This diploma thesis is focused on the problematics of occurence and quantitative determination of hormonal active substances in biological material. The aim of experimental part was to develope and validate the analytical method, suitable for quantification of hormonal active substances in biological materials. Elaborated method is able to be used for screening determination of some steroid hormonal active substances in urine of farming animals, and for performance confirmative analysis of the same substances in the same matrix on required concentration level. Used method is determined for quantification of steroids in low concentration level in urine of farming animals. Among others performs the criteria of confirmative methods intended for monitoring of residues of hormonal active substances in biological material according to Council Directive 2002/657/EC. Determined parameters of the method answer the supposed values. The method proved to be quite suitable and offered required results. Individual analytes were identified,with certainty, they offer expressive, symmetrical and well separated peaks. This diploma thesis was prepared in Institute for State Control of Veterinary Biologicals and Medicaments in Brno.

Genus Phytophthora represents a world-wide spread pathogen with more than hundred recognized species and its devastating effect on plants has a serious economic and ecological impact. This diploma thesis entitled „Proteome and metabolome of genus Phytophthora” briefly summarizes knowledge about this pathogen, including its life cycle and interactions with its host. Twelve species representing six Phytophthora clades that were selected for experimental work are then discussed in details. Phytophthora isolates were characterized on proteome and metabolome level employing an LC-MS untargeted proteome profiling and a GC-MS analysis of volatiles. The results were then processed to identify candidate molecules for a targeted identification of Phytophthora and these results were validated in an independent experiment with P. palmivora and Hordeum vulgare. We found that a proteome profiling can be employed as a tool to differentiate individual Phytophthora species and that the marker peptides can be employed for a targeted monitoring of Phytophthora presence in plants.

The present diploma thesis is focused on the evaluation of steroid metabolome in body fluids in human pregnancy and parturition and also concentrates on the estimation of steroids in men with epilepsy (MWE) regarding the influence of epilepsy and its treatment with antiepileptic drugs (AEDs). The results obtained eventuate to the following conclusions: 1. They support the proposition about the key importance of corticoliberin for the timing of human parturition via stimulation of the steroidogenesis in the fetal zone of the fetal adrenal (FZ). Changes in steroid levels, however, much better predict the timing of parturition than the variation in corticoliberin. 2. The distribution of isoforms of placental oxidoreductases determines the excess of oxidized forms of the sex steroids and NAS in the fetus and the excess of their reduced forms in mother. This mechanism fundamentally affects the steroid bioactivity. The results indicate the role of NAS in pregnancy sustaining, their analgesic effect around parturition and their role in the pathophysiology of postpartum blues.

