considerado uma alternativa para a análise de Δ9-THC, permitindo análises rápidas e de baixo custo para laboratórios forenses. Após a validação do método analítico, os extratos líquidos foram fotografados e as imagens foram submetidas à análise de PCA e de HCA com o software ChemoStat® e com o aplicativo PhotoMetrix PRO®. No capítulo II, são apresentados os resultados desses experimentos, os quais mostram um padrão de gradiente de cor que poderia diferenciar amostras e combinar concentrações quantificadas de Δ9-THC e CBN, ou somente CBN. O uso de quimiometria em análises forenses pode ajudar na triagem, contribuindo na análise de amostras brutas de Cannabis sativa. Por fim, no capítulo III, os resultados da revisão sistemática demonstram que apenas dois dispositivos no mercado apresentam sensibilidade e especificidade satisfatórias. Entretanto, esses testes apresentam também algumas limitações, incluindo um ponto de corte superior ao recomendado por diretrizes internacionais (2ng/mL). Assim, existe a necessidade de mais estudos na área, bem como é destacada a importância de análises confirmatórias. Dessa forma, a presente tese de doutorado consiste em três capítulos de relevância e impacto para a ciência forense, contemplando a importância do desenvolvimento de metodologias eficientes e de custo reduzido de análise para o monitoramento de canabinoides em diferentes matrizes.Cannabis sativa L. (CANNABACEAE) is one of the most consumed drugs in the world. The psychoactive effects of this plant can be mainly associated with the presence of Δ9- tetrahydrocannabinol (Δ9-THC), which is why it is considered the marker of choice for toxicological analyzes according to the legislation of several countries. Considering the current scenario in Brazil, in which the legalization and/or decriminalization of cannabis are under discussion, it becomes evident the need for simple, low-cost and reliable methodologies, allowing the detection, identification and quantification of cannabinoids present in both seizure and biological samples. Thus, this thesis aims to develop and validate a confirmatory analytical methodology for indirect quantification of Δ9-THC using the effective carbon number method, as well as to develop a screening methodology for a possible estimate of the sample age, using multivariate analysis images; both in raw cannabis samples. Additionally, develop a systematic review regarding immunochromatographic screening methods to investigate the presence of cannabinoids (Δ9-THC, cannabidiol and cannabinol) in oral fluid. In chapter I, the results of the proposed analytical methodology for the indirect quantification of Δ9-THC are presented by CBN using gas chromatography coupled to a flame ionization detector (GC/FID), which proved to be viable for both indirect and direct quantification of Δ9-THC, providing similar results in both approaches. Thus, the method can be considered an alternative approach for the analysis of Δ9-THC, allowing for fast and low-cost analysis for forensic laboratories. After validation of the analytical method, the liquid extracts were photographed and the images were subjected to analysis of PCA and HCA with the software ChemoStat® and with the application PhotoMetrix PRO®. In chapter II, the results of these experiments are presented, which show a color gradient pattern that could differentiate samples and combine quantified concentrations of Δ9-THC and CBN, or only CBN. The use of chemometrics in forensic analysis can help in screening, contributing to the analysis of raw cannabis samples. Finally, in Chapter III, the results of the systematic review demonstrate that only two devices on the market have satisfactory sensitivity and specificity. However, these tests also have some limitations, including a cut-off point higher than that recommended by international guidelines (2ng/mL). Thus, there is a need for further studies in the area, as well as the importance of confirmatory analyzes is highlighted. Thus, the present doctoral thesis consists of three chapters of relevance and impact for forensic science, contemplating the importance of developing efficient and low cost analysis methodologies for monitoring cannabinoids in different matrices

