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The effectiveness of highly polar agents in cancer treatment is well recognized, but their physicochemical properties make their analytical determination a demanding task. Their analysis requires peculiar sample preparation and chromatographic separation, which heavily impacts the precision of such an analytical method. As a case study, we chose a polar cytotoxic bleomycin, which is a mixture of complexing congeners with relatively high molecular mass, a fact that creates an added challenge in regard to its detection via electrospray mass spectrometry. These issues combined lead to a deprived method performance, so the aim of this study is manifold, i.e., to optimize, validate, and establish quality performance measures for determination of bleomycin in pharmaceutical and biological specimens. Quantification of bleomycin is done at diametrically different concentration levels: at the concentrations relevant for analysis of pharmaceutical dosage forms it is based on a direct reversed-phase HPLC-UV detection, involving minimum sample pretreatment. On the contrary, analysis of bleomycin in biological specimens requires phospholipid removal and protein precipitation followed by HILIC chromatography with MS/MS detection of bleomycin A2 and B2 copper complexes being the predominant species. This study further attempts to solve the traceability issue in the absence of certified reference standards, determines measurement uncertainty, investigates BLM stability and method performance characteristics, and, last but not least, provides an explanatory example of how a method quality assurance procedure should be established in case of an exceedingly complex analytical method.

Razvoj zdravila je kompleksen in dolgotrajen proces, s katerim iščemo in razvijamo nove metode zdravil in zdravljenja za različne bolezni. Proces odkrivanja zdravila se običajno prične z določitvijo in ovrednotenjem biološke tarče, čemur sledijo različne tehnike za iskanje spojin zadetkov ter spojin vodnic. To lahko vključuje rešetanje velikih knjižnic kemičnih spojin, virtualno rešetanje ali oblikovanje novih spojin na podlagi strukture tarče. Ena izmed pomembnih tehnik pri odkrivanju zdravilnih učinkovin je masna spektrometrija, ki lahko zagotovi pomembne informacije o potencialnih kandidatih za zdravilne učinkovine. Pomembna tehnika je tudi afinitetna selekcijska masna spektrometrija, ki omogoča določitev majhnih molekul, ki se vežejo na biološko tarčo z visoko afiniteto in s tem omogoča razvoj bolj učinkovitih ter selektivnih zdravil. Klik kemija predstavlja pomembno orodje pri sestavljanju enostavnih molekul v kompleksnejše. Gre za učinkovito, hitro in selektivno sintetično metodo za spajanje molekularnih fragmentov pod blagimi reakcijskimi pogoji. Primer klik reakcije je z bakrom(I) katalizirana azid-alkin cikloadicija, ki smo jo uporabili tudi pri sinteznem delu magistrske naloge. Pri magistrski nalogi smo želeli razviti strategijo priprave knjižnice spojin, ne da bi pri tem izolirali posamezne spojine. Sintezni del naloge smo izvedli v treh delih. V prvem delu smo pripravili nekaj monoazidov in diazidov, v drugem delu smo pripravili nekaj monoalkinov in dialkinov, v zadnjem delu pa smo s klik reakcijo (z bakrom(I) katalizirano azid-alkin cikloadicijo) pripravili večje število 1,2,3-triazolov. Nastanek triazolov smo preverili s tekočinsko kromatografijo, sklopljeno z masno spektrometrijo. Končnim produktom smo določili tališče, na koncu pa smo jih testirali tudi na izoliranem encimu InhA in tako določili njihovo zaviralno aktivnost. Tekom magistrske naloge smo pripravili približno 300 spojin v relativno kratkem času (v dveh mesecih). Rezultati testiranj so pokazali, da nobeden od produktov sintez ni učinkovit zaviralec encima InhA. Uporaba afinitetne selekcijske masne spektrometrije tako ni bila smiselna. Za dosego boljših rezultatov bi v prihodnosti lahko uporabili večje število azidov in alkinov, s čimer bi povečali verjetnost zadetka. Prav tako bi bilo smiselno pripravljene zmesi testirati na več tarčah. Drug development involves a complex and lengthy process aimed at discovering and developing new treatment methods and drugs for diverse diseases. This process encompasses the identification and evaluation of biological targets, as well as techniques for identifying lead compounds. Mass spectrometry plays a vital role in drug discovery by providing essential information about potential drug candidates. In particular, affinity selection mass spectrometry enables the identification of small molecules that bind with high affinity to biological targets, facilitating the development of more effective and selective drugs. Click chemistry, exemplified by the copper(I)-catalyzed azide-alkyne cycloaddition, serves as a valuable tool for assembling simple molecules into more complex ones, offering an efficient, rapid, and selective synthetic method. This Master's thesis aimed to develop a strategy for preparing a compound library without isolating individual compounds. The synthesis process consisted of three parts: the preparation of monoazides and diazides, the synthesis of monoalkynes and dialkynes, and the generation of a larger number of 1,2,3-triazoles through the copper(I)-catalyzed azide-alkyne cycloaddition. The formation of triazoles was confirmed using liquid chromatography coupled with mass spectrometry. Additionally, the melting point of the final products (azides and alkynes) was determined, and their inhibitory activity was assessed by testing them on the isolated InhA enzyme. Therefore, affinity selection mass spectrometry was not used, since there was not any active reaction mixture. Approximately 300 compounds were successfully synthesized within a relatively short timeframe (two months) during the Master's thesis. However, none of the synthesized products exhibited effective inhibitory activity against the InhA enzyme. Future work could involve the use of a larger number of azides and alkynes to increase the likelihood of identifying promising compounds as well as testing the products on other enzymes.

The aim of this study was to compare peeled and unpeeled dehydrated apple slices of the red-fleshed ‘Baya Marisa’ and the white-fleshed ‘Golden Delicious’, to analyze the difference in the content of sugars, organic acids, and phenolic compounds during the heat process of dehydration, and to compare it with our previous study on fresh apples of the same cultivar. The purpose of these study was to see how many primary and secondary metabolites are lost in the dehydration process to better understand what is ingested by consumers in terms of nutritional value. A total of 30 phenolic compounds were identified and quantified, some of them for the first time. The total analyzed phenolic content (TAPC) of the unpeeled dehydrated apple slices was 1.7 times higher in ‘Golden Delicious’ than in ‘Baya Marisa’. The unpeeled dehydrated apple slices of ‘Golden Delicious’ had higher total hydroxycinnamic acid (2.7×) and dihydrochalcone (1.2×) content. The peeled dehydrated apple slices of ‘Baya Marisa’ had higher total dihydrochalcone (2.2×) and total flavanol (2.2×) content compared to ‘Golden Delicious’. The content of citric and malic acids was higher in the unpeeled and peeled dehydrated apple slices of ‘Baya Marisa’, compared to ‘Golden Delicious’. The content of ascorbic acid was higher in the unpeeled (1.6×) and peeled (1.8×) dried apple slices of ‘Baya Marisa’. The content of fructose and glucose was 1.4 times higher in the unpeeled dried apple slices of ‘Golden Delicious’.

The consumption of fresh vegetables has been consistently associated with numerous health benefits. However, several factors (such as allelochemicals) influence yield, quality, and metabolites, which inevitably affect the fruit quality and health benefits. The present study was conducted to investigate the yield, quality, metabolic responses, and potential toxicity of Cucumis sativus grown in juglone-containing soils. For the treatments, pure juglone (100 µM, 1 mM) and walnut leaf extracts (100 µM) in soil concentrations found in walnut orchards were used. A total of 36 phenolic compounds were identified and quantified in fruits, leaves, and roots using a mass spectrometer coupled with high-performance liquid chromatography. We concluded that juglone at a concentration of 100 µM or walnut leaf extract at the same juglone concentration does not affect the yield of C. sativus, while juglone at a concentration of 1 mM strongly affects it. In the case of juglone, juglone itself was found only in the roots of C. sativus, but not in the leaves or fruits, so C. sativus fruits are considered safe for cultivation in juglone-containing soils. However, this could prove problematic if the plants grown are tubers or root vegetables. The data suggest that juglone itself inhibits secondary metabolism in the plant, making it more susceptible to stress and pathogen attacks.

Different climatic conditions are known to affect the synthesis of primary and secondary metabolites. Therefore, the phenolic contents in new growing areas could affect the quality and flavor of hazelnuts. The aim of this study was to determine the variability of the phenolic contents of the kernels in different commercial hazelnut cultivars depending on their growing area. Five cultivars (‘Tonda Gentile delle Langhe’, ‘Merveille de Bollwiller’, ‘Pauetet’, ‘Tonda di Giffoni’, and ‘Barcelona’ (syn. ‘Fertile de Coutard’)) grown in different European collection orchards were included in the study. High-performance liquid chromatography coupled with mass spectrometry was used to identify and quantify the phenolic compounds. Thirteen phenols were identified in the hazelnut kernels, including 7 flavanols, 2 hydroxybenzoic acids, 3 flavonols, and one dihydrochalcone. Catechin and procyanidin dimers were the main phenolic compounds found in the hazelnut kernels. The highest contents of catechin and total flavanols were determined in cultivars cultivated in Spain and northern Italy, and the lowest in Slovenia and France. Flavanols were the major phenolic groups independent of the place of cultivation, as they accounted for more than 50% of all phenolic compounds identified. The flavanols were followed by hydroxybenzoic acids, flavonols, and dihydrochalcones. Higher contents of flavanols and flavonols were found in kernels from areas characterized by higher natural irradiation, which stimulates their accumulation. The contents of hydroxybenzoic acids correlated with altitude, which stimulated phenolic acid synthesis. A negative correlation was observed between the dihydrochalcone content and annual rainfall, probably due to hydric stress. Nasl. z nasl. zaslona. Opis vira z dne 16. 11. 2022. Bibliografija: str. 14-15. Abstract.