Q-Med är ett medicintekniskt företag som tillverkar medicintekniska produkter, exempelvis produkter som är baserade på gel som innehåller bland annat hyaluronsyra. Gelen är placerad i en spruta som innehåller en gummikolv som är i kontakt med gelen.Idag används allt mer polymera material för olika tillämpningar, bland annat som medicintekniska produkter och läkemedelsförpackningar i läkemedelsbranschen. Q-Meds produkter betraktas som medicintekniska produkter på många marknader. Medicintekniska produkter och läkemedelsförpackningar innehåller ofta material som består av polymerer, exempelvis gummi och plast. På dessa material ställs olika krav så att dessa inte kontaminerar den produkt de är i kontakt med, då detta kan leda till olika negativa konsekvenser, exempelvis toxiska reaktioner i kroppen eller sänkt effektivitet hos läkemedlet/produkten. I och med att polymera material används kan det förekomma olika föroreningar i dessa material, exempelvis tillsatsämnen och reaktionsprodukter. Om dessa substanser läcker ut brukar dessa kallas för ”leachables” och ”extractables” – det finns inga motsvarande namn på svenska. Organiska och oorganiska föreningar är exempel på vad som kan lakas ut ur gummit.Den här rapporten beskriver en studie av gummi (bromobutylgummi) som är i kontakt med en gelprototyp (Prototyp A och Prototyp A-L) för att se vilka föreningar som kan lakas ut från gummit ur perspektivet leachables/extractables. För att ta reda på vilka föreningar som lakas ut från gummit utsattes denna för olika betingelser, som olika pH, temperatur, lösningsmedel, placebolösning och kontakt med produkt. För att kunna analysera vilka föreningar som lakades ut ur gummit i både gasfas och vätskefas användes Solid Phase Micro Extraction (SPME) och GC-MS som analysmetoder. Tre SPME fibrer med olika egenskaper, exempelvis olika polaritet och selektivitet användes för insamling av extraherbara ämnen.Resultaten visar att olika föreningar lakas ut ur gummit. Vid försök att identifiera dessa med ett referensbibliotek för masspektra gav många av dessa föreningar en relativt låg sannolikhet (< 50 %) jämfört med de föreslagna föreningarna. Detta gör det svårt att avgöra om det är en särskild förening som lakas ut eller om det är något liknande ämne som lakas ut. I gasfasen hittades fler föreningar jämfört med vätskefasen. Detta beror bland annat på att det finns fler ämnen som är villiga att gå upp i gasfas på grund av deras affinitet gentemot provet, det vill säga hur villig en förening är att reagera gentemot en annan förening. Två föreningar som dyker upp i de flesta headspace analyserna är 1-Bromo-3-(2-bromoethyl)heptane och butylated hydroxytoulene (BHT).Slutsatsen är att några enstaka föreningar lakas ut från gummit, så som BHT och 1-Bromo-3-(2-bromoethyl)heptane. Vad det gäller de andra föreningarna som lakas ut är det är svårt att avgöra om dessa föreningar verkligen kommer från gummit eller från någon annan källa, exempelvis fiberns omgivning. Detta beror på att SPME-metoden är en känslig metod som kan fånga upp en rad ämnen från sin omgivning. Därför anses det att fler studier måste göras på detta område, fast med en metod som ger mer pålitliga resultat. / Q-Med is a medical device company that manufactures medical devices, for instance a product that is a gel containing hyaluronic acid. The gel is placed in a syringe component containing a stopper made of rubber that is in contact with the gel.Today, increasingly more polymeric materials for various applications are used, for example medical devices and pharmaceutical packaging in the pharmaceutical industry. The products of Q-Med are considered as medical devices in many markets. Medical devices and pharmaceutical packaging often contains materials composed of polymers, for instance rubber and plastics. For these materials there are different requirements so that they do not contaminate the product that they are in contact with. Contamination may lead to negative consequences, such as toxic reactions and a reduced effectiveness of the drug/product.Since it is polymeric materials that are used there might be some potential impurities in the material, these pollutants are called leachables and extractables. Organic and inorganic compounds are examples of what can leach out of the rubber.In this report the rubber (bromobutyl rubber) that is in contact with a gel prototype (Prototype A and Prototype A-L) is studied to see if the rubber leaches out some compounds of the perspective leachables/extractbales. To know if the rubber leaches out some contaminates the rubber was exposed to various conditions. For instance; different pH, temperature, solvent, placebo solution and contact with the product. In order to analyze which pollutants leached out of the rubber, Solid Phase Micro Extraction (SPME) and GC-MS where used as analytical methods. Three SPME-fibers with different polarity and selectivity where used for the collection of extractable compounds.The results show that the rubber leaches out different compounds. Many of these have a low probability compared to the spectra of masses in the used reference library. This makes it difficult to determine if there is special compound that leaches out or if it is a similar compound that leaches out. When compared, more compounds were found in the gas phase than the liquid phase. This is partly because there are more compounds that are willing to get up in the gas phase, because of the compounds affinity to the sample, for example different polarity. The two main compounds, which are found in the most headspace analyses, are 1-Bromo-3- (2-bromoethyl) heptane and butylated hydroxytoulene.The conclusion is that some compounds, for instance 1-Bromo-3- (2-bromoethyl) heptane and butylated hydroxytoulene leaches out in the gas phase. For other compounds it’s hard to determine if they come from the rubber or from another source from it’s surrounding. This is because the SPME-method is a sensitive method that can absorb a number of compounds from its surroundings. It is therefore considered that more studies must be done in this area but with another method that give more reliable results.

