Hyperspectral imaging in forensic science: An overview of major application areas.

Analysis of evidence is a challenge. Crime scene materials are complex, diverse, sometimes of an unknown nature. Forensic science provides the most critical applications for their examination. Chemical tests, analytical methods, and techniques to process the evidence must be carefully selected by the forensic scientist. Ideally, it may be interpreted, analyzed, and judged in the original context of the crime scene. In this sense, hyperspectral imaging (HSI) has been employed as an analytical tool that maintains the integrity of the samples/objects for multiple and sequential analysis and for counter-proof exams. This paper is an overview of forensic science trends for the application of HSI techniques in the last ten years (2011-2021). The examination of documents was the main area of exploration, followed by bloodstain analysis aging process; trace analysis of explosives and gunshot residue. Chemometric tools were also addressed since they are crucial to obtain the most important information from the samples. There are great challenges in applying HSI in forensic science, but there have been clear technological and scientific advances, and a solid foundation has been built for the use of HSI in real-life cases.

Identifying the most relevant tablet regions in the image detection of counterfeit medicines.

This paper proposes a novel image-based approach to detect counterfeit medicines and identify the most relevant regions of the tablet in the task of classification. Images of medicine tablets undergo an initial pre-processing step which (i) removes the background to find the region of interest, (ii) clusters individual pixels into super-pixels, and (iii) extracts features containing color and texture information. The classification relying on Support Vector Machine (SVM) defines the class the respective image will be inserted into. The task of identifying the relevant regions of the tablets for counterfeiting detection is performed using the concept of support vectors, generating a heat map that indicates the regions that contribute the most to the classification purpose. Two datasets containing images of authentic and counterfeit tablets of Cialis and Viagra were used to validate our propositions, achieving correct classification rates of 100% on both datasets. Regarding the task of identifying the most relevant regions, our proposition outperformed the traditional LIME (Local Interpretable Model-agnostic Explanations) method by yielding more robust explanations.

Analysis of Erythroxylum coca Leaves by Imaging Mass Spectrometry (MALDI-FT-ICR IMS).

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) can determine the chemical identity and spatial distribution of several molecules in a single analysis, conserving its natural histology. However, there are no specific studies on the spatial distribution of alkaloids in Erythroxylum coca leaves by MALDI IMS, preserving the histology of the monitored compounds. Therefore, in this work, positive-ion mode MALDI Fourier-transform ion cyclotron resonance imaging mass spectrometry (MALDI(+)FT-ICR IMS) was applied to identify and analyze the distribution of alkaloids on the surface of coca leaves, evaluating the ionization efficiency of three matrices (α-cyano-4-hydroxycinnamic acid (CHCA), 2-mercaptobenzothiazole (MBT), and 2,5-dihydroxybenzoic acid (DHB)). The last was chosen as the best matrix in this study, and it was studied in five concentrations (0.5, 1.0, 2.0, 4.0, and 8.0 mg·mL-1), where 2 mg·mL-1 was the most efficient. The washing of coca leaves with the organic solvents (acetonitrile, methanol, toluene, and dichloromethane) tested did not improve the performance of the ionization process. Finally, a tissue section, 50 µm thick, was used to study the inner part of the leaf tissue, where alkaloids and flavonoid molecules were detected.

Forensic analysis of anabolic steroids tablets composition using attenuated total reflection Fourier transform infrared microspectroscopy (µATR-FTIR) mapping.