Antibiotiki se uporabljajo za zdravljenje ali preprečevanje bakterijskih okužb. Linkozamidna antibiotika, ki sta preučevana v sklopu te magistrske naloge, sta klindamicin in linkomicin. Namen magistrske naloge je preučiti možnost in vitro tvorbe metabolitov in oksidacijskih produktov klindamicina z uporabo elektrokemijske analize sklopljene z masno spektrometrijo, kar bi predstavljalo veliko cenejši način pridobivanja metabolitov, saj so standardi na trgu zelo dragi in hkrati postavilo izhodišče za nadaljnje raziskave aktivnosti in neželenih učinkov metabolitov. Klindamicin je kloriran, biološko aktivni, semisintetični derivat z višjo in vivo in in vitro aktivnostjo od linkomicina. Po absorpciji se klindamicin večinoma metabolizira do oksidiranega produkta klindamicin sulfoksida in v manjši meri do N-desmetil klindamicina. V člankih je prisotno protislovje, saj nekateri trdijo, da sta metabolita aktivna, drugi pa, da sta metabolita neaktivni spojini. Za pridobitev podatkov za izdelavo masnega spektra klindamicina smo uporabili masno spektrometrijo. Izvedli smo tudi tandemsko masno spektrometrijo z izbiro vrhov pridobljenih iz masnega spektra klindamicina, ki smo jih nadalje fragmentirali. Za proizvodnjo oksidacijskih produktov klindamicina in vitro smo uporabili elektrokemijsko nastavitev Roxy. Iz rezultatov je bilo razvidno, da je pri večjih hitrostih čas napetostnega gradienta krajši. Zamenjava elektrode iz steklastega ogljika z diamantno elektrodo dopirano z borom je pokazala, da daje v našem primeru elektroda iz steklastega ogljika boljše rezultate. V nadaljevanju je bil apliciran enosmerni tok v vrednosti 1 V. Z masno spektrometrijo smo analizirali tudi analizne standarde metabolitov klindamicina. Iz rezultatov je razvidno, da smo uspešno proizvedli N-desmetil klindamicin in v manjši meri klindamicin sulfoksid. Masno spektrometrijo sklopljeno z elektrokemijo smo aplicirali tudi na linkomicin. Dobili smo ustrezen masni spekter, ki je v primerjavi s klindamicinovim brez vrha izotopa klora ob osnovnem vrhu. Rezultatov linkomicina nismo primerjali z analiznimi standardi. Vsi pridobljeni podatki so bili obdelani s programskim jezikom R Studio. Antibiotics are used for treatment or prevention of bacterial infections. Lincosamide antibiotics analysed in the scope of this master's thesis were lincomycin and clindamycin. The aim of this master’s thesis is to examine the possibility of the in vitro production of the metabolites and oxidation products of antibiotic drug clindamycin using electrochemistry coupled to mass spectrometry which would represent much cheaper way of acquiring metabolites as analytical standard metabolites on the market are very expensive and also the basis for further research on the activity of the metabolites or their side effects. Clindamycin is a chlorinated highly biologically active semisynthetic lincosamide derivate with higher in vivo and in vitro activity than lincomycin. When absorbed, the compound is metabolized mainly to the oxidized product clindamycin sulfoxide and to a lesser extent to N-desmethyl clindamycin. There is a contradiction in the articles as some claim the metabolites are active and others that metabolites are inactive compounds. Mass spectrometry was used to obtain mass spectrum data of clindamycin. Additionally, tandem mass spectrometry was performed by selecting the peaks obtained from mass spectrum of clindamycin and fragmenting them to further extent. The Roxy electrochemical setup was used to produce clindamycin oxidation products in vitro. Results showed that at higher scan rates the time of voltage gradient was shorter. Switch of electrode from boron doped diamond electrode to glassy carbon electrode showed that glassy carbon electrode gave better results in our case. Further, direct current of 1 V was applied. Additionally, commercially available metabolites were examined by mass spectrometry coupled to electrochemistry to compare the results. The results show that we successfully produced N-desmethyl clindamycin and to a lesser extent clindamycin sulfoxide. Mass spectrometry coupled with electrochemistry technique was performed for lincomycin as well. We obtained the mass spectrum which has, comparing to clindamycin, no chlorine isotope peak around base peak. We did not compare lincomycin results with analytical standard metabolites. All data obtained were introduced in R Scripts in R Studio program.

Tekočinska kromatografija visoke ločljivosti sklopljena z masno spektrometrijo (HPLC-MS) je tehnika, ki postaja nepogrešljiva na področju farmacije. Združuje dve analitski tehniki, in sicer tekočinsko kromatografijo visoke ločljivosti in zelo občutljivo ter specifično masno spektrometrijo. Tekočinska kromatografija loči komponente zmesi glede na njihovo afiniteto do stacionarne in mobilne faze. Te komponente lahko zazna UV svetloba. Osnova pri masni spektrometriji je ločevanje ioniziranih komponent v vakuumu glede na njihovo razmerje med maso in nabojem. Pri razvoju zdravilnih učinkovin se srečujemo s knjižnicami spojin. Testiramo jih na bioloških tarčah s pomočjo rešetanja visokih zmogljivosti. Biti morajo urejene in dobro oblikovane, da lahko v čim krajšem času testiramo čim večje število spojin. Pomembno je, da so spojine, ko jih shranimo v knjižnico ustrezne kakovosti in v nadaljnje tudi dobro shranjene. Namen magistrske naloge je bil vrednotiti knjižnico spojin, sintetiziranih na Fakulteti za farmacijo s HPLC-UV-MS tehniko. Število vzorcev v knjižnici spojin je bilo 1145. Za separacijo smo uporabili reverzno-fazno kromatografijo. Na masnem spektrometru smo uporabili elektrorazprševalno ionizacijo. Pri vrednotenju rezultatov smo imeli nekaj težav z analizo kromatogramov, na kromatogramih so se pojavili popačeni vrhovi in vrhovi slabe intenzitete. Prav tako je prišlo do napak pri pripravi vzorcev, dekarboksilacije spojin, hidrolize spojin, fragmentacije spojin in nestabilnosti raztopin. Vendar je bila instrumentalna metoda, kljub napakam primerna za večino spojin (90,1 %). Knjižnico smo uredili in v Excel tabeli zbrali vse podatke, ki smo jih pridobili pri interpretiranju rezultatov. Spojinam smo določili retencijski čas, ocenili čistost in odzive pri UV spektroskopiji. Z masno spektrometrijo smo potrdili strukturo spojin, pridobili MS kromatogram in masni spekter spojin. Določili smo nastanek aduktov. S tem smo kvantitativno in kvalitativno ovrednotili knjižnico spojin ter tudi na reprezentativnem vzoru pridobili podatke o obnašanju spojin vodnic v HPLC-UV-MS kromatografskem sistemu. High-pressure liquid chromatography coupled with mass spectrometry is a technique which is becoming indispensable in the pharmaceutical field. It combines two analytical techniques: high-performance liquid chromatography and mass spectrometry. Liquid chromatography separates the components of the compound according to their affinity with the stationary and mobile phase. These components can be detected by UV light. The basis of mass spectrometry is separating ionized components in a vacuum, according to their mass-to-charge ratio. In the development of active substances, compound libraries are one of the key compnents of research. They are tested on various biological targets, using high-throughput screening. They need to be organised and well-formed, to test the largest number of compounds in the shortest possible time. It is of key importance, that the compounds, that are being kept in the library, are of adequate quality and well preserved for future use. The aim of the master's thesis was to assess the library of compounds, which were synthesised at the Faculty of Pharmacy with the HPLC-UV-MS technique. The number of samples in the compound library was 1145. Reverse-phase chromatography was used for the separation and electrospray ionization was used for ionization on the mass spectrometer. Many difficulties were also encountered in the evaluation of results, namely poor mass spectra, decarboxylation of compounds, mistakes in preparation of samples, hydrolysis of compounds, fragmentation of compounds, and instability of solutions. However, despite the errors, the instrumental method was suitable for most of the compounds (90,1%). We organised the library and collected all data, that were gathered when interpreting results, in an Excel table. We determined the retention time of the compounds, assessed the purity and reactions in UV spectroscopy. We confirmed the structure of the compounds with mass spectrometry, attained the MS chromatogram and mass spectra of the compounds. We determined the formation of adducts. This allowed us to quantitatively and qualitatively evaluate the compound library, and also to obtain data on the behaviour of the lead compounds in an HPLC-UV-MS chromatographic system on a representative sample.

V magistrskem delu obravnavam kromatografsko ločbo protamin sulfata na instrumentu za tekočinsko kromatografijo sklopljeno z masno spektrometrijo (LCMS, angl. Liquid Chromatography Mass Spectrometry). Protamin predstavlja skupino močno bazičnih polipeptidov izoliranih iz semenske tekočine keta lososa. V obliki sulfatne soli se uporablja v medicini, med drugim kot pomožna snov v inzulinskih peresnikih, saj upočasni sproščanje inzulina v človeškem telesu. Sestavljen je iz štirih polipeptidov, katerih večinski delež predstavlja aminokislina arginin. Ker so si polipeptidi kemijsko med seboj zelo podobni, je težko doseči njihovo ločbo. Da bi jo dosegla, sem optimizirala parametre ločbe in sicer stacionarno fazo kolone (z uporabo različnih kolon), pH in koncentracijo mobilne faze ter temperaturo ločbe. Ugotovila sem, da v danih pogojih idealne ločbe ne bom dosegla, zato sem na koncu poizkusila še način s snemanjem izbranih ionov (SIM, angl. Selected Ion Monitoring). Poskus je bil uspešen, metoda je na podlagi razlik v masi uspešno ločila vse štiri polipeptide protamin sulfata. Na koncu sem izbrala najboljšo separacijsko metodo in jo uporabila za določitev protamin sulfata v dveh različnih inzulinskih peresnikih. In my master's thesis, I examine the chromatographic separation of protamine sulphate with a liquid chromatography mass spectrometry instrument, LCMS. Protamine consists of highly basic polypeptides, which are isolated from chum salmon semen. Beside its other medical advantages, protamine regulates insulin release into the human body therefore it is used as a sulphate salt in insulin pens. Protamine is composed of four polypeptides that are high in arginine content. Because of the chemical similarity of those polypeptides, it is very difficult to separate them. To achieve that I had been optimising different separation parameters such as stationary phase (by using different columns), pH and concentration of the mobile phase, temperature of the separation. I concluded that with optimising aforementioned parameters I could not reach the ideal separation, so my last option was to try selected ion monitoring, SIM. This experiment was successful, the method adequately separated all four polypeptides based on their mass difference. In the end, I chose the best separation method and used it on two insulin samples to determinate their protamine content.