Commercially available methylphenidate (MPH) exists as a racemic mixture composed of the d- and l-threo enantiomers. Various pharmacokinetic studies of MPH have shown a greater pharmacological potency of the d-threo enantiomer. Furthermore, it was deduced that the stereoselective cleavage of MPH to produce ritalinic acid (RA) by human carboxylesterase results in a higher oral bioavailability of the d-threo enantiomer. As a requirement for pharmaceutical regulation authorities, efforts have been made to determine the differential biological distribution of d- and l-threo MPH and RA enantiomers. In support of these efforts, numerous analytical procedures have been developed for the chiral separation and quantification of MPH enantiomers in a variety of biological matrices. The available methodologies accomplish the enantioseparation and quantification of MPH using gas chromatography, liquid chromatography or capillary electrophoretic techniques coupled with a variety of detectors. The current review discusses the technical procedures involved, and the sensitivity and selectivity of these assays.

Solid Phase Microextraction (SPME) associated with Microwave Assisted Extraction (MAE)^(1-3) , on-line headspace derivatization ^(4-6) and the selectivity of different SPME coatings ⁽⁷⁾ were studied. Trace amounts of Veltol ®, Veltol Plus® and short chain fatty acids in food samples were analyzed by GC/MS. Since SPME is not directly applicable to solid samples, SPME associated with MAE techniques was studied for solids, primarily food samples. The efficiency of classical solvent extraction and MAE was compared. The parameters which affect SPME, were optimized for the determination of Veltol® and Veltol Plus® in food products such as potato chips and coffee. The technique gave a detection limit of 2 ppb for Veltol Plus ® which is 200 times more sensitive than conventional GC technique. Headspace injection is characterized by simple and easy handling of complicated solid and solution matrices. Headspace injection, however, is not suitable for high molecular weight substances or non-volatile compounds. An on-line derivatization headspace technique was studied for short chain fatty acids. These samples are difficult to do by classical GC. The developed technique simplified the conventional derivatization procedures and combined the sample preparation and GC/MS analysis into one step. The thermostatting temperature, time, solvent and matrix effects were investigated. Low calorie fat and some agricultural samples were analyzed. The detection limit for acetic acid is 8 ppb. SPME is a novel sample introduction technique. The behavior of di(methylsiloxane), polyacrylate and Carbowax coatings on SPME fibers for compounds having different functional groups were investigated. The selectivities of the coating, sample pH and the sample temperature were investigated. / Ph. D.

Lambic beer is one of the oldest beer styles still being brewed in the western world today and the only beer that is still brewed through spontaneous fermentation. Lambic beers are only produced within a 500 km radius of Brussels because of the natural microflora found within the air in that region. Little is known about the chemical composition of lambic beers. The objective of this research were (1) to compare SPME and SAFE for the isolation of flavor and aroma compounds,  (2) determine the volatile composition and acids of commercially available lambic gueuze using SPME/GC-MS and HPLC, and (3) determine the major aroma compounds of aging lambic beer using GC-O.  Comparing the two extraction methods, both SPME and SAFE were able to identify a similar number of chemical compounds, however SAFE identified a greater number of acid compounds. A total of 50 compounds were identified within nine commercial brands of lambic beer. HPLC was used in the identification and quantification of acetic and lactic acids. The concentration of acetic acid in the commercial products ranged from 723 mg/L â " 1624 mg/L and lactic acid ranged from 995 â " 2557 mg/L. GC-O was used in the analysis of aged (3-28 months) lambic beer samples. As the samples increased in age, the number of aroma compounds detected by the panelists also increased. Panelists were detected nine aroma compounds in the 3 month old sample, while 17 compounds were detected in the 28 month old sample. The research conduct increased the number of volatile and semi-volatile compounds identified in lambic beer from 27 to 50. / Ph. D.

