Analysis of chemosensory markers in cigarette smoke from different tobacco varieties by GC×GC-TOFMS and chemometrics.

Commercial cigarettes are made from a blend of different tobacco varieties, which in turn are the results of different agronomic practices and post-harvest curing processes. The highly complex mixture of smoke compounds reflects each tobacco variety and the levels of sensory-relevant markers. Therefore, the aim of this work was to identify potential relevant chemosensory markers in the mainstream smoke of four main types of commercial tobaccos and establish any possible relationship between them and the tobacco growing/curing practices. The tobacco samples were segregated into four segments: (1) three curing stages of flue-cured Virginia, (2) three curing stages of air-cured Burley, (3) three geo-regions of sun-cured Oriental and (4) three different process applied to tobacco. One hundred and twenty cigarettes (10 batches per flavour category) were produced and smoked under standard machine-smoking protocols. The mainstream smoke samples collected were extracted and analysed by GC × GC TOFMS. The processed data was analysed by partial least square discriminant analysis (PLS-DA) and the selectivity ratio was used to identify key chemosensory markers responsible for the four segments. All models had sensitivity and specificity equal to unity. Flue-cured Virginia (193 markers) and air-cured Burley (184 markers) showed a similar trend for O-heterocycles markers in the lighter leaf colours and N-heterocycles in the darker leaf colours post-processing, but they had compounds of different flavour descriptions, e. g. sweet and nutty. The three geo-regions of sun-cured Oriental (290 markers) also presented O-heterocycles markers in correlation with leaf sugar contents in addition of sucrose esters markers. The three unusually processed tobacco generated many chemical markers (436 markers), some derived from the so-called Cavendish fermentation process with sweet, spicy and peppery notes, whereas the dark fermented air-cured tobacco presented similar descriptors as air-cured Burley. In addition, some polycyclic aromatic hydrocarbons (PAH) were detected as markers from the fire-curing process. The PLS-DA with selectivity ratio evidenced total of 1098 chemosensory markers in cigarette smoke, in which 173 were tentatively identified.

High-throughput simultaneous quantitation of multi-analytes in tobacco by flow injection coupled to high-resolution mass spectrometry.

The high-throughput screening by flow injection coupled to high-resolution mass spectrometry (HTS-FIA-HRMS) is a powerful technique that enables the identification of several types of samples in a short period of time, either with qualitative or quantitative purposes. Sensory attributes of tobacco are affected by its chemical composition, and it is very important to quantify multi-analytes in a high-throughput methodology. HTS-FIA-HRMS coupled to multivariate analysis was used to create calibration models for 27 analytes, or group of compounds, of tobacco sensory interest. The models were validated by different approaches, including permutation test to avoid overfitting, evaluation of the equipment repeatability by control samples, reproducibility comparison of results from two different equipment and analysts, and with a blind test analysis. All tests demonstrated a good response to the proposed method. No statistical difference between the errors of both equipment was observed, with less than 7% error from the control samples, and a blind test error between 5.96% and 20.10%. The partial least squares (O-PLS) regression models were applied to 815 samples, and a principal component analysis (PCA) was performed from the predicted concentration values, aiming at the non-supervised classification based on tobacco type. We expect that this proposed methodology shows not only the applicability in tobacco samples, but also demonstrates a guideline to an efficient performance of multi-analytes target analysis using the flow injection mass spectrometry with reliable and robust validation steps.

Innovative Approaches for Estimating the Levels of Tobacco-Specific Nitrosamines in Cured Tobacco Samples.

Tobacco-specific nitrosamines (TSNAs), mainly the 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are known carcinogens. Part of the NNK found in smoke is provided from matrix-bound NNK, and its determination is extremely relevant. However, the reference extraction procedure of matrix-bound NNK is time-consuming and labor-intensive and has a limited analytical capacity. Three different methodologies were proposed to predict matrix-bound NNK: simple linear regression (LR) with soluble NNK; multiple linear regression (MLR) considering soluble NNK and characteristic parameters of the samples; and orthogonal partial least-squares (O-PLS) regression using high-throughput screening by flow injection analysis coupled to high-resolution mass spectrometry (HTS-FIA-HRMS) data. Simple linear regression showed a high influence of matrix and leaf origin. Although an existing linearity trend has been observed ( R2 = 0.62) for the global model, higher correlation values were achieved for matrix and country segregation models. Multiple linear regression predicted matrix-bound NNK with more satisfactory efficiency than simple linear regression models. The coefficients of determination were 0.87 and 0.94 for flue-cured Virginia and air-cured Burley, respectively. However, this method has a limited application, since previous information about the sample is required. The proposed method based on HTS-FIA-HRMS and O-PLS has shown the most suitable performance in the prediction of matrix-bound NNK, with errors comparable to the reference method, and a higher throughput. In addition, this approach allows to determine other soluble nitrosamines, namely N'-nitrosoanatabine, N'-nitrosoanabasine, and N-nitrosonornicotine, with relative percentage errors between 5.25 and 11.98%. Therefore, the third approach is the best method for a large number of cured tobacco for accuracy in determination of TSNAs.

