Andvanced coupled techniques in the analysis ofxenobiotics

Prisustvo organskih zagađujućih supstanci (farmaceutski aktivnih komponenata i prirodnih toksina‐mikotoksina) u uzorcima životne sredine i namirnicama je u porastu kao posledica novih industrijskih procesa i ostalih antropogenih aktivnosti, kao i klimatskih promena. Takođe veliku pažnju javnosti privlače i neorganske zagađujuće supstance kao što su teški elementi. S obzirom da zagađujuće supstance imaju negativan uticaj na životnu sredinu i zdravlje ljudi, u svetu se preduzimaju mere u cilju smanjenja stepena izloženosti toksičnim jedinjenjma i posledicama izlaganja. Trenutno, jedan od najvećih izazova, jeste procena rizika povezana sa velikim brojem zagađujućih supstanci u tragovima ili u tzv. ultratragovima, uključujući “novo” otkrivena zagađujuća jedinjenja, a jedan od osnovnih trendova je razvoj i primena brzih i efikasnih metoda za njihovu analizu u ispitivanim uzorcima na bazi naprednih hromatografskih i spektrometrijskih tehnika. Tehnike bazirane na tečnoj hromatografiji sa različitim masenim analizatorima za kvantifikaciju organskih zagađujućih supstanci kao i metode zasnovane na atomskoj apsorpcijonoj spektrometriji za određivanje ultratragova neorganskih zagađujućih supstanci postale su referentne na međunarodnom nivou. Ovakve napredne spregnute tehnike postale su važne za identifikaciju, kvantifikaciju i praćenje različitih zagađujućih supstanci u uzorcima životne sredine i namirnicama i proceni njihovog štetenog uticaja na zdravlje čoveka. S obzirom da su u literaturi retka istraživanja koja se bave razvojem i primenom metoda zasnovanih na naprednim hromatografskim i spektrometrijskim tehnikama i određivanju organskih i neorganskih zagađujućih supstanci u matriksima životne sredine i namirnicama sa prostora zapadnog Balkana, a uzimajući u obzir njihovu važnost, specifični ciljevi disertacije su: • unutrašnja („inhouse“) provera kvaliteta i pouzdanosti postojeće „multi‐rezidualne“ metode zasnovane na UHPLC‐QqLIT‐MS/MS za analizu 81‐e farmaceutski aktivne komponente (PhAC) u otpadnoj, površinskoj, podzemnoj i pijaćoj vodi i po prvi put dobijanje sveobuhvatnih rezultata njihovog prisustva u različitim tipovima vode sa područja Srbije; • unutrašnja („inhouse“) provera kvaliteta i pouzdanosti postojeće „multi‐toksin“ metode za analizu 8 Fusarium mikotoksina u uzorcima ozime pšenice različitih sorti zasnovane na HPLC‐QqQ‐MS/MS radi određivanja regionalnih razlika između žitnih regiona kao i otpornosti ispitivanih sorti pšenice na kontaminaciju Fusarium toksinima; • modifikacija postojeće „multi‐toksin“ metode zasnovane na UHPLC‐QqQ‐MS/MS za analizu 11 osnovnih mikotoksina u uzorcima brašna i njena unutrašnja („inhouse“) provera kvaliteta i pouzdanosti, kao i provera kroz interlaboratorijsko poređenje, radi dobijanja podataka za procenu štetnog uticaja ispitivanih mikotoksina na zdravlje populacije; • razvoj „multi‐toksin“ (višekomponentne) i „multi‐matriks“ (za više matriksa) metode bazirane na UHPLC‐QqQ‐MS/MS za analizu 10 mikotoksina u različitim vrstama koštuničavog voća čija provera kvaliteta je zasnovana na intralaboratorijskoj proveri tačnosti i preciznosti dobijenih rezultata;                                                                                  • primena postojeće analitičke procedure zasnovane na naprednoj tehnici pripreme (mikrotalasnoj digestiji) različitih uzoraka biljnog i životinjskog porekla i provera kvaliteta metode identifikacije i kvantifikacije zasnovane na atomskom apsorpcionom spektrometru sa grafitnom kivetom (GFAAS) radi dobijanja sveobuhavatnih rezultata o prisustvu teških elemenata (arsena, olova i kadmijuma) radi procene izloženosti stanovništva Srbije toksičnim neorganskim elementima. Postignuti rezultati predstavljaju jedinstvene rezultate za područje Srbije dobijene primenom naprednih spregnutih tehnika koje imaju značajnu ulogu u praćenju prisustva većeg broja organskih i neorganskih zagađujućih