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Application of Mass Spectrometry for the Determination of Oxidative Stress Markers and Mycotoxins
Čumová, Martina ; Večeřa, Zbyněk (referee) ; Hajšlová, Jana (referee) ; Vávrová, Milada (referee) ; Čáslavský, Josef (advisor)
The first topic presented in the dissertation thesis is determination of isoprostanes as markers of oxidative stress and other compounds affected by presence of oxidative stress. Isoprostanes iPF2-III, iPF2-VI, iPF2-VI, astaxanthin and polyunsaturated fatty acids (PUFA), especially arachidonic acid (AA) were monitored in Atlantic salmon eggs (Salmo salar). Methods for the determination of these compounds have been developed and optimized using chromatographic separation coupled to conventional or mass spectrometric detection. Freshly laid eggs, eyed embryos and non-viable eggs were used to test a general hypothesis that egg viability can be affected by susceptibility to oxidative stress, either through the specific fatty acid concentration and/or the antioxidant capacity of the eggs. Levels of isoprostanes and arachidonic acid (AA) were significantly higher in non-viable eggs than in control (eyed embryos) as well as relative abundance of PUFA. While no difference of isoprostanes was found between freshly laid and control those from the Atlantic stock except iPF2-VI which was observed under the LOQ in the control. Higher levels of PUFA and AA in comparison with the control were observed in the freshly laid eggs. However, the only statistically significant difference was observed in the amount of astaxanthin. Different levels of PUFA and astaxanthin may be related to their biochemical consumption during the development of eggs. This work evaluated potential effect on the viability of eggs Salmo salar due to the presence of oxidative stress. The monitoring of mycotoxins in food and feed was the subject of the second topic. Mycotoxins are secondary metabolites produced by fungi. They are ubiquitous undesirable natural contaminants that are toxic for humans and animals. Today are known more than 500 mycotoxins. However, only few of them are regulated by the European Union. The European Food Safety Authority (EFSA) was asked by the European Commission to provide a scientific opinion on other mycotoxins for which statutory limits could be developed. In this study is proposed simultaneous screening allowing fast, reliable and sensitive approach, identification and quantification of 17 mycotoxins in food and feed sample. The method includes both mycotoxins regulated by the EU and selected mycotoxins required by the EFSA (aflatoxins, deoxynivalenol, nivalenol, zearalenone, fumonisin, ochratoxin A, T-2 toxin, HT-2 toxin, enniatins and beauvericin). Analytes are isolated by the modified QuEChERS method. For separation and target mycotoxins detection, ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC –MS/MS) was employed. The method also allows determination of ergot alkaloids (ergocornine, ergosine, ergocryptine, ergocristine and their respective epimers). The developed method was used either for monitoring mycotoxins and ergot alkaloids in feed and raw materials and barley and malt prepared from it.
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Preparation of hyaluronan oligosaccharides with N-acetylglucosamine at both ends of the chain using enzymatic catalysis
Gadová, Martina ; Hermannová,, Martina (referee) ; Šílová, Tereza (advisor)
This bachelor thesis deals with preparation of odd-numbered hyaluronan oligosaccharides with N-acetylglucosamine at both ends of the chain using enzymatic catalysis by -Glucuronidase. The theoretical part focuses on present knowledge of hyaluronic acid and it fragments. It focuses on properties, enzymatic hydrolysis, application in different branches of science, but also analysis of oligosaccharides. One chapter is devoted to properties of enzyme -Glucuronidase. The experimental part deals with preparation of oligosaccharides type HANN. The reaction conditions for preparation of HA oligosacharides were optimized (pH, temperature) and the progress of enzymatic hydrolysis and the mixture composition after digestion were monitored by method UHPLC.. After optimization the mixture of oligosaccharides HANN was prepared and consenquently it was analysed by methods UHPLC and LC-MS. All the data were acquired in company Contipro Pharma, a.s., Dolní Dobrouč, Czech republic.
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Methylation of viral RNA
Šimonová, Anna ; Macíčková Cahová, Hana (advisor) ; Sýkora, David (referee) ; Elleder, Daniel (referee)
Viruses are the major force that shapes the evolution of both pro- and eukaryotic organisms. They have a simple inner organization and contain only a few, usually well-described RNAs. In the case of +(ss)RNA viruses, their genomic RNA serves also as mRNA. This makes them a perfect model system for searching for new mRNA modifications as well as for understanding the role of already known modifications. In this work, Human Immunodeficiency Virus type 1 (HIV-1) from the Retroviridae family was used as a model system. In the following study, four representatives from the Picornaviridae family were tested for RNA methylation profile. To get the information, a combination of two techniques was developed, liquid chromatography- mass spectrometry (LC-MS) and sequencing techniques. Results of LC-MS reveal a surprisingly high amount of 1-methyladenosine (m1 A) in RNA isolated from HIV-1. Nevertheless, the m1 A mapping sequencing technique confirm m1 A position only in co-packed tRNA. This led to the recalculation of HIV-1 virion RNA composition. In the case of Picornaviridae, LC-MS revealed m1 A and 5-methylcytidine (m5 C) in two insect viruses (Sacbrood virus, SBV and Deformed wing virus, DWV). RNA seq techniques (m1 A mapping and bisulfite sequencing) confirmed the presence of m1 A and m5 C only in tRNA....