Razvili smo metodo tekočinske kromatografije visoke ločljivosti sklopljene s tandemsko masno spektrometrijo za določitev askorbinske kisline in njenega izomera, eritorbinske kisline. Za separacijo smo uporabili Aminex HPX-87H kolono in izokratsko elucijo z 0,1 % mravljično kislino v vodi. Kvantifikacijo smo izvedli z uporabo ESI ionizacije v negativnem načinu s prekurzorskim ionom m/z 174,9 in produktnim ionom m/z 115 za obe kislini. Da se izognemo lažno pozitivnim rezultatom, smo pri vsaki meritvi spremljali še produktni ion m/z 87, ki je služil kot kontrola. Študirali smo stabilnost standardov, linearnost, meje zaznave in ponovljivost instrumenta. Ugotovili smo, da standardi na sobni temperaturi zelo hitro razpadajo in se odločili za uporabo reducenta TCEP. Območje linearnosti je bilo od meje zaznave do 2000 ppb, meja zaznave za askorbinsko kislino 69 ppb, za eritorbinsko pa 61 ppb. RSD meritev je bil pod 10 %. Preučevani parametri metode so bili v sprejemljivih območjih kar je vodilo do zanesljive določitve askorbinske in eritorbinske kisline. Metodo smo ovrednotili na različnih realnih vzorcih: šumečih in navadnih tabletah ter na sokovih. Rezultati analize tablet so odstopali od deklarirane vrednosti največ ±10 %. Preverili smo tudi homogenost tablet in ugotovili dobro homogenost tako znotraj tablet kot med tabletami. Pri vseh analiziranih vzorcih je bila koncentracija eritorbinske kisline pod mejo zaznave, kar je zelo pomembno zaradi njene nizke aktivnosti v primerjavi z askorbinsko kislino. A high-performance liquid chromatography coupled with tandem mass spectrometry method for determination of ascorbic acid and its isomer, erythorbic acid was developed. The separation was performed on Aminex HPX-87H column using isocratic elution with 0,1 % formic acid in water. Quantification was done using ESI ionization in negative mode with m/z 174,9 as a precursor ion and m/z 115 as a product ion for both acids. In order to avoid false positive results, the product ion m/z 87 was also recorded at each measurement and was used as a confirmation. The stability of standards, linearity, limits of detection and instrument repeatability were studied. The standards were found to degrade very quickly at room temperature. Because of that, TCEP was used as a reducing agent. The linear range was from the limit of detection to 2000 ppb while the limits of detection were 69 ppb and 61 ppb for ascorbic and erythorbic acid, respectively. RSD of measurements was below 10 %. The studied parameters were within the acceptable ranges which lead to the reliable determination of ascorbic and erythorbic acid. The method was evaluated on different real samples: effervescent and ordinary tablets and on juices. The results for tablets deviated from the value on the label by a maximum of ±10 %. The tablets were found to be homogeneous with good homogeneity both within and between tablets. The concentration of erythorbic acid was below the limit of detection in all the analyzed samples which is very important due to its low activity compared to ascorbic acid.

Paenibacillus larvae je po Gramu pozitivna sporogena bakterija, ki povzroča hudo gnilobo čebelje zalege (HGČZ). Poznanih je pet genotipov ERIC, ki se med seboj razlikujejo tudi fenotipsko. Izolate P. larvae smo izolirali ob izbruhih bolezni v Sloveniji v letih 2017 – 2019. Skupaj z izolati smo pridobili tudi pripadajoče epidemiološke podatke. Za identifikacijo izolatov P. larvae smo validirali metodo masne spektrometrije MALDI TOF in s tem pohitrili in pocenili bakteriološko preiskavo. Kolonije P. larvae so na gojiščih rasle v obliki dveh osnovnih morfotipov. Ugotovili smo, da genotip ERIC I fenotipsko večinoma izraža morfotip I – sive hrapave kolonije, genotip ERIC II pa izraža morfotip II – gladke sive ali pigmentirane kolonije. Z metodo ERIC-PCR smo prvič tipizirali in ugotovili pojavnost genotipov ERIC v Sloveniji. Prevladoval je genotip ERIC II (70,2 %). Pri nekaterih izolatih genotipa ERIC I smo ugotovili nekoliko spremenjen profil pomnožkov ERIC, zato smo 59 izbranim izolatom genotipa ERIC I sekvencirali tudi celotne genome (WGS). Kljub odstopanjem od klasičnega profila ERIC I, smo po tipizaciji z metodo WGS in silico MLST pri vseh izolatih ugotovili isti sekvenčni tip ST2. Ko smo z metodo wgMLST primerjali zaporedja lokusov v celotnih genomih z zaporedji, dostopnimi v javnih podatkovnih zbirkah, so se vsi naši izolati filogenetsko razvrstili v klad ERIC I in gručo ERIC I-ST2. Z molekularnimi in fenotipskimi metodami smo potrdili prisotnost različnih morfotipov in genotipov P. larvae v enem kužnem krogu, celo v istem čebelnjaku. Rezultati anketnega vprašalnika so statistično potrdili značilne klinične znake za genotipa ERIC I in II, kljub številnim drugim naključnim dejavnikom, ki lahko vplivajo na izražanje klinične slike HGČZ, in jih v naši raziskavi nismo spremljali. V celotnih genomih izolatov P. larvae smo ugotovili prisotnost gruč genov za sintezo inhibitornih substanc. Z disk difuzijsko metodo smo ugotovili, da posamezni sevi genotipa ERIC I izločajo protimikrobne snovi, ki inhibirajo rast nekaterih po Gramu pozitivnih bakterij in kvasovke Malassezia pachydermatis. Vpeljane diagnostične metode in rezultati raziskave predstavljajo pomemben doprinos k razumevanju genetske raznolikosti povzročitelja P. larvae in proučevanju načinov širjenja HGČZ. Paenibacillus larvae is a Gram-positive sporogenic bacterium that causes American Foulbrood (AFB). Five ERIC genotypes are described, which also differ phenotypically. In 2017–2019, P. larvae isolates were obtained during AFB outbreaks in Slovenia. Together with the isolates, we also obtained the corresponding epidemiological data. For the determination of P. larvae by bacteriological examination, we validated the MALDI TOF mass spectrometry method, thus speeding up and reducing the cost of the investigation. Two morphotypes of P. larvae colonies were observed. We found that the ERIC I genotype phenotypically mostly shows one morphotype – gray rough colonies, and the ERIC II genotype another – gray smooth or pigmented colonies. For the first time, ERIC-PCR was used for P. larvae typing in Slovenia and the frequency of ERIC genotypes could be determined. ERIC II was the predominant genotype (70,2 %). Some ERIC I isolates exhibited a slightly altered ERIC I banding pattern, thus 59 isolates were selected for the whole-genome sequencing (WGS). Despite showing non-classical ERIC I pattern, WGS-typing using the in silico MLST revealed that all of the investigated isolates belonged to the sequence type ST2. When comparing the whole-genome loci using the wgMLST analysis of the investigated isolates supplemented with all the publicly available P. larvae genomes, all Slovenian isolates fell within the ERIC I clade and phylogenetically clustered with the remaining ERIC I-ST2 genomes. Using molecular and phenotypic methods, we confirmed the presence of different morphotypes and genotypes of P. larvae in the same AFB zone or even in the same apiary. Results of the questionnaire statistically confirmed the presence of clinical signs characteristic for genotypes ERIC I and II, despite many other random factors that may affect the expression of AFB clinical picture and were not monitored in the scope of our study. In the genomes of P. larvae isolates, we identified the presence of gene clusters for the synthesis of inhibitory substances. Using the diffusion method, the ability to inhibit the growth of some Gram-positive bacteria and the yeast Malassezia pachydermatis was identified for individual ERIC I strains. The implemented diagnostic methods and study results represent an important contribution to the understanding of P. larvae genetic characteristics and AFB transmission routes.

This study focuses on the feasibility of collaborative robot implementation in a medical microbiology laboratory by demonstrating fine tasks using kinesthetic teaching. Fine tasks require sub-millimetre positioning accuracy. Bacterial colony picking and identification was used as a case study. Colonies were picked from Petri dishes and identified using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry. We picked and identified 56 colonies (36 colonies of Gram-negative Acinetobacter baumannii and 20 colonies of Gram-positive Staphylococcus epidermidis). The overall identification error rate was around 11%, although it was significantly lower for Gram-positive bacteria (5%) than Gram-negative bacteria (13.9%). Based on the identification scores, it was concluded that the system works similarly well as a manual operator. It was determined that tasks were successfully demonstrated using kinesthetic teaching and generalized using dynamic movement primitives (DMP). Further improvement of the identification error rate is possible by choosing a different deposited sample treatment method (e.g., semi-extraction, wet deposition).