The use of falsified and unregistered drugs is a worldwide public health problem. Because these global market products usually do not follow the Good Manufacturing Practices required by health legislation, its composition may be completely different from the original or may contain relevant concentrations of impurities and toxic contaminants. Since anabolic steroids are among the main irregular therapeutic classes seized in Brazil, here we propose a new methodology for analyzing these products, in tablets form, using Attenuated Total Reflection Fourier Transform Infrared Microspectroscopy (µATR-FTIR) mapping. Spectra were acquired from solid tablets by attenuated total reflection, through point mapping methodology. In data processing, a characteristic absorption band for each Active Pharmaceutical Ingredient (API) was integrated and plotted to create its distribution map. This technique was applied in an unprecedented way for the forensic analysis of anabolic steroids and proved to be effective in distinguishing falsified products based on the detection of their APIs. It was possible to detect APIs in 26 out of 30 samples, five of which were classified as falsified only through µATR-FTIR analysis. We were able to create distribution maps of the detected substances associating the microspectroscopic results with characteristic band integration method, which can be used to detect substances and to study samples' homogeneity. We concluded that this methodology is promising for the analysis of anabolic steroid tablets, and can be used in a complementary way with techniques already consolidated in forensic laboratory routine for a better classification of questioned samples between authentic and falsified ones.

Geographic origin determination of Brazilian Cannabis sativa L. (Marihuana) by multi-element concentration.

The Marihuana Polygon production of Cannabis sativa L. supplies the northeastern region of Brazil and represents 30% of the nation's market. The international trend of indoor cultivation is also occurring in Brazil, and the Brazilian Federal Police (BFP) has been increasing its apprehension of cannabis seeds sent by mail. The present work aims to assess the utility of the multi-element composition of different cannabis plant parts and soil samples where the plants were cultivated to determine their geographic origin. Statistical tools were applied to classification of marijuana samples from distinct geographic regions within northeastern Brazil, including indoor cultivated samples. The multi-element quantification was determined using inductively-coupled plasma - optical emission spectrometry (ICP-OES), and the data were compared by the Kruskal-Wallis H test, and subsequently, multiple discriminant analysis (MDA). The results of the multi-element concentration of cannabis plant samples were also subjected to a principal component analysis (PCA) and an orthogonal partial least squares discriminant analysis (OPLS-DA). The cannabis plant samples from the Marihuana Polygon could be clearly separated from those cultivated indoors, and the distance between them was detectable. The MDA revealed that phosphorus, calcium, magnesium, selenium, and arsenic concentrations were used as variables for this separation. Our results demonstrate that multi-element composition analysis can be used to indicate the origin or cultivation location of cannabis plants. Routine laboratory analyses consisting of multi-element composition combined with statistical analyses provide a reliable tool by which C. sativa movement, cultivation, and interdiction efforts in Brazil may be assessed.

In vitro model to study cocaine and its contaminants.

Cocaine is one of the most popular illicit drug worldwide. Due its great addictive potential, which leads to euphoria and hyperactivity, it is considered a public health concern. At the central nervous system, the drug acts inhibiting catecholamine re-uptake. It is now known that in addition to the toxicity of the drug itself, the contaminants present in the street drug have raised concern about the harmful effects on health. Toxicological in vivo and in vitro studies have demonstrated the toxic effects of cocaine correlated with the generation of reactive oxygen species (ROS), which in turn lead to oxidative damage to the cells. Therefore the aim of this work was to propose an in vitro model that reunites the main parameters of toxicity of the cocaine already observed in the literature so far, and we tested this model using cocaine and seizure cocaine sample (SCS), kindly provided by Federal Police of Brazil. For that, we used a C6 glioblastoma cells and evaluated cell death, oxygen reactive species induction, oxidation of macromolecules as membrane lipids and DNA and loss of mitochondrial membrane potential after cocaine exposure. The results showed that cocaine can decrease cellular viability in a dose-dependent way in the C6 cell immortalized and astrocytes primary culture. Cocaine also induced cellular death by apoptosis. However, in the seizure cocaine sample (SCS), the predominant cell death was due to necrosis. Using dichlorofluorescein (DCF) assay, we confirmed ROS production after cocaine exposition. In agreement with these findings, occurred an increasing in MDA production, as well as increased superoxide dismutase (SOD) and catalase (CAT) activity. The induction of DNA damage was observed after cocaine. Our results demonstrate the occurrence of mitochondrial dysfunction by depolarization of mitochondrial membrane as a consequence of cocaine treatment. In summary, these results demonstrated that cocaine can induce reactive oxygen species formation, leading to oxidative stress. As a consequence of this unbalance, DNA damage, lipidic peroxidation and loss of mitochondrial membrane occurred, which could be an answer to cell death observed.