Immunochromatography screening devices for cannabinoids in oral fluid sample

Cannabis sativa L. is one of the most consumed drugs in the world and recent studies have associated its use with an increase in the number of traffic accidents in different countries. In many countries, like Brazil, simple and reliable methodologies are still needed for the detection of drugs on site, mainly cannabinoids, considering its prevalence of use and oral fluid (OF) has been proved as an appropriate biological matrix for this purpose. Considering that, this work aims to review previous studies on immunochromatographic devices for on-site detection of cannabinoids in OF, discussing their sensitivity, specificity, cut-offs values and confirmatory methods. This data shows the importance of choosing a screening device and it reinforces the need for its implementation in Brazil. The research was conducted on 5 databases and all original articles, published in the last 10 years, were selected. A total of 32 articles were found, providing data for 17 screening devices of distinct brands. Only 2 screening devices showed satisfactory sensitivity and specificity in the evaluated studies (≥80% and ≥90% respectively). However, it should be considered that the screening devices still have some limitations, such as a higher cut-off than those recommended by international guidelines (cut-off > 2 ng/mL), therefore demonstrating the need for more studies in the area and the importance of confirmatory analysis usually fulfilled by LC-MS/MS, GC-MS/MS or GC-MS. Thus, the screening analyzes should not be evaluated by itself, but in association with confirmatory results and observational traits (behavioral changes), for a better understanding of the traffic scenario

Main compounds and major methods in latent fingermark aging analysis : a short review

Fingermarks are a complex biological matrix with variability factors that change in the same donor, depending on the moment of collection, and in different donors, according to age, sex and routine. Thus, they can still undergo alterations related to the deposition surface, the environment, and the variable that connects all those mentioned: the action of time. In Forensic Science, time is a important variable to situate the crime events. This review proposes a classification in the temporal estimation research of fingermarks, dividing them into Temporal Preservation Analysis (TPA) and Temporal Aging Analysis (TAA). In TPA studies, the components in fingermark residues undergo a few changes over time, tending to stability after a certain period. Those are interesting targets to identify possible exogenous components, such as firearm residues, illegal substances and contaminants related to particularity of forensic cases. In TAA studies, a time estimation related to the fingermarks age can be established. In this case, the time elapsed from its deposition until the forensic processing will vary according to the component classes degradation. Endogenous and exogenous substances that are demonstrably present in one donor and that undergo changes over time will, resulting be demonstrated by a decrease in intensity and/or formation of other substances, and those are good targets for this type of study. The same analysis can have both proposals and the instrumental method available will enable the extraction of information relevant to the sample. The purpose of this study is to demonstrate how important is to identify fingermarks components as evidence beyond the ridge pattern and to list the main instrumental methods used in the analysis of fingermark degradation

Evaluation of basil extract ( Ocimum basilicum L.) on oxidative, anti-genotoxic and anti-inflammatory effects in human leukocytes cell cultures exposed to challenging agents

Ocimum is one of the most important genera of the Lamiaceae family. Several studies about basil and its popular use reveal many characteristics of the herb, including its use as antioxidant, anti-aging, anti-inflammatory, anti-carcinogenic, anti-microbial, and cardiovascular agents, among others. In this paper, we evaluated genotoxic, oxidative, and anti-inflammatory parameters from the extract of Ocimum basilicum in different concentrations, using human leukocytes cultures exposed to challenging agents. Our results confirm that the O. basilicum extract acts as an antioxidant and effectively reverts or subjugates the effects of high oxidizing agents such as hydrogen peroxide. These actions are attributed to its composition, which is rich in polyphenols and flavonoids as well as compounds such as rosmarinic acid, all of which have well-known antioxidant activity. We also show that our basil extract presents anti-inflammatory properties, the mechanism of which is a composed interaction between the inhibition of pro-inflammatory mediator and the stimulation of anti-inflammatory cytokines. Although pharmacodynamics studies are necessary to evaluate the activities in vivo, our results demonstrated that basil could act as an antioxidant and anti-inflammatory and a possible alternative for medicinal treatment

APPLICATION OF MULTIVARIATE ANALYSIS ON DIGITAL IMAGES OF CANNABIS SATIVA L EXTRACTS

Cannabis sativa L is one of the most used drugs in the world. Information about the plant’s age and storage can help forensic scientists to identify and to track samples. The ratio between the cannabinoids tetrahydrocannabinol (THC) and cannabinol (CBN) has been related to the degradation of cannabis with time. Thus, this study aimed to test Multivariate Image Analysis (MIA) to evaluate cannabis extracts concerning its colors. Initially, 52 samples of Cannabis sativa L. extracts were analyzed by Gas Chromatography coupled to Flame Ionization Detector (GC/FID) to quantify THC and CBN. Afterwards, the extract samples were photographed and analyzed by two different multivariate analysis tools: ChemoStat®, a free chemometrics software, and PhotoMetrix PRO®, an app for mobile devices. Using exploratory analysis of principal component analysis (PCA) and hierarchical cluster analysis (HCA). It was observed that the more intense the color for an extract, the higher concentration of THC and CBN it has, while the lighter color extracts correspond to samples with no THC. The results suggest to propose a simple method for previous clustering of samples that may precede chromatographic analyzes, assist in chemical profile studies or simply aggregate samples of similar profiles for analyzed together