V doktorskem delu sem preučeval pomen singletnega kisika in hidrotrioksi radikala pri pretvorbah organskih aerosolov. Kemijske pretvorbe s singletnim kisikom sem preučeval eksperimentalno, pretvorbe s hidrotrioksi radikalom pa teoretično s semiempiričnimi kvantnomehanskimi metodami in metodami, ki so osnovane na teoriji gostotnega funkcionala. Kot modelno spojino sem izbral α-pinen, ki je količinsko najbolj pomemben monoterpen in eden glavnih prekurzorjev sekundarnih organskih aerosolov. Razvil sem enostaven postopek za oksidacijo terpenov v tekoči fazi s singletnim kisikom, ki sem ga sintetiziral fotokemično z žarnice z volframovo nitko in acetonitrilno raztopino metilensko modrega. Za oksidacijske produkte, štiri hidroperokside α-pinena, sem razvil ločbi s tekočinsko kromatografjo (LC) in plinsko kromatografijo (GC), ter za zaznavo uporabil ultravijolični (UV), plamensko ionizacijski (FID) in masnospektrometrični (MS) detektor. Za analizo nestabilnih sistemov je primerna tudi kvantitativna nuklearna magnetna resonanca (NMR), ki kot absolutna tehnika omogoča določitev absorpcijskih koeficientov, in sem jo v doktorskem delu uporabil za identifikacijo hidroperoksidnih izomerov α-pinena. Za njihovo kvantifikacijo sem razvil GC-FID metodo s predkolonskim sililiranjem in kvantifikacijo z odzivnimi faktorji na osnovi efektivnega števila ogljikovih atomov. Z razvitim analiznim pristopom sem zaobšel težave z reaktivnostjo analitov, njihovo temperaturno neobstojnost, neznane in nizke absorpcijske koeficiente in šibko MS ionizacijo. Linearnost metode sem preveril med 1 in 90 mg/L, kjer so bili korelacijski koeficienti nad 0,99. Metoda je ponovljiva z relativnimi standardnimi odmiki pod 5 %. Celoten postopek, od sinteze hidroperoksidov do njihove kvantifikacije, je prenosljiv tudi na druge terpenske hidroperokside. Metodo sem uporabil za spremljanje staranja terpentina, pri katerem je masni delež raziskovanih hidroperoksidov α-pinena narasel na 5 %. Pri radikalski oksidaciji in oksidaciji s singletnim kisikom sem opazil razliko v deležu posameznih hidroperoksidov in jo uspel mehanistično razložiti. Poleg tega mi je uspelo dokazati, da omenjeni hidroperoksidi nastajajo tudi pri fotokemičnih reakcijah s PM10 delci. V raztopini hidroperoksidov sem z LC-MS in GC-MS opazil dimerizacijo hidroperoksidnih derivatov α-pinena in jo uspel opisati s semiempirično kvantnomehansko metodo PM6. Do sedaj so bile določitve 1O2 v zraku zelo redke, v znanstvenem članku je bila objavljena le določitev z α-terpinenom. Visoke koncentracije so lahko posledica reakcij z drugimi oksidanti, ki reagirajo na podoben način kot 1O2, zato je za potrditev pravilnosti določenih koncentracij potreben razvoj novih metod. Pri razvoju nove metode sem preizkusil tri različne kemijske pasti: N,N–dimetil-4-nitrozoanilin, sečno kislino in furfurilni alkohol. Kot najprimernejši reagent se je izkazal furfurilni alkohol, s katerim sem določil koncentracije singletnega kisika v raztopini med 2x10-12 in 4,5x10-12 mol/L. Pri meritvah v zraku so potrebne še nadaljnje izboljšave metode. Pomembnost hidrotrioksi radikala (•OOOH) sem ovrednotil in silico. Dokazal sem, da •OOOH ne nastaja pri reakciji α-pinena z radikalsko obliko ozona. Na dvojni vezi poteka adicija •OOOH, saj je abstrakcija alilnega vodika termodinamsko in kinetično neugodna. Hidrotrioksi radikal je manj reaktiven od hidroksilnega in hidroperoksi radikala, najbolj kritična pa je njegova majhna stabilnost, zato verjetno ne igra pomembne vloge v atmosferski kemiji. In my PhD thesis, I investigated the importance of singlet oxygen and hydrotrioxy radicals in the chemical transformation of aerosols. Chemical transformations involving singlet oxygen were studied experimentally and transformations involving hydrotrioxy radicals were studied theoretically, using semiempirical quantum mechanical methods and methods based on density functional theory. Alfa-pinene was chosen as a model compound because it is very abundant in the atmosphere and important for the formation of secondary organic aerosols. I have developed a simple procedure for liquid phase oxidation with singlet oxygen using a light bulb with tungsten filament and an acetonitrile solution of methylene blue. For the oxidation products, four α-pinene hydroperoxides, I have developed a liquid chromatography and a gas chromatography separation with an ultraviolet (UV), flame-ionization (FID), and mass spectrometry (MS) detection. The quantitative NMR technique, as an absolute technique, allows the determination of absorption coefficients, and was used to identify the hydroperoxide isomers of α-pinene. The analytes had low absorption coefficients, weak MS ionization, and low thermal stability. To overcome these limitations, I proposed a GC-FID approach involving pre-column silylation and quantification via the effective carbon number approach. The linearity of the method was confirmed between 1 and 90 mg/L, with correlation coefficients above 0.99. The method is reproducible with relative standard deviations below 5%. The entire process, from the synthesis of the hydroperoxides to their quantification, is transferable to other terpene hydroperoxides. The validated method was applied to study the aging of turpentine in which the mass fraction of α-pinene hydroperoxides increased to 5%. It was found that the differences in abundance of each hydroperoxide were due to radical oxidation or a singlet oxygen oxidation mechanism. Furthermore, I was able to show that hydroperoxides are also formed by photochemical reactions with PM10 particles. In solution, the dimerization of α-pinene hydroperoxides was observed by LC-MS and GC-MS and the mechanism was explained by the PM6 semiempirical quantum mechanical method. So far, determinations of 1O2 in the air have been very rare. Only one determination using the chemical trap α-terpinene was published in a scientific article. Since high concentrations may result from reactions with oxidants that react in a similar manner to 1O2, the development of new methods is needed to confirm the accuracy of determinations. In developing the new method, I tested three 1O2 chemical traps: N, N-dimethyl-4-nitrosoaniline, uric acid, and furfuryl alcohol. Furfuryl alcohol proved to be the most suitable reagent and was used to determine concentrations of singlet oxygen in solution, ranging from 2x10-12 to 4.5x10-12 mol/L. Further improvements to the method are required for air measurements. The importance of the hydrotrioxy radical (•OOOH) was investigated in silico. It was shown that •OOOH is not formed by the reaction of α-pinene with the radical form of ozone. The addition of •OOOH occurs at the double bond because the allylic hydrogen abstraction is thermodynamically and kinetically unfavorable. The hydrotrioxy radical is less reactive than the hydroxyl and hydroperoxy radicals and has very low stability, so it is unlikely to play a significant role in atmospheric chemistry.

Desnosučne aminokisline so v majhnih količinah prisotne tako v sintetičnih kot tudi v nekaterih naravnih peptidih in proteinih. Določevanje vezavnih mest desnosučnih aminokislin je pomembno za podrobno analizo in razumevanje teh peptidov in proteinov. V diplomskem delu sem predstavil metode za analizo proteinov, ki vsebujejo desnosučne aminokisline. Vse metode za določevanje vezavnih mest desnosučnih aminokislin temeljijo na masni spektrometriji, ki je ključna tehnika za analizo zgradbe proteinov. Cilj eksperimentalnega dela je bil razviti metodo za ločbo proteinov, ki vsebujejo desnosučne aminokisline. Metodo sem razvijal na dveh tandemskih masnih spektrometrih, ki sta bila sklopljena s tekočinskima kromatografoma. D-amino acids are found in small quantities both in natural and synthetic peptides and proteins. Determination of their bonding sites is important for detailed analysis and understanding of these peptides and proteins. In my diploma thesis I presented methods for analysis of proteins that consist of D-amino acids. All methods for determination of bonding sites of D-amino acids are based on mass spectrometry, which is the key technique for analysing the structure of proteins. The goal of the experimental part was to develop a method to separate proteins consisting of D-amino acids. The method was developed with two tandem mass spectrometers, which were coupled with liquid chromatography instruments.

Pred masnospektrometrično analizo je N-glikane potrebno sprostiti iz glikoproteinov, nato pogosto sledi derivatizacija N-glikanov za izboljšanje občutljivosti masne spektrometrije, učinkovitejšo ionizacijo spojin in zaščito funkcionalnih skupin. Ker se z masno spektrometrijo ne da razlikovati med vsemi izomeri, se te loči s sklopitvijo s separacijsko tehniko. Najpogosteje se za ionizacijo uporabljata laserska desorpcija s pomočjo matrice in elektrorazprševanje, ki povzročita malo ali nič fragmentacije. Iz masnega spektra se tako lahko določi molekulska masa analita. Informacijo o strukturi N glikana se dobi iz tandemskega masnega spektra na podlagi ionskih fragmentov. Z visokoločljivostno masno spektrometrijo je možno razlikovati med izobarnimi spojinami. N-glikane se lahko detektira tudi z drugimi tehnikami, vendar le masna spektrometrija poda točno molekulsko maso in informacijo o strukturi analita. Before mass spectrometry analysis, N-glycans must be released from glycoproteins, usually followed by N-glycan derivatization. Derivatization improves sensitivity in mass spectrometry, ionization efficiency and protects functional groups. A lot of isomers cannot be distinguished by mass spectrometry analysis, therefore coupling with separation technique is needed to separate isomers. Matrix-assisted laser desorption/ionization and electrospray ionization are most common ionization modes, which cause little fragmentation or none at all. Molecular mass of analyte can be determined from mass spectrum. N-glycan structural information can be provided by ion fragments in tandem mass spectrum. Isobaric compounds can be differentiated by high-resolution mass spectrometry. N-glycans can be detected by other detection techniques as well, but accurate molecular mass and structural information can be provided by mass spectrometry only.

V magistrskem delu sem se ukvarjal z določitvijo hlapnih komponent v medu z GC-MS. Najprej sem optimiziral metodo za njihovo določitev. Poiskal sem primeren način za pripravo vzorcev. Pri tem sem ugotovil, da dodatek nasičene vodne raztopine NaCl pomaga pri ekstrakciji spojin. Za tem sem optimiziral še čas in temperaturo ekstrakcije ter temperaturni program. S pripravljeno metodo sem določil hlapne komponente v cvetličnem, gozdnem, akacijevem, lipovem, hojevem, smrekovem in kostanjevem medu. V vseh skupaj sem določil prisotnost 148 spojin. Največ različnih je vseboval lipov med. Z masnimi spektri, ki sem jih dobil in njihovo primerjavo z NIST-ovo knjižnico sem uspel identificirati nekatere izmed spojin, ostalim pa sem določil strukturne fragmente, iz katerih so sestavljene. Ugotovil sem, da gre predvsem za alkohole, aldehide, kisline in estre. Pogosto se pojavljajo terpeni in terpenoidi. Za bolj gotovo identifikacijo sem z isto metodo analiziral še nekatere standarde. Ugotovil sem, da nekateri izmed analiziranih vzorcev medu vsebujejo furfural, 2-metilbutanojsko kislino, stiren, benzil alkohol, fenilacetaldehid, acetofenon in fenilocetno kislino. Noben vzorec pa ne vsebuje dekanojske kisline, dodekanola in kinolin-5-ola. In my master's thesis, I studied determination of volatile compounds in honey with the use of GC-MS. I first optimised the method for their determination. I was looking for a suitable way to prepare the samples. In doing so, I found out that the addition of saturated aqueous NaCl solution aids in the extraction of the compounds. After that I optimised the extraction time and temperature, as well as the temperature program. Using the prepared method, I determined the volatile compounds in floral, forest, acacia, lime, fir, spruce and chestnut honey. I determined the presence of 148 compounds, with lime honey containing the most variety. With the obtained mass spectra and their comparison with the NIST library, I was able to identify some of the compounds. In others I determined the structural fragments of which they are composed. I found out that these compounds are mainly alcohols, aldehydes, acids and esters. There are many terpenes and terpenoids. For a more definite identification, I used the same method to analyse some standards. I found that some of the honey samples analysed contained furfural, 2-methylbutanic acid, styrene, benzyl alcohol, phenylacetaldehyde, acetophenone and phenylacetic acid. However, no sample contained decanoic acid, dodecanol or quinolin-5-ol.

