Electrospray ionization mass spectrometry (ESI-MS) is a fast and sensitive technique that is ideal for detecting low concentration species of interest within complex mixtures. Because ESI-MS simply transfers charged species to the gas phase, only ions pre-formed in solution are visible. Accordingly, the charged tag, 3-(4-(bromomethyl)benzyl)-1-methylimidazolium hexafluorophosphate, was designed and synthesized to allow selective detection of phenols in petroleum fractions. Pressurized sample infusion (PSI) was optimized and used for time dependent analyses. PSI ESI-MS was applied to measure O-alkylation of the phenol species leading to rate information about the overall reaction along with dynamic information about reaction progress. The relative intensity of the charged tag was used to determine semi-quantitatively the presence of phenols in different petroleum fractions. Other derivatization methods in petroleomics were also explored. 1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide methiodide (EDT) derivatization followed by PSI ESI-MS analysis was applied for the selective measurement and identification of naphthenic acids in petroleum fractions. The reactions of standard naphthenic acids and EDT were studied by PSI ESI-MS to assess the efficiency of EDT and the rate of reactions. The same petroleum fractions were analysed by cold Electron ionization (Cold EI) gas chromatography-mass spectrometry (GC/MS) and classical EI GC/MS. The combination spectra from the subtraction from cold EI spectra to classical EI spectra provide us a new dimension to cold EI analysis of complex matrices. Meanwhile, a Python program was written to rapidly screen cold EI GC/MS data for routine tasks, such as retention time comparison on different instrument parameters for single petroleum sample and spectrum comparison on the same retention time for different petroleum samples. / Graduate