supstanci u izabranim uzorcima životne sredine i namirnica, (regulisanih postojećim zakonodavstvom) radi procene stepena zagađenosti ili u slučaju jedinjenja koja nisu regulisana zakonodavstvom radi sticanja novih saznanja o njihovom prisustvu i proceni mogućeg negativnog uticaja na životnu sredinu i zdravlje populacije.The presence of organic pollutants in environmental samples and food (pharmaceutically active components and natural toxins‐mycotoxins) is increased as a result of new industrial processes and other anthropogenic activities, as well as climate change. Similarly heavy elements as inorganic pollutants have attracted worldwide attention. Since, these pollutants have negative impact on environment and human health, extremely efforts are undertaken in the world to reduce the level of exposure to these pollutants and consequences of the exposure. Currently, one of the highest challenges is to assess the risk associated with a large number of pollutants in trace or ultra trace levels, including "new" (emerging) discovered pollutants, and one of the main trends is development and implementation of fast and efficient methods for their analysis on the basis of advanced chromatographic and spectrometric techniques. Therefore, coupled techniques have become important for the identification, quantification and monitoring of various pollutants in environmental samples and food and assessment of their hazard impact on human health. Since, there are scarce data about the development and application of advanced methods based on chromatographic and spectrometric techniques for determination of organic and inorganic pollutants in environmental matrices and food from the Western Balkan, and taking into account their importance, specific objectives of the dissertation were: • internal ("in‐house") quality control of the existing "multi‐residual" method based on UHPLC‐QqLIT‐MS/MS for analysis of 81 pharmaceutically active components (PhAC) in wastewater, surface, underground and drinking water due to obtained for the first time comprehensive results of their presence in different types of water from Serbia; • internal ("in‐house") quality control of the existing "multi‐toxin" method for the analysis of 8 Fusarium mycotoxins in samples of different winter wheat cultivars based on HPLC‐QqQ‐MS/MS to determine the differences among wheat-growing regions as well as the resistance of the analysed wheat cultivars towards Fusarium toxins; • modification of existing "multi‐toxin" method based on UHPLC‐QqQ‐MS/MS for analysis of 11 principal mycotoxins in samples of flour and its internal ("in‐house") quality control as well as verification through the interlaboratory comparison, in order to obtain data for assessing the hazard effect of these mycotoxins on the health of the population; • the development of "multi‐toxin" and "multi‐matrix" method based on UHPLC‐QqQMS/ MS for the analysis of 10 mycotoxins in various types of nuts based on intralaboratory verification of the accuracy and precision of the obtained results; • application of analytical procedure based on advanced preparation technique (microwave digestion) and atomic absorption spectrometer with a graphite furnace (GFAAS) and its verification in order to obtain comprehensive results on the presence of heavy elements (arsenic, lead and cadmium) in different samples of plant and animal origin to assess the exposure of the Serbian population to toxic inorganic elements. The obtained results are unique for the Serbia. They are obtained by applying advanced coupled techniques that have a significant role in monitoring the presence of a numerous organic and inorganic pollutants in analyzed samples of the environment and food. The presented results contribute to the assessment of pollution degree and in the case of new (emerging) not regulated polutant they might give new information about the possible negative impact on the environment and health of the population