Bor, običajno podan kot borov oksid (B2O3), je ena izmed sestavin borosilikatnega stekla, ki je pomembna komponenta v proizvodnji steklene volne. Ker so surovine, ki vsebujejo bor, zelo drage, se bor vzdržuje na točno določeni koncentraciji, ki se jo preverja s titracijsko metodo. Titracija je časovno zelo potraten proces in je primerna le za vzorce, ki vsebujejo več kot 0.35 masnih odstotkov B2O3. Bolj optimalna rešitev je meritev z masnim spektrometrom z induktivno sklopljeno plazmo (ICP-MS), saj je priprava vzorcev enostavnejša, analizira se lahko več vzorcev hkrati, s tem pa se tudi zmanjša možnost napak in prihrani veliko časa. Ključna komponenta ICP-MS-a je stabilna argonova plazma, ki pri zelo visoki temperaturi molekule razbije na posamezne ione, le - ti pa potujejo naprej skozi kvadrupol in se ločijo na osnovi razmerja med maso in nabojem (m/z). Zaradi občutljivosti naprave, se ICP-MS uporablja predvsem za analizo elementov v sledovih, zato je ta analizna tehnika primerna za določevanje bora v naših vzorcih. V diplomskem delu smo optimizirali obstoječo metodo za določitev koncentracije bora v stekleni ter kameni volni s pomočjo ICP-MS. Rezultate realnih vzorcev smo primerjali s koncentracijami bora v standardnih referenčnih materialih. Rezultati meritev bora so pokazali, da z obstoječo metodo pri stokratni redčitvi vzorca zelo dobro merimo višje koncentracije bora (nad 0.35%) v vzorcih in bi lahko v tem primeru ICP-MS analizo uporabljali namesto časovno zamudne titracije. ICP-MS je primeren tudi za merjenje vzorcev z nizko koncentracijo bora (pod 0.35%). Poleg metode ICP-MS, ki se uporablja za analizo tekočih vzorcev, smo uporabili tudi metodo laserske ablacije, sklopljene z metodo ICP-MS. Vzorci pri tej analizni tehniki so v trdnem stanju, pripravljeni pri dveh različnih temperaturah, in sicer pri 750 ° in 1150 °C. Rezultati so pokazali, da LA-ICP-MS lahko nadomesti tekočinsko ICP-MS pri vzorcih z visoko koncentracijo bora, saj so bile relativne razlike med rezultati, pridobljenimi v internem laboratoriju in rezultati, pridobljenimi na zunanjem inštitutu majhne (do 3.4%). Boron oxide (B2O3) is one of the components of borosilicate glass, which is used as an important raw material in the production of glass mineral wool. Raw materials containing boron are very expensive and this is one of the main reasons that the concentration of boron is maintained at a certain level. For boron determination usually the titration method is used. Since this is a very time-consuming procedure and has high boron detection limit (0.35%), determination of boron by using the inductively coupled plasma-mass spectrometry (ICP-MS) would be a better solution. Furthermore, sample preparation for ICP-MS measurements is easier and more samples can be analysed at the same time. Stable argon plasma is a key component of the ICP-MS. At very high temperatures molecules are ionised. Ions are traveling through quadrupole and are separated according to their mass and charge (m/z) ratio. Due to its high sensitivity, the ICP-MS is mainly used for trace analyses and is appropriate technique for boron determination in our samples. In the diploma thesis, the existing ICP-MS method for boron determination in glass and rock mineral wool was optimised. The results of real samples were compared with boron concentrations in standard reference materials. The results showed that high concentrations of boron (above 0.35%) could be accurately determined by current method and 100-fold dilution of the sample. It can be concluded that the solution mode ICP-MS can be used instead of time-consuming titration. In addition, with that method also concentrations below 0.35% can be determined. Beside the solution mode ICP-MS, laser ablation inductively coupled plasma-mass spectrometry (LA-ICP-MS) was used. Solid state samples were prepared at two different temperatures, 750 °C and 1150 °C. The results showed that LA-ICP-MS can replace the solution mode ICP-MS when boron concentrations are high, since the relative differences between the results obtained in the internal laboratory compared to the results obtained in the external laboratory were low (up to 3.4%).

Endometrial (EC) and ovarian (OC) cancers are generally considered as post-menopausal, hormone-dependent pathologies with increasing incidence and mortality rate. Estrogen intracrine action is regarded vital in the cancer initiation and progression. Estrogens can form locally from androgen and estrogen precursors, through the aromatase and the sulfatase pathway, respectively. Here we intend to elucidate the contribution of the sulfatase pathway to active estrogen formation in model cell lines of EC and OC, by means of liquid chromatography-tandem mass spectrometry (LC-MS/MS). For this purpose we developed a robust LC-MS/MS method with a picomolar sensitivity for the following analytes: estrone (E1), estrone sulfate (E1-S), estradiol (E2), and estradiol sulfate (E2-S). Control and model cell lines of EC and OC were incubated with different E1-S concentrations for 8, 24, 48, and 72 hours, and the formed metabolites analyzed by LC-MS/MS. Our data indicate that the sulfatase pathway contributes to the estrogen formation actively, yet differently in EC and OC model cell lines and the respective control lines. More specifically, greater levels of E1 and E2 are synthesized in the EC and OC model cell lines comparing to the respective control lines, altogether implying an active role of estrogen in EC and OC carcinogenesis, acting as pro-proliferative hormones and/or genotoxic agents. Rak endometrija (angl. endometrial cancer/EC) in jajčnikov (angl. ovarian cancer/OC) splošno obravnavamo kot hormonsko odvisni patologiji, ki sta značilni za obdobje po menopavzi, z naraščujočo pojavnostjo in smrtnostjo. Intrakrino delovanje estrogenov razumemo kot ključno pri začetku in napredovanju raka. Estrogeni se lahko tvorijo lokalno iz androgenih in estrogenih prekurzorjev, po aromatazni in sulfatazni poti. V tem delu nameravamo razjasniti prispevek sulfatazne poti pri nastanku aktivnih estrogenov v modelnih celičnih linijah EC in OC, s pomočjo tekočinske kromatografije sklopljene z masno spektrometrijo (LC-MS/MS). V ta namen smo razvili robustno LC-MS/MS metodo s pikomolarno občutljivostjo za sledeče analite: estron (E1), estron sulfat (E1-S), estradiol (E2), in estradiol sulfat (E2-S). Kontrolne in modelne celične linije EC in OC smo inkubirali z različnimi koncentracijami E1-S, 8, 24, 48, in 72 ur in nastale metabolite analizirali z LC-MS/MS. Naši podatki kažejo, da sulfatazna pot aktivno prispeva k nastanku estrogenov, vendar različno pri EC in OC modelnih celičnih linijah v primerjavi z ustreznimi kontrolnimi celičnimi linijami. Bolj podrobno, EC in OC modelni celični liniji sintetizirata več E1 in E2 v primerjavi z ustrezno kontrolno linijo. Zadnje nakazuje aktivno vlogo estrogenov kot pro-proliferativnih in/ali kot genotoksičnih spojin pri karcinogenezi EC in OC.

Advanced oxidation processes (AOPs) have been introduced to deal with different types of water pollution. They cause effective chemical destruction of pollutants, yet leading to a mixture of transformation by-products, rather than complete mineralization. Therefore, the aim of our study was to understand complex degradation processes induced by different AOPs from chemical and ecotoxicological point of view. Phenol, 2,4-dichlorophenol, and pentachlorophenol were used as model pollutants since they are still common industrial chemicals and thus encountered in the aquatic environment. A comprehensive study of efficiency of several AOPs was undertaken by using instrumental analyses along with ecotoxicological assessment. Four approaches were compared: ozonation, photocatalytic oxidation with immobilized nitrogen-doped TiO2 thin films, the sequence of both, as well as electrooxidation on boron-doped diamond (BDD) and mixed metal oxide (MMO) anodes. The monitored parameters were: removal of target phenols, dechlorination, transformation products, and ecotoxicological impact. Therefore, HPLC–DAD, GC–MS, UHPLC–MS/MS, ion chromatography, and 48 h inhibition tests on Daphnia magna were applied. In addition, pH and total organic carbon (TOC) were measured. Results show that ozonation provides by far the most suitable pattern of degradation accompanied by rapid detoxification. In contrast, photocatalysis was found to be slow and mild, marked by the accumulation of aromatic products. Preozonation reinforces the photocatalytic process. Regarding the electrooxidations, BDD is more effective than MMO, while the degradation pattern and transformation products formed depend on supporting electrolyte.

Analiza materialov predmetov kulturne dediščine predstavlja prvi korak pri načrtovanju kakršnega koli konservatorskega-restavratorskega posega. V okviru magistrskega dela sem v realnih vzorcih odvzetih z umetniških slik iz 20. stoletja z uporabo infrardeče spektroskopije in plinske kromatografije sklopljene z masno spektrometrijo identificiral uporabljena veziva. Najprej sem si z izbiro ustreznih modelnih vzorcev pripravil knjižnico IR spektrov, MS spektrov in kromatogramov, s pomočjo katerih sem nato interpretiral rezultate analiz realnih vzorcev odvzetih iz umetniških slik. Poleg identifikacije veziv v realnih vzorcih sem s pomočjo infrardeče spektroskopije identificiral tudi nekatere anorganske primesi (pigmente in polnila). Na modelnih vzorcih damar smole, čebeljega voska in njunih mešanic sem z infrardečo spektroskopijo preučeval tudi vpliv povišane temperature za različna časovna obdobja. Analysis of the materials of cultural heritage objects is the first step toward any kind of a conservation-restoration procedure. In this work I have characterized binder materials in samples taken from 20th century paintings using infrared spectroscopy and gas chromatography coupled with a mass spectrometry. With appropriate selection of model samples the library of infrared spectra, MS spectra and chromatograms was prepared for further interpretation of the results of the real samples from paintings. Together with the identification of the binder materials in the samples also some inorganic compounds (pigments and fillers) were identified using infrared spectroscopy. I have also employed infrared spectroscopy to study the effects of increased temperature on model samples of dammar resin, beeswax and their mixtures.