La consommation excessive d’alcool présente des risques élevés pour l’individu et pour la société ; elle est fréquemment associée à une augmentation du risque d’accidents, d’actes de violence, et peut également conduire à court et/ou à long terme à de graves maladies et à des problèmes sociaux. Dès lors, l’utilisation de marqueurs fiables permettant de détecter une consommation excessive d’alcool, ponctuelle ou chronique, s’avère nécessaire pour prévenir des conséquences néfastes de l’abus d’alcool. L’éthylglucuronide (EtG) est un marqueur d’alcoolisation utilisé en toxicologie clinique (alcoologie) et médicolégale. Par rapport aux marqueurs indirects d’alcoolisation (CDT, &#947;-GT), ce métabolite mineur de l’éthanol est très spécifique et est quantifiable dans diverses matrices biologiques. La production d’EtG est catalysée par des enzymes de la famille des UDP-glucuronosyl-transférases (UGT). Cependant, les UGT impliquées dans la glucuronoconjugaison de l'éthanol, ainsi que les sources potentielles de variabilité interindividuelle de la production d'EtG, sont encore mal connues. Nos travaux ont ainsi consisté à (1) développer et valider une méthode de dosage de l’EtG dans différentes matrices biologiques par chromatographie en phase gazeuse couplée à la spectrométrie de masse en tandem, (2) identifier les UGT humaines impliquées dans la glucuronoconjugaison de l’éthanol et étudier la contribution relative de chaque isoforme active au niveau hépatique, (3) étudier l’impact de substances fréquemment utilisées par les consommateurs d’alcool sur la production d’EtG in vitro, (4) étudier l’impact de polymorphismes génétiques fonctionnels des UGT sur la production hépatique d’EtG, et enfin (5) évaluer l’impact de la consommation de cannabis et d’autres drogues sur la production d’EtG à l’aide de prélèvements post-mortem. Ces travaux ont notamment permis de montrer que (1) l'éthanol est glucuronoconjugué principalement par le foie, puis dans une moindre mesure par les reins et par l'intestin, (2) les UGT1A9 et 2B7 sont les deux enzymes majoritairement impliquées dans la glucuronoconjugaison de l’éthanol, quel que soit l’organe considéré, (3) la morphine, la codéine, la nicotine et la cotinine n’entraînent aucune modification des taux de production d’EtG in vitro ; le lorazépam et l'oxazépam augmentent légèrement cette production (p = 0,2 et 0,065, respectivement) ; le cannabidiol inhibe la glucuronoconjugaison de l’éthanol par un mécanisme non-compétitif (CI50 = 1,17 mg/L; Ki = 3,1 mg/L), alors que le cannabinol augmente cette glucuroconjugaison de manière concentration-dépendante (p <0,05), (4) les SNP c.-900G>A affectant l’UGT2B7 et IVS1+399T>C affectant l’UGT1A9 augmentent légèrement la production d’EtG in vitro. Enfin (5) le rapport des concentrations sanguines EtG/éthanol apparaît significativement plus élevé chez des co-consommateurs de cannabis et/ou d’autres drogues que chez des consommateurs d’alcool seul. L’ensemble de ces résultats démontre l’existence de plusieurs facteurs pouvant potentiellement influencer la production d’EtG et devraient donc être pris en considération lors de l’interprétation de sa concentration in vivo. / Alcohol abuse is frequently associated with an increased risk of road accidents and violence, and can also lead to serious social and health problems. Therefore, the use of reliable markers to detect excessive punctual and/or chronic consumption of alcohol is necessary to prevent the harmful consequences of alcohol abuse. Ethylglucuronide (EtG) has been proposed as a marker of alcohol consumption in a variety of clinical and forensic contexts. Compared with the indirect markers (e.g. CDT, &#947;-GT), this minor metabolite of ethanol is very sensitive and specific, and is quantifiable in various biological matrices. It is formed by conjugation of ethanol with uridine 5’-diphosphate glucuronic acid (UDP-GA) via the action of UDP-glucuronosyltransferase (UGT) enzymes. However, the knowledge of the UGTs involved in the glucuronidation of ethanol, and the potential sources of the interindividual variability of EtG production are still not clearly established. The aims of our work were (1) to develop and validate a method for the determination of EtG in different biological matrices by gas chromatography coupled with tandem mass spectrometry, (2) to identify the human UGT isoforms involved in the glucuronidation of ethanol, and then to evaluate qualitatively and quantitatively their specific contribution in the formation of EtG, (3) to study the impact of the co-administration of drugs frequently used by ethanol consumers on the in vitro production of EtG, (4) to study the impact of functional genetic polymorphisms of two UGTs on the hepatic production of EtG, and finally (5) to study the impact of the consumption of cannabis and other drugs on the production of EtG using post-mortem samples. The main results of our study showed that (1) ethanol is primarily glucuronidated by the liver and, to a lesser extent, by kidneys, (2) UGT1A9 and 2B7 were identified as the main human UGTs involved in ethanol glucuronidation, (3) morphine, codeine, nicotine, and cotinine did not modify EtG in vitro formation rate; lorazepam and oxazepam produced a minor, but not significant, increase of EtG formation. Only cannabinol and cannabidiol significantly affected ethanol glucuronidation; cannabinol significantly increased the glucuronidation of ethanol in a concentration-dependent manner, whereas cannabidiol inhibited the glucuronoconjugaison of ethanol by a non-competitive mechanism (CI50 = 1.17 mg / L; Ki = 3.1 mg / L), (4) the SNP c.-900G>A and IVS1+399T>C affecting UGT2B7 and UGT1A9, respectively, seem to increase the in vitro production of EtG, and (5) cannabis and/or drugs consumption (mainly opioids, benzodiazepines, and paracetamol) seem to be associated with ratios of blood concentrations of EtG/ethanol significantly higher than those observed among only alcohol consumers. Taken together, these results show the existence of several factors that could potentially influence the production of EtG, and that should be taken into account when interpreting its concentration in vivo.

Gunshot residue (GSR) is the term used to describe the particles originating from different parts of the firearm and ammunition during the discharge. A fast and practical field tool to detect the presence of GSR can assist law enforcement in the accurate identification of subjects. A novel field sampling device is presented for the first time for the fast detection and quantitation of volatile organic compounds (VOCs). The capillary microextraction of volatiles (CMV) is a headspace sampling technique that provides fast results (< 2 min. sampling time) and is reported as a versatile and high-efficiency sampling tool. The CMV device can be coupled to a Gas Chromatography-Mass Spectrometry (GC-MS) instrument by installation of a thermal separation probe in the injection port of the GC. An analytical method using the CMV device was developed for the detection of 17 compounds commonly found in polluted environments. The acceptability of the CMV as a field sampling method for the detection of VOCs is demonstrated by following the criteria established by the Environmental Protection Agency (EPA) compendium method TO-17. The CMV device was used, for the first time, for the detection of VOCs on swabs from the hands of shooters, and non-shooters and spent cartridges from different types of ammunition (i.e., pistol, rifle, and shotgun). The proposed method consists in the headspace extraction of VOCs in smokeless powders present in the propellant of ammunition. The sensitivity of this method was demonstrated with method detection limits (MDLs) 4-26 ng for diphenylamine (DPA), nitroglycerine (NG), 2,4-dinitrotoluene (2,4-DNT), and ethyl centralite (EC). In addition, a fast method was developed for the detection of the inorganic components (i.e., Ba, Pb, and Sb) characteristic of GSR presence by Laser Induced Breakdown Spectroscopy (LIBS). Advantages of LIBS include fast analysis (~ 12 seconds per sample) and good sensitivity, with expected MDLs in the range of 0.1-20 ng for target elements. Statistical analysis of the results using both techniques was performed to determine any correlation between the variables analyzed. This work demonstrates that the information collected from the analysis of organic components has the potential to improve the detection of GSR.