Comparison of Cocaine/Crack Biomarkers Concentrations in Oral Fluid, Urine and Plasma Simultaneously Collected From Drug Users.

The use of oral fluid (OF) as an alternative specimen for drug analysis has become very popular in forensic toxicology. Many clinical studies have evaluated the correlations between concentrations of cocaine and its metabolites in OF and other matrices, but results have shown high variability. In addition, there are no data available regarding the correlations between biomarkers of crack-cocaine use in different matrices. This study evaluated the relationship between concentrations of cocaine/crack-cocaine biomarkers in OF, urine and plasma samples collected from cocaine users. All samples were analyzed for the presence of cocaine (COC), benzoylecgonine (BZE) and anhydroecgonine (AEC) by a validated liquid chromatography-mass spectrometry method. Median COC, BZE and AEC concentrations ranged from 4.20 to 33.26 ng/mL, from 13.03 to 3,615.86 ng/mL and from 7.40 to 1,892.5 ng/mL across matrices, respectively. The relationship between drug concentrations in OF versus plasma (OF/P) and OF versus urine (OF/U) was evaluated by their coefficients of determination (R2). Least-squares regression analyses demonstrated significant correlations between OF/P and OF/U for cocaine and BE (P < 0.05), with R2 = 0.17, 0.07 for cocaine and R2 = 0.73, 0.45 for BE, respectively. The correlation coefficients (r) found for BZE, COC and AEC in OF/P and OF/U were 0.85 and 0.67 (P < 0.05); 0.41 and 0.26 (P < 0.05); and 0.30 and -0.37 (P > 0.05), respectively. Many factors contribute to the variability of drug correlation ratios in studies involving random samples, including uncertainty about the time of last administration and dosage. Overall, we found significant R2 values for COC and BZE in OF/P and OF/U, but not for AEC. Despite the good correlations found in some cases, especially for BZE, the large variation in drug concentrations seen in this work suggests that OF concentrations should not be used to estimate concentrations of COC, BZE or AEC in plasma and/or urine.

A genetic algorithm-based framework for wavelength selection on sample categorization.

In forensic and pharmaceutical scenarios, the application of chemometrics and optimization techniques has unveiled common and peculiar features of seized medicine and drug samples, helping investigative forces to track illegal operations. This paper proposes a novel framework aimed at identifying relevant subsets of attenuated total reflectance Fourier transform infrared (ATR-FTIR) wavelengths for classifying samples into two classes, for example authentic or forged categories in case of medicines, or salt or base form in cocaine analysis. In the first step of the framework, the ATR-FTIR spectra were partitioned into equidistant intervals and the k-nearest neighbour (KNN) classification technique was applied to each interval to insert samples into proper classes. In the next step, selected intervals were refined through the genetic algorithm (GA) by identifying a limited number of wavelengths from the intervals previously selected aimed at maximizing classification accuracy. When applied to Cialis®, Viagra®, and cocaine ATR-FTIR datasets, the proposed method substantially decreased the number of wavelengths needed to categorize, and increased the classification accuracy. From a practical perspective, the proposed method provides investigative forces with valuable information towards monitoring illegal production of drugs and medicines. In addition, focusing on a reduced subset of wavelengths allows the development of portable devices capable of testing the authenticity of samples during police checking events, avoiding the need for later laboratorial analyses and reducing equipment expenses. Theoretically, the proposed GA-based approach yields more refined solutions than the current methods relying on interval approaches, which tend to insert irrelevant wavelengths in the retained intervals. Copyright © 2016 John Wiley & Sons, Ltd.

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.

Seized cannabis seeds cultivated in greenhouse: A chemical study by gas chromatography-mass spectrometry and chemometric analysis.