Lignin je kompleksen heterogen aromatski biopolimer, ki se nahaja v stenah rastlinskih celic kot ena izmed komponent lignoceluloze. Velike količine lignina nastajajo kot stranski produkt v papirni industriji in industriji biogoriv. Ker predstavlja bogat naravni vir aromatskega ogljika, bi lahko potencialno predstavljal odličen vir za proizvodnjo goriv in kemikalij z dodano vrednostjo. Kljub temu pa še vedno velja za odpadni produkt in je v veliki meri porabljen kot nizkokakovostno gorivo. Predvsem zaradi energetske krize je v zadnjih letih zanimanje za lignin in njegovo valorizacijo precej zraslo, in sicer še posebej za bolj okolju prijazno razgradnjo lignina z mikroorganizmi. Raziskave so bile doslej večinoma osredotočene na glivno razgradnjo lignina, ki pa je zaradi kompleksnosti njihovih encimov manj primerna za uporabo v industriji. Ligninolitični encimi so v bakterijah sicer precej redkejši kot v glivah, a bi lahko predstavljali novo, boljšo možnost za valorizacijo lignina. Pri tej magistrski nalogi smo se osredotočili na bakterijsko razgradnjo lignina. Naš cilj je bil preučiti ligninolitično aktivnost 5 bakterijskih sevov, ki proizvajajo lakazo, pomemben encim z delovanjem na lignin. Tako smo si zastavili raziskovalno vprašanje: Ali je v katerem od vzorcev prišlo do biorazgradnje? Če je odgovor »da«, kateri produkti so pri tem nastali? Po pregledu literature smo izbrali 20 fenolnih spojin, ki bi se potencialno lahko pojavile kot produkt bakterijske razgradnje lignina. Za 13 izmed njih smo nato razvili dve analizni metodi za tarčno analizo razgradnih produktov lignina. Metodi temeljita na analizi vzorcev z reverznofazno tekočinsko kromatografijo, sklopljeno s tandemsko masno spektrometrijo. Pred LC-MS/MS analizo smo vzorce pripravili z ekstrakcijo na trdnem nosilcu, z uporabo ekstrakcijskih plošč na sistemu za nadtlačno ekstrakcijo. V naslednjem koraku smo analizno metodo testirali na realnih vzorcih. Ti so bili predhodno pripravljeni tako, da smo kot vir ogljika za posamezne bakterijske seve na gojiščih uporabili lignin. Pri tem smo predpostavili, da z določitvijo katerega od potencialnih produktov razgradnje lahko potrdimo, da je v posameznem vzorcu do biorazgradnje zares prišlo. Odsotnost potencialnih produktov razgradnje v vzorcih pa ne pomeni, da do biorazgradnje ni prišlo, pač pa samo, da se ni zgodila na načine, kot so opisani v literaturi. Ker v vzorcih nismo uspeli potrditi prisotnosti nobenega izmed iskanih analitov, smo izvedli še netarčno analizo, in sicer v načinu IDA (Information Dependent Acquisition, oziroma »od podatkov odvisna analiza«). Dobljeni kromatogrami nakazujejo očitno razliko med kontrolnimi in dejanskimi vzorci, kar nakazuje razgradnjo lignina. Nalogo smo predstavili tudi na 12. študentski konferenci Mednarodne podiplomske šole Jožefa Stefana. Lignin is a complex and heterogenous aromatic biopolymer that can be found in plant cell walls as one of the components of lignocellulose. Large quantities of lignin are daily generated as a by-product in paper and biofuel industries. To date, lignin is still treated as waste and is mostly burned as a low-quality fuel, even though it could potentially be a great source for producing value-added chemicals and fuels as it is an abundant source of renewable aromatic carbon. As a result of the energy crisis, the interest in lignin and its valorization has grown considerably in recent years, mainly focusing on more environmentally friendly lignin degradation by microorganisms. The majority of research has so far been concentrating on fungal lignin degradation. However, fungi unfortunately seem to be unsuitable for large-scale application because of the complexity of their enzymes. Ligninolytic enzymes are very rare in bacteria, but those that possess this physiological trait present a new, better option than using fungi to valorize lignin. Our research focuses on bacterial lignin degradation, with the objective to study the ligninolytic activity of 5 laccase-producing bacterial strains, laccase being an important lignin-modifying enzyme. Our research question was: ''Did biodegradation occur in any of the samples? In case the answer is affirmative, which products were generated by the degradation?'' Based on the existing literature we defined 20 phenolic compounds, which could potentially appear as products of bacterial lignin degradation. 13 of those compounds were then included in two analytical methods that we developed for targeted screening of lignin degradation products. The analytical methods were based on determination by reversed-phase liquid chromatography coupled to tandem mass spectrometry. Prior to the LC-MS/MS analysis, we purified the samples by solid-phase extraction using extraction plates on a positive pressure extraction system. The analytical method was then tested on real samples, containing lignin treated with either of the laccase-producing bacterial strains, presuming that detection of any of the potential degradation products in a sample would prove biodegradation. The absence of those potential degradation products does, however, not mean that biodegradation did not take place. It merely suggests the absence of the specific degradation pathways, found in the literature. Since the target analysis did not confirm the presence of the expected analytes, we also performed nontargeted analysis using information-dependent acquisition-mediated LC-MS/MS screening. Acquired chromatograms showed significant differences between lignin samples and blanks, confirming its degradation. We presented our research at the 12th Jožef Stefan international postgraduate school students' conference.

Kromatografija je ena izmed najpogosteje uporabljanih analitskih tehnik za ločevanje in identifikacijo spojin. Poznamo različne kromatografske tehnike, ki se razlikujejo na podlagi različnih mobilnih in stacionarnih faz. Najbolj razširjena med njimi je tekočinska kromatografija, ki temelji na tekoči mobilni fazi in trdni ali tekoči stacionarni fazi med katerima se porazdeljujejo analiti. Kromatografija je zaradi svoje univerzalnosti in vsestranske uporabe v farmacevtski industriji nepogrešljiva. Pomembno vlogo v razvoju, raziskavah in proizvodnji zdravil ima sam razvoj analiznih metod, ki je precej kompleksen, saj zahteva upoštevanje velikega števila faktorjev in izkušenj. V prvem in glavnem delu magistrske naloge smo razvili analizno metodo s tekočinsko kromatografijo visoke ločljivosti sklopljeno z masnim spektrometrom, s katero je možno ločiti čim večje število različnih spojin vodnic v čim krajšem času. Ko smo uspeli razviti ustrezno analizno metodo z optimalnimi kromatografskimi pogoji, smo v nadaljevanju naloge preverili metabolno stabilnost naših spojin. In sicer tako, da smo jih testirali ob času 0 in po 30 minutah inkubiranja v prisotnosti S9 frakcije metabolnih encimov. Spojine, ki so po 30 minutah izražale podvrženost metabolizmu (površina vrha se jim je glede na začetno vrednost zmanjšala za vsaj 10 %) smo nato testirali v časovnih točkah 0, 15, 30, 60 in 120 minut. Preučevali smo 17 spojin, med katerimi se jih je 12 pri testiranju ob časih 0 in 30 minut izkazalo za metabolno stabilne, z razpolovnim časom večjim od 5-ih ur. 5 spojin pa je z S9 encimsko frakcijo izražalo podvrženost metabolizmu. Posameznim spojinam smo na podlagi spremembe površine pod krivuljo v UV kromatogramu določili razpolovne čase. V zadnjem delu naloge smo nastalim metabolitom poizkušali predlagati možno strukturo. Strukture nastalih metabolitov smo predlagali na podlagi določene molske mase spojine. Pri določenih spojinah metaboliti zaradi nizkih odzivov v UV kromatogramu niso bili vidni in smo jih lahko spremljali samo z masnim detektorjem. Chromatography is one of the most commonly used analytical techniques for the separation and identification of compounds. We know different chromatographic techniques that differ based on different mobile and stationary phases. The most common of these is liquid chromatography which is based on the liquid mobile phase and the solid or liquid stationary phase between which the analytes are partitioned. Chromatography is indispensable due to its universality and versatility in the pharmaceutical industry. An important role in the development, research, and production of medicines is played by the development of analytical methods which is quite complex as it requires consideration of a large number of factors and experience. In first and the main part of our project we developed an analytical method with high performance liquid chromatography coupled with mass spectrometer which is able to separate as many different conductor compounds as possible in the shortest possible time. Once we were able to develop an appropriate analytical method with optimal chromatographic conditions, we further verified the metabolic stability of our compounds by testing them at time 0 and after 30 minutes of incubation. Compounds that expressed metabolic instability after 30 minutes (their peak surface area decreased by at least 10 % compared to the initial one) were then tested at 0, 15, 30, 60, and 120 minute time points. We studied 17 compounds, of which 12 proved to be metabolically stable when tested at times 0 and 30 minutes, with a half-life greater than 5 hours. Other 5 compounds expressed their susceptibility to metabolism with the S9 enzyme fraction. The half-lives of individual compounds were determined in a UV chromatogram based on the change in area under the curve. In the last part of the task, we tried to suggest a possible structure to the resulting metabolites. The structures of the resulting metabolites were proposed based on the determined molecular mass. For certain compounds, the metabolites were not visible in the UV chromatogram due to low responses and could only be monitored by mass detector.

Odkrivanje spojin s protimikrobnimi lastnostmi je odločilnega pomena za preprečevanje rastoče odpornosti proti antibiotikom. Za iskanje zdravil farmacevtska industrija uporablja pristop iskanja ligandov, ki vežejo (in s tem inhibirajo) tarčni protein. Da bi zaznali tudi interakcije z drugimi celičnimi komponentami in se s tem izognili spojinam s potencialno toksičnim vplivom, v našem laboratoriju uporabljamo pristop iskanja proteinskih tarč malih molekul. V diplomskem delu smo želeli določiti proteinske tarče male molekule 4-(2-aminoetil)-1-(piridin-2-il)-1H-pirzol-5-ol, za katero smo ugotovili, da zavira rast laboratorijskega seva DH5α bakterije Escherichia coli. Spojino smo imobilizirali na agarozni nosilec in pripravljeno kolono uporabili za afinitetno kromatografijo, pri kateri smo iz lizata E. coli izolirali interagirajoče proteine. Eluirane frakcije smo nato analizirali z NaDS-PAGE in poslali na analizo z masno spektrometrijo. Ugotovili smo, da dve intenzivni lisi na gelu predstavljata treonin dehidrogenazo in Fe–S podenoto sukcinat dehidrogenaze. Glede na literaturo je možen mehanizem bakteriostatičnega delovanja tarčne spojine inhibicija treonin dehidrogenaze, ki tako ni sposobna sinteze signalnih molekul. Inhibicija sukcinat dehidrogenaze verjetno ne vpliva na rast bakterije E. coli. The discovery of compounds with antimicrobial properties is crucial to prevent the growing resistance to antibiotics. To search for drugs, the pharmaceutical industry utilises the approach of finding ligands that bind (and thus inhibit) the target protein. In order to detect interactions with other cellular components and thus avoid compounds with potentially toxic side effects, we used a small molecule to protein targeting approach in our laboratory. In this thesis, we aimed to identify the protein targets of the small molecule 4-(2-aminoethyl)-1-(pyridin-2-yl)-1H-pyrzol-5-ol, which was shown to inhibit the growth of Escherichia coli laboratory strain DH5α. The compound was immobilized on agarose beads and the prepared column was used for affinity chromatography with which we isolated interacting proteins from the E. coli lysate. Eluted fractions were analyzed by SDS-PAGE and sent for protein identification by mass spectrometry. Two prominent bands on the gel were identified as threonine dehydrogenase and the Fe-S subunit of succinate dehydrogenase. According to the literature, the possible mechanism for bacteriostatic action of the compound is inhibition of threonine dehydrogenase which is involved in the biosynthesis of signalling molecules. Inhibition of succinate dehydrogenase is unlikely to affect E. coli growth.

Namen magistrskega dela je bil ugotoviti ali iz rezultatov določevanja hlapnih organskih spojin (HOS) liofiliziranih tartufov lahko razlikujemo med posameznimi vrstami tartufov in geografskim izvorom ter ali postopek sočasne liofilizacije večjega števila vzorcev v liofilizatorju vpliva na aromatski profil HOS posameznega vzorca. V naključno izbranih vzorcih tartufov (54) različnih vrst (Tuber aestivum, Tuber brumale, Tuber magnatum in Tuber melanosporum) smo določevali HOS po razviti metodi mikroekstrakcije na trdnem nosilcu iz plinaste faze nad vzorcem (HS-SPME) s plinsko kromatografijo sklopljeno z masno spektrometrijo (GC-MS). Ugotovili smo, da se aromatski profil HOS svežih tartufov po liofilizaciji zelo spremeni. Pri razlikovanju vrst tartufov smo z linearno diskriminantno analizo (LDA) določili vrednost navzkrižne validacije za sveže in liofilizirane tartufe > 80 %, kar kaže na to, da posamezne vrste lahko razlikujemo glede na aromatski profil HOS. Po liofilizaciji smo lahko ločevali vse preučevane vrste (T. aestivum, T. brumale, T. magnatum in T. melanosporum). Navzkrižna validacija pri razlikovanju geografskega porekla svežih tartufov je bila 30 %, medtem ko pri liofiliziranih 41 %. Kljub nizki vrednosti, rezultati kažejo, da bi bilo razlikovanje porekla glede na aromatski profil HOS možno. Sočasna liofilizacija večjega števila vzorcev v liofilizatorju vpliva na aromatski profil HOS tartufov. The aim of this theses was to evaluate whether the species and the geographical origin of freeze-dried truffles could be differentiated according to their aromatic profiles based on volatile organic compounds (VOCs), and whether the simultaneous freeze-drying of large number of samples in a lyophilizer affects the aromatic profile of each sample. VOCs were evaluated on randomly selected truffle samples (54) of different species (Tuber aestivum, Tuber brumale, Tuber magnatum and Tuber melanosporum) using a combination of headspace solid-phase microextraction (HS-SPME) and gas chromatography coupled with mass spectrometry (GC-MS). The results demonstrate that the aroma of freeze-dried truffles of all species investigated was deeply modified as compared to the fresh truffles. The cross-validation, which was determined with linear discriminant analysis (LDA) of fresh and freeze-dried truffles used in the differentiation of species was > 80%, which indicates that individual species can be distinguished according to their aromatic profile. After freeze-drying, we were able to distinguish all the species tested (T. aestivum, T. brumale, T. magnatum and T. melanosporum). Cross-validation of distinguishing the geographical origin of fresh truffles amounted to 30%, while that of freeze-dried 41%. Despite the low cross-validation score, the results indicates possible differentiatiojn between geographical origin based on aromatic profile. The analysis confirmed that the simultaneous freeze drying of a large number of samples in the lyophilizer influences changes in the aromatic profile of truffles.