In certain European countries and the United States of America, canines have been successfully used in human scent identification. There is however, limited scientific knowledge on the composition of human scent and the detection mechanism that produces an alert from canines. This lack of information has resulted in successful legal challenges to human scent evidence in the courts of law. The main objective of this research was to utilize science to validate the current practices of using human scent evidence in criminal cases. The goals of this study were to utilize Headspace Solid Phase Micro Extraction Gas Chromatography Mass Spectrometry (HS-SPME-GC/MS) to determine the optimum collection and storage conditions for human scent samples, to investigate whether the amount of DNA deposited upon contact with an object affects the alerts produced by human scent identification canines, and to create a prototype pseudo human scent which could be used for training purposes. Hand odor samples which were collected on different sorbent materials and exposed to various environmental conditions showed that human scent samples should be stored without prolonged exposure to UVA/UVB light to allow minimal changes to the overall scent profile. Various methods of collecting human scent from objects were also investigated and it was determined that passive collection methods yields ten times more VOCs by mass than active collection methods. Through the use of polymerase chain reaction (PCR) no correlation was found between the amount of DNA that was deposited upon contact with an object and the alerts that were produced by human scent identification canines. Preliminary studies conducted to create a prototype pseudo human scent showed that it is possible to produce fractions of a human scent sample which can be presented to the canines to determine whether specific fractions or the entire sample is needed to produce alerts by the human scent identification canines.

Biochar is a material whose properties, composition and impact on the environment are often discussed nowadays. The reason is its application as a soil conditioner. This leads to the need for qualitative as well as quantitative knowledge of its composition. This diploma thesis focuses on the qualitative analysis of biochar leachates processed by various extraction techniques. To approximate the real conditions, six aqueous mediums with different pH values is used, which a solid sample of biochar is exposed to. The prepared leachates are after that processed by extraction techniques, which are solid phase microextraction (SPME), single drop microextraction (HS-SDME), solid phase extraction (SPE) and liquid-liquid extraction (LLE). Analysis of the extracts was performed by gas chromatography-mass spectrometry (GC/MS). The measurements showed that the HS-SDME technique did not show a response. SPE was a rapid technique, but compared to other techniques, it acquired a smaller range of substances. Liquid-liquid extraction, a time-consuming technique, did not obtain as much analytes as SPME. By comparing these applied techniques, the SPME technique is the best choice. The most common determined analytes were those from the group of ketones and carboxylic acids and their derivatives.

The subject of this thesis was the determination of the profile of volatile aromatic compounds of varietal wines and the subsequent statistical analysis, aiming to find varietal markers of selected varieties – individual substances, their typical contents, mutual correlations. The HS-SPME-GC-MS technique was used to determine the aromatic profile. The main focus of this work was the analysis of the varieties Pálava, Hibernal, Johanniter and Solaris. The data obtained were statistically processed using descriptive and multivariate statistical methods. On the basis of these analyses, models were developed which, from the contents of 14 selected compounds of the aromatic profile, allow the Piwi varieties Hibernal, Johanniter and Solaris to be distinguished from each other and from the Pálava variety and, for the Pálava variety wines, to determine their origin in the Znojmo or Mikulov wine sub-region.

Echinacea purpurea is a rich source of biologically active compounds, which are attributed to several therapeutic effects. This thesis is focused on the optimization of the extraction process, considered these substances. Maceration was chosen as a type of extraction, due to its simplicity. The Optimal conditions were solid-solvent ratio 1: 5, 40% ethanol, 60 °C, 60 minutes. An extract from the dried flowers of Echinacea purpurea was prepared by this optimized process. The content of total phenolic compounds was set on 365.18 ± 3.51 [mg_GAEl^(-1)] and the antioxidant activity was 507.50 ± 6.89 [mg_TEACl^(-1)]. The percentage of radical scavenging activity ABTS•+ was 95.45 ± 1.30. The antimicrobial activity of the extract was tested against 3 microorganisms – Escherichia coli, Bacillus cereus and Candida glabrata. There was not detected inhibitory effect on the growth of microorganisms. In a total, 39 volatile compounds were identified, and the biggest group were represented sesquiterpenes (70.9%). The obtained results confirm that Echinacea extract has antioxidant activity and contains several interesting aromatic substances, In the future, it can be used as a natural resource of nutritionally and sensory valuable compounds, e.g., into various foods and delicacies.