AVALIAÇÃO DOS EFEITOS ANTI-GENOTÓXICOS DE Phyllanthus niruri (EUPHORBIACEAE) EM LEUCÓCITOS HUMANOS EXPOSTOS A AGENTE AGRESSOR

Em muitos lugares ao redor do mundo as plantas medicinais ou parte delas são usadas de forma paliativa ou curativa para o tratamento de diversas enfermidades, devido a dificuldade ao acesso a tratamentos convencionais. Nesse contexto, preparações a base de Phyllanthus niruri, que é popularmente conhecida como “Quebra-Pedra” ou “Erva-Pombinha” tem sido muito usadas no tratamento de diversas enfermidades como hipertensão, diabetes, cálculos renais, entre outros. O presente estudo visou avaliar a capacidade anti-genotóxica do extrato hidroalcoólico de Phyllanthus niruri, através da análise fitoquímica e dosagem de compostos, avaliação de parâmetros genotoxicológicos e  oxidativos. Os resultados mostram que o Phyllanthus niruri possui uma grande quantidade de compostos antioxidantes, tendo estes ação na proteção em nível de DNA, onde o extrato mostrou-se capaz de aumentar a viabilidade celular e de parâmetro oxidativos, mostrando ser capaz de diminuir a peroxidação lipídica.Descritores: Phyllanthus niruri; Cultura Celular; Leucócitos; Genotoxicidad

The Main Compounds and Major Methods in Latent Fingermark Aging Analysis: a Short Review

Fingermarks are a complex biological matrix with variability factors that change in the same donor, depending on the moment of collection, and in different donors, according to age, sex and routine. Thus, they can still undergo alterations related to the deposition surface, the environment, and the variable that connects all those mentioned: the action of time. In Forensic Science, time is a important variable to situate the crime events. This review proposes a classification in the temporal estimation research of fingermarks, dividing them into Temporal Preservation Analysis (TPA) and Temporal Aging Analysis (TAA). In TPA studies, the components in fingermark residues undergo a few changes over time, tending to stability after a certain period. Those are interesting targets to identify possible exogenous components, such as firearm residues, illegal substances and contaminants related to particularity of forensic cases. In TAA studies, a time estimation related to the fingermarks age can be established. In this case, the time elapsed from its deposition until the forensic processing will vary according to the component classes degradation. Endogenous and exogenous substances that are demonstrably present in one donor and that undergo changes over time will, resulting be demonstrated by a decrease in intensity and/or formation of other substances, and those are good targets for this type of study. The same analysis can have both proposals and the instrumental method available will enable the extraction of information relevant to the sample. The purpose of this study is to demonstrate how important is to identify fingermarks components as evidence beyond the ridge pattern and to list the main instrumental methods used in the analysis of fingermark degradation

The Main Compounds and Major Methods in Latent Fingermark Aging Analysis: a Short Review

Fingermarks are a complex biological matrix with variability factors that change in the same donor, depending on the moment of collection, and in different donors, according to age, sex and routine. Thus, they can still undergo alterations related to the deposition surface, the environment, and the variable that connects all those mentioned: the action of time. In Forensic Science, time is a important variable to situate the crime events. This review proposes a classification in the temporal estimation research of fingermarks, dividing them into Temporal Preservation Analysis (TPA) and Temporal Aging Analysis (TAA). In TPA studies, the components in fingermark residues undergo a few changes over time, tending to stability after a certain period. Those are interesting targets to identify possible exogenous components, such as firearm residues, illegal substances and contaminants related to particularity of forensic cases. In TAA studies, a time estimation related to the fingermarks age can be established. In this case, the time elapsed from its deposition until the forensic processing will vary according to the component classes degradation. Endogenous and exogenous substances that are demonstrably present in one donor and that undergo changes over time will, resulting be demonstrated by a decrease in intensity and/or formation of other substances, and those are good targets for this type of study. The same analysis can have both proposals and the instrumental method available will enable the extraction of information relevant to the sample. The purpose of this study is to demonstrate how important is to identify fingermarks components as evidence beyond the ridge pattern and to list the main instrumental methods used in the analysis of fingermark degradation