The aim of the present study was to characterize human milk microbiota (HMM) with 16S rRNA gene amplicon next-generation sequencing and cultivation/matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry (MS) identification approaches. We analyzed 31 human milk samples from healthy Slovenian mothers. To check the accuracy of MALDI-TOF MS identification, several colonies representing most abundant genera and those, which could not be reliably identified by MALDI-TOF, were subjected to Sanger sequencing of their 16S rRNA gene. We showed that cultivation/MALDI-TOF MS was a suitable tool for culture-dependent determination of HMM. With both approaches, Staphylococcus and Streptococcus were found as predominant genera in HMM and the abundance of Staphylococcus was associated with decreased microbial diversity. In addition, we characterized factors that might influence HMM. The use of a breast pump was significantly associated with composition of HMM, lower microbial load, and higher abundance of cultivable staphylococci. Moreover, our study suggests that administration of probiotics to the suckling infant might influence HMM by increased abundance of lactobacilli and the presence of viable probiotic bacteria in human milk. However, since our study was observational with relatively small sample size, more targeted studies are needed to study possible transfer of probiotics to the mammary gland via an external route and the physiological relevance of these events.

Measurements of carbon dioxide and their stable isotopes are propulsive research tool in ecology and environmental science as they can give us insight into carbon cycle. They are widely used to investigate both natural and anthropogenic carbon sources in the atmosphere, hydrosphere and geosphere, as well as the exchange between these reservoirs. In this paper, we provide a basic overview of two different analytical measurement techniques, isotope ratio mass spectrometry (IRMS) and laser-absorption spectroscopy (LAS) which have been developed and utilized for monitoring of CO2 isotopologues in ecosystem. We present the basics for each technique, however with the emphasis on LAS measurement technique we are targeting readers who are not familiar with this topic. A major objective of this paper is to illustrate the current value and future potential of various analytical instruments in a wide range of applications deployed in the terrestrial ecosystem. Finally, we draw a conclusion from recent research campaigns by presenting a decision tree to better understand and choose a correct application combination for a selected scale of ecosystem. Nasl. iz nasl. zaslona. Opis vira z dne 24. 10. 2019. Bibliografija: str. 9-12.

Microplastic pollution is globally widespread; however, the presence of microplastics in soil systems is poorly understood, due to the complexity of soils and a lack of standardised extraction methods. Two commonly used extraction methods were optimised and compared for the extraction of low-density (polyethylene (PE)) and high-density microplastics (polyethylene (PET)), olive-oil-based extraction, and density separation with zinc chloride (ZnCl2). Comparable recoveries in a low-organic-matter matrix (soil; most >98%) were observed, but in a high-organic-matter matrix (compost), density separation yielded higher recoveries (98 ± 4% vs. 80 ± 11%). Density separation was further tested for the extraction of five microplastic polymers spiked at different concentrations. Recoveries were >93% for both soil and compost, with no differences between matrices and individual polymers. Reduction in levels of organic matter in compost was tested before and after extraction, as well as combined. Double oxidation (Fenton’s reagent and 1 M NaOH) exhibited the highest reduction in organic matter. Extracted microplastic polymers were further identified via headspace solid-phase microextraction–gas chromatography–mass spectrometry (HS-SPME–GC–MS). This method has shown the potential for descriptive quantification of microplastic polymers. A linear relationship between the number of particles and the signal response was demonstrated for PET, polystyrene (PS), polyvinyl chloride (PVC), and PE (R2 > 0.98 in alluvial soil, and R2 > 0.80 in compost). The extraction and identification methods were demonstrated on an environmental sample of municipal biowaste compost, with the recovery of 36 ± 9 microplastic particles per 10 g of compost, and the detection of PS and PP.

Pivo zaradi svojih lastnosti velja za mikrobiološko varno. Kljub temu pa je mikrobiološki kvar piva pogost problem v pivovarski industriji in razlog ekonomskih izgub. Med mikrobiološke povzročitelje kvarjenja spadajo tako grampozitivne kot gramnegativne bakterije in divje kvasovke, kontaminacija pa lahko povzroči motnost, kislost in spremembo okusa piva. Z naraščajočo priljubljenostjo nepasteriziranih, brezalkoholnih piv in piv z nizko vsebnostjo alkohola pa to predstavlja še toliko večjo skrb za pivovarsko industrijo. Za učinkovito odpravljanje težav je potrebna hitra in zanesljiva identifikacija bakterijskih vrst in tudi razlikovanje sevov iste vrste. Identifikacija bakterij v pivu večinoma poteka z uporabo standardnih fenotipskih testov po izolaciji na selektivnih gojiščih, kar je časovno potraten in manj specifičen postopek. Posledično se zaradi dolgotrajne analize povečajo tudi stroški skladiščenja in potreba po večji skladiščni kapaciteti. Ena od možnih načinov za hitro in zanesljivo identifikacijo mikroorganizmov v pivu je identifikacija z MALDI-TOF masno spektrometrijo (angl. matrix assisted laser desorption/ionization time-of-flight mass spectrometry). V diplomskem delu so predstavljeni najbolj pogosti mikrobiološki povzročitelji kvarjenja piva in njihova rezistenca na hmeljne spojine kot ključen faktor za zmožnost rasti v pivu. Predstavljena je tudi mikrobiološka identifikacija teh povzročiteljev z MALDI-TOF masno spektrometrijo v primerjavi z ustaljenimi metodami. Beer is, according to its properties, considered as microbiologically safe beverage. However, there are numerous reported events of beer spoilage due to the presence of microorganisms, which are a big problem in the brewing industry and reason for important economic losses. Beer spoilers belong to groups of Gram-positive, Gram-negative bacteria and wild yeasts and their presence can lead to beer turbidity, acidity and unwanted flavour. With the increased popularity of unpasteurized, nonalcoholic and low-alcoholic beers, beer-spoilage organisms present even greater problem for the brewing industry. Effective problem solution therefore demands quick and reliable identification of beer spoilers to species and/or strain level. However, bacterial identification in most laboratories is still carried out by traditional methods such as incubation on culture media and subsequent characterization through biochemical and other tests, which are labour-intensive and very time-consuming tasks. This increases storage expenses and the need for bigger storage capacity. One of the approaches for quick microbiological identification is the implementation of MALDI-TOF MS (matrix assisted laser desorption/ionization time-of-flight mass spectrometry) instrument into standard microbiological practice. The thesis presents main beer spoilers and their resistance to hop compounds as the key factor for their growth in beer. It also presents microbiological identification by MALDI-TOF mass spectrometry and compares it with the standard methods in the brewing microbiological laboratory.

Razgradnja odpadne embalaže je v današnjem času zelo aktualna tema, saj se delež komunalnih in industrijskih odpadkov iz dneva v dan povečuje. V zadnjem času so zato raziskave na področju alternativnih, okolju prijaznih metod recikliranja odpadkov zelo intenzivne. Polietilen (PE) predstavlja najbolj razširjeno plastiko na svetovni ravni, ki se nahaja predvsem v embalaži, kot so plastične vrečke, folije, plastenke, tudi v kozmetičnih izdelkih, prehrambeni industriji in energetiki. Sub- in superkritična voda je okolju prijazno topilo in odličen reakcijski medij za depolimerizacijo plastike v različne uporabne produkte. S tem namenom smo v diplomski nalogi proučevali razgradnjo odpadne barvne embalaže iz PE v superkritični vodi (SCW). Postopek smo izvajali v visokotlačnem, visokotemperaturnem šaržnem reaktorju, kjer smo odpadni barvni PE v razmerju z vodo 1/5 (g/mL) izpostavljali temperaturam 425 ºC in 450 °C v časovnih intervalih od 15 do 240 minut. Pri razgradnji odpadne barvne embalaže iz PE v SCW smo dobili štiri faze: oljno fazo ali vosek, vmesno, vodno ter plinsko fazo. Produkte v oljni in plinski fazi smo analizirali z GC/MS metodo, v vodni fazi pa smo določevali totalni ogljik (TC). Ugotovili smo, da se odpadna barvna embalaža iz PE v SCW ni popolnoma razgradila pri temperaturi 425 ºC pri časih 15 in 30 minut. Nastala oljna faza je vsebovala veliko ogljikovodikov, kjer so pri nižjih temperaturah in krajših časih prevladovali dolgi ogljikovodiki, pri višjih temperaturah in daljših časih pa so se pojavljali aromatski ogljikovodiki. Plinska faza je vsebovala različne pline, predvsem alkane in alkene. The decomposition of packaging waste is a very popular topic nowadays because the quantity of municipal and industrial waste is fastly growing. Lately, intensive research work has been devoted to alternative and eco-friendly methods of recycling. Polyethylene (PE) is the most widely used plastic in the world, which is mainly found in packaging, such as plastic bags, foils, bottles, even in cosmetic products, in food industry and energy. Sub- and supercritical water is an eco-friendly solvent and a great reaction medium for the depolymerization of plastic into various useful products. For this purpose, the degradation of colored packaging waste of PE in supercritical water (SCW) has been studied. The procedure was carried out in a high pressure, high temperature batch reactor at the temperatures of 425 °C and 450 °C. The mixture of coloured PE waste and water in a ratio 1/5 (g/mL) was exposed at various intervals ranging from 15 to 240 minutes. During the decomposition of colored packaging waste of PE in SCW four phases were obtained: oil phase or wax, intermediate, water and gas phase. The degradation products in the oil and gas phases were analyzed by gas cromatography and mass spectrometry. The content of total carbon was determined in the aqueoous phase. It was found that colored packaging waste of PE was not completely decomposed at 425 °C for 15 and 30 minutes. The obtained oil phase was composed of many hydrocarbons, where long-term hydrocarbons predominated at lower temperatures and shorter times, and aromatic hydrocabrons appeared at higher temperatures and longer times. The gas phase contained various gases, especially alkanes and alkenes.