This diploma thesis deals with the comparison of aroma compounds in goat and cow milk. The content of aroma active substances in samples of goat and cow milk, which were analyzed by HS-SPME-GC-MS method, is monitored. In the theoretical part, milk is characterized, its formation in the dairy mammary gland is described, its composition including an overview of aromatic active substances, physical and chemical as well as its sensory properties, milk defects and differences between cow's and goat's milk are described. Further, gas chromatography in combination with solid phase microextraction technique in tandem with mass spectrometry is described. In the experimental part, 13 samples of goat and 8 samples of cow milk were subjected to HS-SPME-GC-MS analysis. Samples were analyzed fresh and after heat treatment. Altogether, 78 volatile substances were identified in the samples. Ketones were the most numerous, aldehydes, alcohols and carboxylic acids were the most abundant chemical groups. There were differences between samples in number as well as in content of the identified compounds, 64 in goat milk and 48 in the cow milk.

Plectranthus amboinicus is a herb that was found to have a lot of bioactive compounds, most of them are polyfenolic compounds. Extraction is neccesary to obtain bioactive compounds and to use them subsequently in food or cosmetic industry. Three methods of extraxtion – maceraction, PHWE and PFE, were used in this study. The best yield of extracted polyfenols was observed by maceration. Maceration was, in the next step, optimized using mathematical statistical method of planned experiment (DOE). The optimal conditions found for extraction were: temperature 60 °C, solvent 40% ethanol, time 90 minutes, solid-solvent ratio 1:10. Total phenolic compunds content in this extract was 0,18 ± 0,05 mgGAE·ml-1, which means 1,83 ± 0,53 mgGAE·g-1 of plant. The antioxidant activity was 148,69 ± 26,92 gTEACml-1. Percentage of radical scavenging activity ABTS•+ was 30,03 ± 5,44 %. Further, 27 of volatile aroma compounds were identified in optimal extract, the most important were: -selinene (16,67 %), trans--bergamotene (14,22 %), caryophyllene (14,11 %), 3-carene (8,22 %), -copaene (7,55 %), -terpinene (6,28 %), humulene (5,87 %), cadina-1(10)-4-diene (5,19 %).

The presented dissertation is a study focused on the characterization of sensory quality, especially the flavour of model samples of natural cheeses. Attention is aimed to the composition and content of volatile (sensory active) substances as important markers of flavour and free/bound fatty acids as their precursors. The headspace solid phase microextraction in conjunction with gas chromatography with mass spectrometry was used to determine volatiles. For the determination of fatty acids, the lipids were isolated from the sample with a solvent mixture of diethyl ether and petroleum ether, followed by esterification using a methanolic solution of boron trifluoride as a catalyst and final analysis by gas chromatography with flame ionization detection. The experimental part was divided into two experiments; in experiment I, Moravský bochník type cheeses (45 % fat in dry matter., 60 % dry matter) were analysed, in experiment II, Edam type cheeses (45 % fat in dry matter, 50 % dry matter). Model samples of cheeses were made at Tomas Bata University in Zlín. For all samples, a classical mesophilic culture containing Lactococcus spp. and Leuconostoc spp. was used, this was always supplemented with a suitable monoculture of thermophilic bacteria; in the case of Moravský bochník cultures of Streptococcus thermophilus and Lactobacillus helveticus and in the case of Edam cheeses Lactobacillus casei and Lactobacillus plantarum. The essence of both experiments was to assess the influence of the used microbial cultures on the above-mentioned parameters, at the same time their changes during the ripening of the cheeses were monitored. The results showed that the addition of thermophilic cultures, as well as the maturation process did not affect the number of fatty acids or volatiles, but significant differences were found in their content. The content of bound fatty acids decreased during cheese ripening due to ongoing lipolytic changes while increasing the content of free fatty acids and their subsequent degradation to volatile substances, which ultimately contribute to the taste and aroma of the cheese. Ketones and acids were the most quantitatively present in all samples. During maturation, contrary to expectations, the content of identified volatiles also decreased. The addition of the thermophilic cultures didn’t have a clear effect and varied depending on the maturation phase. In the first phase, although they showed more pronounced lipolytic activity, the subsequent metabolic processes - the breakdown of free fatty acids into volatile substances, compensated for these differences. These results confirm a significant share of the basic mesophilic culture in the formation of volatile substances, in particular the flavour of cheeses.