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Biopharmaceuticals from the MS point of view
Hubálek, Martin ; Lenčo, J.
The course aims to introduce biotherapeutics, which are becoming an increasingly important part of the pharmacotherapy of serious diseases. Mass spectrometry plays an important role for their quality control in the pharmaceutical industry. We will introduce the audience to biopharmaceuticals, which in this context are peptides, proteins, monoclonal antibodies or oligonucleotides. From the analyst's point of view, we will focus mainly on antibody drugs, looking at the possibilities of their separation and analysis by mass spectrometry, at the level of intact protein molecules, antibody subunits or peptides.
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Analysis of bilirubin's relevant photoproducts and their biological significance in the issue of neonatal jaundice
Křepelka, David ; Kozlík, Petr (advisor) ; Zelenka, Jaroslav (referee)
Neonatal jaundice occurs in almost 60% of full-term and 80% of premature babies, where a slightly increased concentration of bilirubin protects against oxidative stress just after birth. When a specific bilirubin level is exceeded in serum (usually above 340 µmol·l-1 ), bilirubin-could induce kernicterus. These negative states are prevented by blue-green light phototherapy (420-510 nm), which converts bilirubin into more polar photoproducts that are more easily excreted via bile and/or urine. Published data have shown that newborns with indicated phototherapy may develop clinical problems later in life (higher incidence of e.g. asthma, allergies, type 1 diabetes was observed at a later age). A possible reason for the occurrence of these diseases is the specific biological activity of photoproducts. This study aims to purify an unknown photoproduct (band P), formed after 8 hours of irradiation of a bilirubin solution with blue light, and to establish a quantitative analytical method for its measurement in relevant matrices. This product was isolated and separated by a thin layer and subsequent column flash chromatography. In the next part, an LC-MS/MS method was developed for quantification of band P. Finally, urine, plasma, and feces samples collected from 15 newborns before and after phototherapy...
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Monitoring process contaminants in coffee roasting using LC-MS
Ilyushchenkova, Alexandra ; Juglová, Zuzana (referee) ; Diviš, Pavel (advisor)
The bachelor thesis focuses on monitoring changes in the concentration of process contaminants during coffee roasting using liquid chromatography with mass detection. Green coffee was roasted at 220 °C for 14 minutes with two-minute intervals. Samples for analysis were taken from 10. by 24. minutes of roasting. Practical monitoring of the acrylamide’s formation during coffee roasting was not carried out, since it was not possible to detect acrylamide in coffee using a mass detector, even after its derivatization with thiosalicylic acid. The relevant data were taken from other scientific articles, which reported a linear increase in concentration with the roasting time up to the maximum value and a subsequent exponential decrease at the end of roasting. By monitoring 5-hydroxymethylfurfural, it was found that during roasting, this substance is gradually formed in coffee due to the decomposition of carbohydrates, however, with increasing roasting time, the degradation of this substance occurs quite quickly. The concentration of 5-hydroxymethylfurfural was also determined in coffee samples purchased in the trade network. Concentrations of 5-hydroxymethylfurfural in these samples ranged from 0,3 to 0,38 mg/kg, which are corresponded to those reported in other expert studies.
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Comparison of different tandem mass detection scans in the analysis of cannabidiol
Tichá, Tereza ; Kozlík, Petr (advisor) ; Kubíčková, Anna (referee)
The aim of this bachelor thesis was to compare different tandem mass spectrometer scans in order to develop an LC-MS/MS method suitable for the determination of cannabidiol in rat serum. In this bachelor thesis the mass spectrometer and high-performance liquid chromatography method were optimized. The optimum parameters were as follows: chromatographic column Acquity UPLC BEH C18 50 × 2,1 mm, 1,7 μm from Waters (Wexford, Ireland). The mobile phase consisted of methanol and water of LC-MS purity. The flow rate of the mobile phase was 0,4 ml/min, the column temperature 40 řC, the temperature in an autosampler 15 řC, the sample injection volume was 1 μl and the analysis time was 7 minutes under gradient elution conditions. The MRM transitions of highest intensity were monitored. For CBD, the MRM transition in positive mode was 315,2 → 193,1 (Q1 = -10 V, CE = -24 V, Q3 = -20 V), for isotopically labeled cannabidiol D3 was chosen 318,4 → 196,0 (Q1 = -10 V, CE = -23 V, Q3 = -20 V). The calibrations in solvent and rat serum were performed in MRM and SIM mode. No calibration was constructed for the SCAN mode due to its low selectivity, which made it able to detect cannabidiol only from concentration of 312,5 ng/ml. The highly selective MRM was able to operate over the entire observed concentration range of...
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