Near infrared spectroscopy combined with chemometrics for growth stage classification of cannabis cultivated in a greenhouse from seized seeds.

Cannabis sativa L. (cannabis, Cannabaceae), popularly called marijuana, is one of the oldest plants known to man and it is the illicit drug most used worldwide. It also has been the subject of increasing discussions from the scientific and political points of view due to its medicinal properties. In recent years in Brazil, the form of cannabis drug trafficking has been changing and the Brazilian Federal Police has exponentially increased the number of seizures of cannabis seeds sent by the mail. This new form of trafficking encouraged the study of cannabis seeds seized germinated in a greenhouse through NIR spectroscopy combined with chemometrics. The plants were cultivated in a homemade greenhouse under controlled conditions. In three different growth periods (5.5weeks, 7.5weeks and 10weeks), they were harvested, dried, ground and directly analyzed. The iPCA was used to select the best NIR spectral range (4000-4375cm-1) in order to develop unsupervised and supervised methods. The PCA and HCA showed a good separation between the three groups of cannabis samples at different growth stages. The PLS-DA and SVM-DA classified the samples with good results in terms of sensitivity and specificity. The sensitivity and specificity for SVM-DA classification were equal to unity. This separation may be due to the correlation of cannabinoids and volatile compounds concentration during the growth of the cannabis plant. Therefore, the growth stage of cannabis can be predicted by NIR spectroscopy and chemometric tools in the early stages of indoor cannabis cultivation.

Physical profile of counterfeit tablets Viagra® and Cialis®

The profile of tablets containing an active pharmacological ingredient can be obtained using different sets of properties, including physical and chemical aspects. The first measurements carried out on tablets are the physical characteristics also called post-tabletting batch (post-TB) characteristics. These data may be valuable to assist in the detection of pharmaceutical product forgery and may also be used in a forensic intelligence perspective when inserted into databases. This work is focused on the physical characteristics of Cialis® and Viagra® tablets seized by the Brazilian Federal Police in the Rio Grande do Sul state. Using the F-test (ANOVA), all samples of counterfeit Viagra® (n = 28) and Cialis® (n = 40) were well distinguished from authentic samples by the following post-TB characteristics: length (major and minor), thickness, and mass. Using the exploratory statistical technique of Hierarchical Cluster Analysis (HCA), tablets with similar physical profiles were grouped. This result may indicate a common illicit production. We observed the validity of using post-TB properties to generate - in a fast and reliable manner and with no sample preparation - a technological profile that joins itself to the other analytical methods assisting in routine forensic detection of counterfeit Viagra® and Cialis®.

Perfil para medicamentos na forma farmacêutica comprimidos contendo uma substância ativa pode ser obtido usando diferentes conjuntos de propriedades, incluindo aspectos físicos e químicos. As primeiras medições realizadas em comprimidos são de características físicas, também chamadas características pós-compressão. Tais dados podem ser valiosos para auxiliar na detecção de falsificações de medicamentos e ser utilizados em uma perspectiva de inteligência forense, quando inseridos em bancos de dados. Este trabalho está focado nas características físicas dos comprimidos de Cialis® e Viagra® apreendidos pela Polícia Federal no Estado brasileiro do Rio Grande do Sul. Com o emprego do Teste de Fisher (ANOVA), todas as amostras falsificadas de Viagra® (n = 28) e de Cialis® (n = 40) foram diferenciadas das amostras autênticas pelas seguintes características pós-compressão: comprimento (maior e menor), espessura e massa. Utilizando-se a Análise Hierárquica de Cluster (AHC), os comprimidos com perfis físicos semelhantes foram agrupados, o que pode indicar uma produção ilícita em comum. Observou-se a validade da utilização das características pós-compressão para gerar, de um modo rápido, confiável e sem preparo de amostra, um perfil tecnológico que se una aos demais métodos analíticos utilizados na rotina forense de detecção de falsificações de Cialis® e de Viagra®.