Mogućnost primene alternativnih žita (prosa i ječma) za poboljšanje tehnološkog kvaliteta hleba od brašna loših tehnoloških osobina

The aim of the study was to examine does it possible to improve bread properties of the bread based on wheat flour of poor technological quality by substitution of barley and millet flour. For that purpose were examined chemical properties of raw materials (Wheat flour of extremely poor technological quality (WFPQ), dehulled wholegrain barley flour (DWBF) and decorticated millet flour (DMF)), as well as rheological properties of doughs (WFPQ flour and mixtures of DWBF and DMF with WFPQ in ratios 10:90 and 30:70) and end-use quality of control and composite breads. The substitution of the small amount of WFPQ with DMF (10%) improved most of the bread properties, whereas the sensory properties stay almost the same. Also, substitution of the high amount of DMF (30 %) neither showed effects of improvement and worsening for to the most of technological properties. On the other hand, substitution with DWBF has negative impact on the most technological properties of composite bread.Cilj ovog istraživanja je bio da se ispita mogućnost primene brašna od ječma i prosa za poboljšavanje tehnoloških osobina hleba proizvedenog od brašna lošeg tehnolološkog kvaliteta supstitucijom određenog procentualnog udela u osnovnoj recepturi hleba. Zbog toga je ispitan hemijski sastav sirovina (pšeničnog brašna izuzetno lošeg tehnološkog kvaliteta (WFPQ), integralnog brašna od oljuštenog ječma (DWBF) i integralnog brašna od oljuštenog prosa (DMF)), reološke osobine testa (od WFPQ i smešama od DWBF i DMF sa WFPQ u odnosu 10:90 i 30:70) i tehnološke osobine kontrolnog i mešanih hlebova (zapremina hleba, spec. zapremina hleba, visina vekne, boja kore i sredine, Texture Profile Analyses (TPA) sredine hleba i senzorna analiza hleba). Zamenom male količine WFPQ sa DMF (10%) poboljšane su tehnološke osobine hleba: zapremina, visina vekna, boja kore i TPA pokazatelji; dok su senzorne osobine hleba ostale skoro iste kao kod kontrolnog hleba. Takođe, zamenom veće količine DMF (30%) nije se dobilo niti poboljšanje niti pogoršavanje većine tehnoloških pokazatelja. Supstitucijom DWBF u osnovnoj recepturi hleba poboljšali su se neki reološki pokazatelji i boja kore, dok je većina tehnoloških pokazatelja mešanog hleba bila lošija od kontrolnog hleba. Može se očekivati da će zamenom veće količine WFPQ sa DMF i DWBF doći do boljeg poboljšavanja nutritivnih osobina hleba nego u slučaju zamene male količine DMF i DWBF jer su hemijske analize pokazale da su DMF i DWBF boljih nutritivnih osobina od WFPQ

CONTAMINATION OF CULTIVATED VEGETABLES BY HEAVY ELEMENTS FROM FLOODED ARABLE SOIL: HUMAN EXPOSURE

The consumption of vegetables is one of the most important pathways for heavy elements to harm human health. Direct deposition of contaminants from the atmosphere onto plant surfaces and accumulation of heavy elements in flooded arable soil are of great concern because of the potential health risk to the local population. Thus, the present study was carried out to analyse distribution and soil-plant transfer of Pb, Cd, As, Co, Cr, Ni, Cu, Mn and Fe in potato, carrot, celery, parsnip and onion in order to evaluate their potential effects on human health. Total content of heavy elements in 26 vegetable samples collected from different flooded fields was analysed by atomic absorption spectrometry with a graphite furnace (AASGF) after microwave digestion of the analysed samples. Average concentrations of lead (Pb) and cadmium (Cd) in some of the investigated vegetable samples were higher than maximum allowable concentrations set by EC/Serbian regulation. On the other hand, arsenic (As) was not detected in any of the analysed samples. Accumulation and translocation of analysed elements were varied from element to element as well as among selected vegetable crops. The results showed that the parsnip had highest uptake for the most analysed elements (Fe (107 mg/kg) ˃ Mn (6.98 mg/kg)˃ Cu (1.94 mg/kg)˃Ni (0.34 mg/kg)˃ Pb (0.13 mg/kg) compared with the other investigated crops. The bioaccumulation factor (BA) for analysed elements in different vegetables was found in order of Cd (0.08)˃Fe(0.07)˃Cu (0.06)˃Pb (0.01)=Ni˃Mn (0.001), indicating that analysed crops are categorized as excluder (BA<1). The total health risk associated with the consumption of investigated vegetables grown in studied flooded arable soil was assessed using target hazard quotient (THQ). The THQ values estimated for investigated crops were notably below the safe limit of 1, except for Mn (2.10) and Fe (11.65)