Skozi razvoj in potrebo po čim krajšem in učinkovitem iskanju novih zdravilnih učinkovin je v ospredje stopila zmožnost sinteze knjižnic strukturno različnih spojin. Za odkrivanje novih zdravilnih učinkovin se trenutno najpogosteje uporablja metodo visoke zmogljivosti rešetanja (High Throughput Screening – HTS), iz katere so se na osnovi načrtovanja strukture in lastnosti molekul razvili še drugi sorodni koncepti sintezne kemije. V zadnjem desetletju se je tako znatno povečalo število novih odkritih spojin, vendar se kljub temu še vedno poslužujemo klasičnega pristopa rešetanja, zato poleg HTS knjižnic uporabljamo tako imenovane hišne (in-house) knjižnice. Namen magistrske naloge je bil razviti in optimizirati metodo tekočinske kromatografije visoke ločljivosti (HPLC) sklopljene z masnim detektorjem za identifikacijo ter kvantitativno določanje spojin knjižnice Fakultete za farmacijo. Glavni poudarek je bil na razvoju standardne HPLC metode, ter na velikem številu pripravljenih spojin, na katerih smo preverili učinkovitost naše metode. Predvsem smo raziskali vpliv različnih kromatografskih pogojev, kot je sestava mobilne faze, izbira primerne kromatografske kolone ter izbira ustreznega gradienta, kar bi omogočalo univerzalno uporabo. Z uporabo HPLC/MS metode smo bili sposobni identificirati 80 % preiskovanih spojin. Metoda je tako primerna za kvalitativno in kvantitativno vrednotenje. Ključni del raziskovalnega dela je bilo ročno preverjanje pridobljenih rezultatov, saj sodobno metode še ne omogočajo avtomatiziranega vrednotenja tako pridobljenih podatkov. Podatke smo ovrednotili in identificirali za osnovno spojino tudi prisotne nečistote. Z uporabo masne spektrometrije smo lahko tudi predvideli, kaj se je s preiskovano spojino zgodilo ter katere kemijske reakcijo so potekle pred ali po analiziranju spojin. Ostalim 20 % preiskovanih spojin niso ustrezali kromatografski pogoji, zato bi jih bilo potrebno obravnavati individualno. Focus on the ability of synthesize libraries of structurally different compounds was given through discovery and need for quick and effective search of new active substances. For the discovery of new active substances, is most commonly used the High Throughput Screening (HTS) method, from which other related concepts of synthetic chemistry have been developed based on the design of the structure and properties of molecules. In the last decade, the number of new detected compounds has increased significantly, but nevertheless, we continue to use the classic approaches. In addition to HTS libraries we use the so-called in-house libraries. The aim of this thesis was to develop and optimize HPLC method coupled with a mass detector to identify and quantify the compounds from the library of Faculty of pharmacy. The main focus was on the development of the standard HPLC method, and on the large number of prepared compounds, for which we checked the effectiveness of our method. In particular, we investigated the impact of various chromatographic conditions, such as the composition of the mobile phase, the choice of an appropriate chromatographic column, and the choice of the appropriate gradient, which would allow the universal use. Using the HPLC / MS method, we were able to identify 80% of the investigated compounds. Method is therefore suitable for qualitative and quantitative evaluation. A key part of the research work was to manually verify the results obtained, since modern methods do not allow automated evaluation of data. The data were statistically evaluated and identified for the main compound and also its disintegrating products. By using the mass spectrometry, we could also predict what happened to the investigated compound and which chemical reaction has occurred before or after analyzing the compounds. The chromatographic conditions were not suitable for the remaining 20% of the investigated compounds, thus should be considered individually.

Neposredna hitra identifikacija bakterij iz pozitivnih hemokultur je nepogrešljiva informacija tako za laboratorijsko osebje kot za lečečega zdravnika na kliniki. Polimikrobne hemokulture predstavljajo večje tveganje za smrt in daljše okrevanje bolnikov. Želeli smo ovrednotiti analizo masnih spektrov iz pozitivnih hemokultur s pomočjo standardne MALDI Biotyper Compass programske opreme in jih primerjati z modulom Sepsityper za zaznavanje mešanih kultur, pri čemer smo za referenco uporabili kulturo na gojišču. Hkrati smo želeli v laboratorijskem poskusu z zadostnim številom dveh bakterijskih vrst v različnih razmerjih ugotoviti optimalno končno razmerje za pravilno identifikacijo obeh z modulom Sepsityper. Ugotovili smo, da modul Sepsityper ni ustrezen za karakterizacijo polimikrobnih hemokultur. Ta nam je namreč podal le 4 pravilne identifikacije izmed 46/58 polimikrobnih hemokultur, za katere je uspel pridobiti določen spekter. V poskusu določitve meje zaznave je bila najbolj optimalna meja zaznave, ki je modulu Sepsityper omogočila ločiti dve bakterijski vrsti, 1:1, najnižja pa 8:1. Pri razmerju 20:1 modul Sepsityper ni več ločil bakterijskih vrst. Direct fast identification of bacteria in positive blood culture presents key information for laboratory personel as well as for the coresponding clinician. Polymicrobial bacteremia correlate with a higher death risk and present a longer period of recovery than do monomicrobial bacteremias. Using the module Sepsityper we wanted to obtain information about the possible presence of more than one microorganism in blood culture. We thus wanted to evaluate the analysis of mass spectra of positive blood cultures with the standard MALDI Biotyper Compass and compare it to the analysis of mass spectra obtained via module Sepsityper for identification of mixed cultures. Our gold standard was the coresponding culture of hemoculture on agar plates. We also wished to determine the optimal ratio between two bacterial species for which the Sepsityper module would still give us a sufficient result. We concluded that the Sepsityper module isn't appropriate for the characterization of polymicrobial spectra. Through the Sepsityper settings we identified only 4 out of 46/58 polymicrobial blood cultures for which it determined some spectra, i.e. got a response. In our experiment, the optimal ratio for differentiating between two bacterial species was 1:1, with the lowest being 8:1 and at a ratio of 20:1 Sepsityper module could not differentiate the two any longer.

Razvoj na področju avtomobilske industrije in transporta je v zadnjem desetletju usmerjen predvsem v zamenjavo pogonskega agregata, torej motorja z notranjim izgorevanjem, z okolju prijaznejšimi elektromotorji. Uporaba elektrike kot vira energije pa zahteva ustrezne rešitve prenosa elektrike, za kar skrbijo drsni obroči. Tudi drsni obroči morajo za ustrezno opravljanje svoje funkcije prenosa elektrike med gibanjem izpolnjevati materialne, trdnostne in funkcionalne zahteve. Vsebina diplomske naloge je tako vezana na opis delovanja drsnih obročev in preučitev zahtev kupca o potrebnih lastnostih na novo razvitega drsnega obroča za pogon električnega osebnega avtomobila. Na podlagi teh zahtev je potrebno nato definirati in izvesti preizkuse lastnosti, s katerimi zagotovimo izpolnjevanje zahtev izdelka. Namen diplomskega dela je bil predstaviti teoretične osnove delovanja in izvedb drsnih obročev, opisati preizkuse lastnosti in funkcionalnosti drsnih obročev ter predstaviti rezultate preizkušanja na novo razvitega drsnega obroča vključno z alternativno tehnologijo tesnjenja kontaktne žice. V diplomskem delu so tako predstavljeni rezultati preizkusov tesnosti žice z uporabo različnih materialov žice in s primerjavo različnih tehnologij tesnjenja žice ter rezultati preizkušanja tesnosti po izpostavljenosti agresivnim medijem. V drugem delu so prestavljeni rezultati preizkušanja zvarov na zvarjencih, ki so sestavni del drsnega obroča, z uporabo dveh različnih testnih metod. Rezultati diplomskega dela kažejo, da je nova tehnologija izdelave tesnil iz tesnilne mase primerna za uporabo v primeru drsnih obročev, medtem ko na mestu zvara električnih kontaktov najboljšo trdnost, določeno z dvema različnima testnima metodama, dosežemo z uporabo spajkalne paste in časa varilnega pulza 125 ms. In the last decade research and development in automotive industry has been focused mainly on replacing internal combustion engines with more environmentally friendly means of power generation, including electrical motors. Use of electric energy, however, requires proper methods of energy transfer, especially when it comes to the rotating assemblies. One of the methods for transferring electric energy from a stationary to a rotating part is the use of a slip ring. Like every part of transport vehicles, slip rings also have to fulfill several material, structural, and functional requirements, in order to function properly and transfer electric power or signals during rotational movement. Based on the customer specification, tests have to be defined and implemented in the development of a new slip ring. The purpose of this work was to theoretically describe slip rings, to describe tests that slip rings or similar electro-mechanical products have to undergo and to present testing procedures defined and implemented on a new slip ring aimed to be fitted in an electric car. The aim was to present the results of the implemented tests and compare different technologies used on the product. Therefore, in this bachelor thesis, the results of tightness of electric contacts are presented, that were derived from tests using different wire materials and using different sealing technologies. In the second part the results of weld strength testing used on welded electric contacts are presented. Results of this thesis show that sealing with a flexible sealant is suitable for slip ring applications. Furthermore, the best electrical contacts weld strength, determined by two different test methods is obtained when using soldering paste and welding pulse of 125 ms.

Several phenolic acids (PAs), caffeic, vanillic, syringic, p-coumaric and ferulic acid, found in Slovenian red wines were studied using gas chromatography and mass spectrometry (GC-MS). For isolation of the PAs from wine samples, solid phase extraction (SPE) using hydrophilic modified styrene - HLB cartridges was used. The bound PAs were extracted after basic hydrolysis and o-coumaric acid was used as the internal standard (ISTD). The method developed was validated and the linear concentration range for all analytes was from 1 to 100 mg L$^{-1}$ with correlation coefficients above 0.999. We show that the method is repeatable (RSD

V članku je opisana analiza delovne atmosfere v lakirnici ene izmed pohištvenih proizvodenj v Sloveniji. Adsorbcija substanc iz zraka je potekala na aktivnem oglju, desorbcija pa s stresanjem v ogljikovem disulfidu. Z uporabo GC-MS metode smo v vzorcih določili prisotnost benzena, toulena, butacilena in izomer ksilena. The article describes the analysis of the working atmosphere in a varnish works of one of the furniture factory in Slovenia. The adsorbtion of air substances was carried out on activated carbon and the desorption by means of shaking in carbon disulphide. By the application of the GC-MS method, the presence of benzene, toulene, butyl acetate and xylene isomers was determined in the samples.