Indiana University-Purdue University Indianapolis (IUPUI)

High gravity (HG) brewing has become the most used strategy for maximizing fermenter productivity in commercial brewing. While HG brewing has many benefits, the additional stress placed on the yeast due to the higher concentration of fermentable sugars in the wort can negatively impact fermentation performance and flavor compound formation. A proper dissolved oxygen (DO) level is vital to guarantee adequate yeast performance during HG fermentations. Dissolved oxygen is vital to yeast viability throughout the fermentation process, as yeast requires oxygen to synthesize vital cell membrane components needed for continued anaerobic growth and cell division. Previous research has demonstrated the importance of DO in wort for regular gravity fermentation and flavor compound production. However, the impact of dissolved oxygen during HG brewing on fermentation performance and how this will impact the production of flavor compounds have not been fully researched. The objectives of this research were to analyze the impact of wort aeration timing and concentration on fermentation performance, flavor stability, and the formation of volatile flavor compounds, determined using gas chromatography. Gas chromatography analysis was modeled after the ASBC Method Beer-48. Flavor stability and staling was analyzed during aging under normal and accelerated conditions utilizing TBA analysis. Pre-pitch oxygen treatments at levels greater than 8 ppm dissolved oxygen significantly increased attenuation when compared to the unoxygenated controls. Post-pitch oxygenation significantly increased attenuation, with DO treatments at levels of 8 ppm showed the most significant decrease in wort specific gravity. Aldehyde, ester, and higher alcohol production were all significantly affected by DO concentration. Aldehyde production decreased with increased DO concentration. Ester production increased from 0 to 8 ppm DO treatment and decreased at DO treatments greater than 8 ppm. Higher alcohol production increased from 0 to 10 ppm and decreased with DO treatments greater than 10 ppm. Greater concentrations v of DO resulted in greater TBA index values after normal and accelerated aging, with accelerated aging producing greater TBA index values than normal aging.

Date palm (Phoenix dactylifera L.) is a fruit-bearing tree with numerous potential sustainable applications. Since ancient times, it has been considered a stable, secure, and sustainable food. This work provides comprehensive metabolic profiling of both parts, flesh and seed, of four P. dactylifera cultivars; Ajwa, Anbara, Sukkari, and Degelt Nour, which originated from two countries, Saudi Arabia, Tunisia. The analysis performed using mass spectrometry-untargeted metabolomics approaches, included a combination of LC-MS and GC-MS coupled to multivariate statistical analysis to reveal the differences in metabolite compositions among date varieties. The LC-MS seed results showed several classes of metabolites that belong to the flavonoids, phenolic acids, and amino acids derivatives, including citric acid, malic acid, lactic acid, hydroxyadipic acid, caffeic acid, which were at high concentrations in AJS followed by DNS and ARS. The LC-MS flesh analysis displayed that DNF had a high level of Isoquercitrin (flavonoid compound) and sinapic acid, and AJF was high concentrations level in hydroxyadipic acid and ascorbic acid. GC-MS concluded that seed samples of four date varieties are richer in metabolites classes than the flesh samples. The metabolites contributed to the seed metabolite compositions included several classes of amino acids, hydrocinnamic acids (caffeic, ferulic and sinapic acids), antioxidant phenolics, and long-chain fatty acids. The PCA and its loading analysis demonstrated the discriminating metabolites that were responsible for date varieties segregation, as HCA displayed the metabolic patterns and groups of metabolites that drive the clustering of the date samples, two groups of dates clustered together (AR and AJ) and (SR and DN). These clusterings are based on the similarities and differences observed in the metabolite compositions of the studied date samples that could be explained by differences in various metabolic responses and the environmental conditions, genotypes and geographical regions. This extensive date palm information would increase the potential of date fruits and seeds as low-cost sources of natural diet that may provide nutritious and bioactive components in the food and pharmaceutical fields to produce value-added products.