Cannabis sativa L. is cultivated in most regions of the world. In 2013, the Brazilian Federal Police (BFP) reported 220 tons of marijuana seized and about 800,000 cannabis plants eradicated. Efforts to eradicate cannabis production may have contributed to the development of a new form of international drug trafficking in Brazil: the sending of cannabis seeds in small amounts to urban centers by logistics postal. This new and increasing panorama of cannabis trafficking in Brazil, encouraged the chemical study of cannabis seeds cultivated in greenhouses by gas-chromatography coupled with mass spectrometry (GC-MS) associated with exploratory and discriminant analysis. Fifty cannabis seeds of different varieties and brands, seized by the BFP were cultivated under predefined conditions for a period of 4.5 weeks, 5.5 weeks, 7.5 weeks, 10 weeks and 12 weeks. Aerial parts were analyzed and cannabigerol, cannabinol, cannabidiol, cannabichromene Δ9-tetrahydrocannabinol (THC) and other terpenoids were detected. The chromatographic chemical profiles of the samples were significantly different, probably due to different variety, light exposition and age. THC content increased with the age of the plant, however, for other cannabinoids, this correlation was not observed. The chromatograms were plotted in a matrix with 50 rows (samples) and 3886 columns (abundance in a retention time) and submitted to PCA, HCA and PLS-DA after pretreatment (normalization, first derivative and autoscale). The PCA and HCA showed age separation between samples however it was not possible to verify the separation by varieties and brands. The PLS-DA classification provides a satisfactory prediction of plant age.

Toxic and nutrient elements in yerba mate (Ilex paraguariensis).

Toxic and nutrient elements were investigated in yerba mate (Ilex paraguariensis) from South America. Fifty-four brands of commercialised yerba mate from Argentina, Brazil, Paraguay and Uruguay were analysed for Al, Ba, Ca, Cu, Fe, K, Mg, Mn, P, Sr, and Zn, using inductively coupled plasma optical emission spectrometry (ICP-OES), and Li, Be, Ti, V, Cr, Ni, Co, As, Se, Rb, Mo, Ag, Cd, Sb, La, Ce, Pb, Bi and U using inductively coupled plasma mass spectrometry (ICP-MS). Antimony, Se, Ag and Bi were not detected in any sample whereas the limits of detection (LODs) of these elements were 0.19, 0.40, 0.003 and 0.001 µg g(-1), respectively. Analysis of variance (ANOVA) revealed that the concentrations of Cd, Ti, Ni, As, Mo, U, Li and Be in yerba mate were not statistically different with regard to the country of origin, while those of the other investigated elements differed.

Authentication of yerba mate according to the country of origin by using Fourier transform infrared (FTIR) associated with chemometrics.

This study deals with the development of a method for classification of yerba mate (Ilex paraguariensis) using attenuated total-reflectance Fourier transform infrared (ATR-FTIR) and multivariate analysis. Fifty-four brands of yerba mate from southern South America were analysed in order to classify the commercialised yerba mate according to the respective country of yerba mate processing. The yerba mate was ground in a cryogenic mill, and the reflectance was directly measured in the region ranging from 4000 to 650 cm(-1). Different pre-processing algorithms and three methods of multivariate analysis were investigated, including principal component analysis (PCA), partial least square discriminant analysis (PLS-DA) and support vector machine discriminant analysis (SVM-DA). The yerba mate classification was 100% correct when the reflectance spectra were pre-treated (derived at first order, normalised by standard normal variation, smoothed and mean centred) and analysed using the SVM-DA method.

Descargas elétricas transtorácicas em ratos albinos: alterações ultra-estruturais do miocárdio atrial / Electrical transthoracic discharges in albine rats: ultrastructural changes in atrial myocardium

O objetivo principal deste estudo foi avaliar qualitativamente a integridade da ultra-estrutura atrial miocárdica depois de descargas elétricas transtorácicas. O equipamento para descargas elétricas foi adaptado para pequenos animais, mantendo-se as mesmas características daqueles utilizados em seres humanos. Descargas elétricas da ordem de 300 mV foram aplicadas na região precordial de 30 ratos. Por seleção não preferencial, os animais foram divididos em três grupos com dez animais cada um, para compor as diferentes análises biológicas: o grupo controle – no qual não se executou nenhum tipo de procedimento; o grupo experimental – em que os animais receberam dez eletrochoques. A seguir, os animais desses dois grupos foram sacrificados. O grupo experimental pós-descarga recebeu dez eletrochoques e foi sacrificado uma semana depois. Após anestesia foram retirados fragmentos do fígado, suprarenal e do átrio esquerdo para análise. Resultados: O tecido miocárdico foi analisado por microscopia eletrônica onde se verificou que um expressivo número de mitocôndrias teve a sua parede celular alterada ou lesada no grupo de descarga elétrica. Nos demais grupos, observou-se a integridade da citoarquitetura atrial. Em todos os fragmentos hepáticos e adrenais examinados não ocorreu depleção glicogênica ou lipídica, respectivamente. Conclusão: Os resultados evidenciaram que após descargas elétricas transtorácicas houve dano celular do tecido miocárdico em nível ultra-estrutural, mesmo não tendo sido observada a ocorrência da reação de alarme, ressaltando a importância da proteção celular, antes e após os procedimentos clínicos relacionados à cardioversão elétrica.