The influence of wheat genotype and environmental factors on gluten index and the possibility of its use as bread quality predictor

Gluten index is an indicator of gluten strength as well as a parameter which simultaneously defines its quantity and quality. If compared to the farinographic and extensographic methods, gluten index determining is faster, less complicated and requires smaller amount of flour. The aim of this study has been to determine the significance of the influence of genotype and environmental factors on the value of gluten index and usability of these parameters as indicators and predictors of bread quality. Five local varieties of winter wheat (Simonida, NS 40S, Rapsodija, Pobeda, Zvezdana) were grown in a macro-experiment carried out in three locations (Novi Sad, (Cack, Sombor) during two seasons (2011/2012 and 2012/2013). Gluten index value determination was achieved by using ICC standard method 155 (1996), with a slight modification that included mechanical washing of gluten (Theby). Statistical analysis was performed by using IBM SPSS Statistics 20. Variance analysis revealed a statistically significant effect of genotype on gluten index value, whereas the influence of environmental factors, as well as the interaction of two factors, had no statistical significance. There was no correlation between the values of gluten index and meteorological factors such as average temperature and total precipitation during the phenophase of grain filling, and in the period from the beginning of flowering until the harvest. However, medium-strong negative dependence was found between gluten index and the index of heat stress, as well as between gluten index and the number of days with temperatures above 30 degrees C. Weak negative dependence was recorded between gluten index and bread volume. Based on the value of gluten index, gluten varieties used in our study can be described as strong. Genotype proved to be the only cause of statistically significant variation of gluten index

Solid-phase extraction as promising sample preparation method for compound of emerging concerns analysis

Today’s environment is under the influence of numerous substances and most of them are not covered by current national and international regulations. Substances that are classified as contaminants of emerging concern, CECs, are not regulated nor systematically controlled in the environment, and they belong to the different chemical classes such as pesticides in current use, pharmaceutically active compounds (PhACs), personal care products, illicit drugs, hormones, micro- and nano-plastics, per- and poly-fluoroalkyl substances (PFAS), and many others. Once released CECs end up in surface water where they can be either accumulated or transported to the other environmental compartments, i.e. soil (by irrigation), underground water, or drinking water. The aim of the research was to investigate the efficiency of different selected solid-phase extraction (SPE) cartridges for preparation of water samples for simultaneous analysis of several CECs. For this purpose, a model mix solution of selected CECs in concentration relevant to environmental appearance was used. The selected CECs (14 PhACs, 11 pesticides in current use, and 4 PFAS) represented emerging contaminants with different properties, including polarities, and varying adverse effect on the environment and human health. Different types of sorbents were investigated: (i) commercial single-layer HLB, (ii) homemade multi-layer I: HLB plus a mix of WAX, WCX, and PPL, and (iii) multi-layer II: mix of WAX, WCX, and PPL plus HLB. The results revealed that the single-layer sorbent showed better efficiency in extraction of the analyzed CECs. Multi-layer sorbent should be further investigated to elucidate the possible reason for the poor extraction of some compounds, as development of a balanced extraction for a wider range of contaminants with different polarities, especially for non-target analysis of chemical residues, is required in order to capture the occurrence of the full profile of micropollutants

Application of laccases, produced by ganoderma species, for the detoxification of some aniline and phenol derivatives

The objective of this work was to collect isolates of the white rot fungus Ganoderma from decaying woods and investigate their laccase producing and detoxification abilities against aniline and phenol compounds. Sporocarps were collected and the tissue culturing technique was used for fungal isolation. In these experiments specific medium, containing benomyl, dichloran and guaiacol was used. Laccase producing of the isolates were tested in liquid media containing different inducers. Crude enzyme extracts were prepared and characterized. The best laccase-producing strains were identified with partial ITS sequence analysis. Their secreted laccases were investigated with SDS-PAGE and the molecular weights of these enzymes were estimated. Degradation of 7 different aniline and phenol derivates (2,4-dichlorophenol, 2-methyl-4- chlorophenol, 3-chloroaniline, 4-chloroaniline, 2,6-dimethylaniline, 3,4-dichloroaniline and 3-chloro-4- methylaniline) were investigated, observing high degradation rates in most cases