<p>Rod Hypericum obuhvata oko 500 različitih vrsta klasifikovanih u 36 sekcija. Najpoznatiji predstavnik roda je kantarion (Hypericum perforatum, Hypericaceae). Hemijska karakterizacija kantariona je bila predmet mnogobrojnih istraživanja koja su pokazala prisustvo različitih klasa jedinjenja, kao i &scaron;irok spektar biolo&scaron;kog potencijala. Međutim, ispitivanja su otkrila prisustvo istih klasa jedinjenja i u drugim predstavnicima roda Hypericum, opravdavajući hemijska istraživanja i određivanje biolo&scaron;kih aktivnosti. S obzirom da se kantarion retko plantažno uzgaja, već uglavnom sakuplja iz prirode, neretko usled nedovoljne obučenosti sakupljača lekovitog bilja dolazi do zamene propisanog biolo&scaron;kog izvora droge Hyperici herba (H. perforatum subsp. perforatum, Hypericaceae) morfolo&scaron;ki sličnim predstavnicima roda, &scaron;to posledično utiče na sadržaj aktivnih principa i biolo&scaron;ki potencijal. Zbog toga su ciljevi ove doktorske disertacije: utvrđivanje uticaja geografskog porekla i stadijuma ontogenetskog razvoja biolo&scaron;kog izvora na kvalitativni i kvantitativni hemijski sastav, biolo&scaron;ki potencijal vodeno-alkoholnog ekstrakta taksona roda Hypericum, kao i utvrđivanje kvaliteta (stepen usitnjenosti, biolo&scaron;ki izvor, hemijski sastav i biolo&scaron;ki potencijal) komercijalno dostupnih čajeva na bazi kantariona koji se nalaze u slobodnoj prodaji. Biljni materijal je obuhvatio uzorke 32 taksona roda Hypericum kao i 51 uzorak monokomponentnih čajeva na bazi kantariona prikupljenih na teritoriji centralnog dela Balkanskog poluostrva, centralne Evrope, Rusije i Turske tokom 2011-2016. godine. U većini ispitanih ekstrakta kvantifikovan je visok sadržaj ukupnih fenola i flavonoida. Rezultati određivanja sadržaja hipericina u ispitivanim ekstraktima pokazali su njegovo prisustvo kod uzoraka svih taksona prikupljenih za vreme cvetanja, pri čemu je to bila i fenofaza u kojoj je dostignut maksimalan sadržaj. Hiperforin je takođe detektovan u većini uzoraka prikupljenih za vreme cvetanja, prateći dinamiku akumulacije sličnu hipericinu. Sa druge strane apigenin i epikatehin nisu detektovani u velikom broju ispitanih ekstrakta, dok je prisustvo naringenina kvantifikovano u manje od 50% uzoraka. Amentoflavon je bio prisutan u manje od 30% ispitanih uzoraka. Visok sadržaj kvercetina i rutina je određen u svim ispitanim ekstraktima, pri čemu u slučaju kvercetina nije zabeleženo značajnije variranje tokom ontogenetskog razvoja, dok je maksimalna koncentracija rutina određena u uzorcima prikupljenim pre, ili za vreme cvetanja. U većini uzoraka detektovane su galna, hlorogenska, kafena i p-hidroksibenzojeva kiselina (naročito u periodu koji prethodi cvetanju), dok ferulna kiselina nije kvantifkovana kod vi&scaron;e od 80% ispitanih ekstrakta. Rezultati su pokazali snažnu antioksidantnu sposobnost ispitivanih ekstrakata. Uočena je snažnija inhibicija monoaminooksidaze A u odnosu na monoaminooksidazu B, kao i vi&scaron;i anti-&alpha;-glukozidazni od anti-&alpha;-amilaznog potencijala. Sposobnost inhibicije acetilholinesteraze je ocenjena kao umerena. Ispitivanje antiproliferativnog potencijala ekstrakata vrsta roda Hypericum, je pokazalo izrazit sinergistički efekat sa antineoplastičnim lekom bleomicinom (radiomimetik) kao i visoku selektivnost prema ćelijama karcinoma cerviksa i melanoma, za razliku od umerenog antiproliferativnog potencijala i niske selektivnosti samih ekstrakata prema malignim ćelijama. Zabeležen je značajan broj neusitnjenih (in toto) komercijalno dostupnih uzoraka čajeva na bazi kantariona, dok su kao biolo&scaron;ki izvor ispitanih uzoraka čajeva identifikovane različite podvrste H. perforatum. Sličnost hemijskog profila i biolo&scaron;kog potencijala velikog broja ispitanih taksona sa H. perforatum ukazuju na mogućnost njihove eksploatacije. Međutim, zavisnost navedenih parametara od geografskog porekla biljnog materijala ističe značajan uticaj abiotičkih ekolo&scaron;kih faktora na kvalitet droge. Hemijski sastav i morfolo&scaron;ko ispitivanje uzoraka monokomponentnih čajeva na bazi kantariona ukazuju na neophodnost kontrole njihovog kvaliteta.</p> / <p>The genus Hypericum includes more than 500 different species classified in 36 sections. The most known representative of genus is St. John&#39;s wort (Hypericum perforatum, Hypericaceae). Chemical characterization of St. John&#39;s wort was the subject of many conducted researches which demonstrated the presence of different classes of compounds, as well as a wide spectrum of biological potential. However, the studies have also shown the presence of the same compounds in other representative of the genus Hypericum, justifying the evaluation of their chemical composition and biological potential. Considering the fact that St. John&#39;s wort is rarely being cultivated, rather collected from nature, unqualified collectors may substitute the official biological source of Hyperici herba (H. perforatum subsp. perforatum, Hypericaceae) for morphologically similar representatives of the genus, consequently affecting the quantities of active principles and biological potential of the drug. The aims of this PhD thesis were to evaluate the influence of geographical origin and stage of ontogenetic development of biological source on qualitative and quantitative chemical composition and biological potential of water - alcoholic extract of different Hypericum taxa, as well as to determine the quality (grinding degree, biological source, chemical composition and biological potential) of commercially available St. John&rsquo;s wort teas on the market. The plant material included samples of 32 taxa of genus Hypericum, as well as 51 commercially available sample of monocomponent teas based on St. John&#39;s wort collected at territory of central part of Balkan Peninsula, central Europe, Russia and Turkey, during period 2011 - 2016. High amounts of total phenolics and flavonoids were quantified in the most of the examined extracts. Quantification of hypericin showed its presence in all of the samples collected during flowering period, while that was also the ontogenetic stage of its maximum accumulation. Hyperforin was also quantified in most of the samples collected during flowering period, following the hypericin trend of accumulation. On the other hand, apigenin and epicatechin were not detected in large number of the examined extracts, while naringenin was detected in less than 50% of samples. Amentoflavone was present in less than 30% of the examined samples. High amounts of quercetin and rutin were determined in all of the examined samples. No significant changes in the amounts of quercetin during ontogenetic development were noticed, while the highest amounts of rutin were determined in samples collected before, or during flowering. Gallic, chlorogenic, caffeic and p-hydroxybenzoic acid were detected in most of the examined samples (especially in period before opening of the flowers), while ferulic acid was not quantified in more than 80% of samples. The results have demonstrated strong antioxidant potential of the examined extracts. Stronger potential of the examined extracts to inhibit monoamine oxidase A than monoamine oxidase B, as well as stronger anti-&alpha;-glucosidase than anti-&alpha;-amylase potential were recorded. The obtained anticholinesterase activity of the examined extracts was moderate. The examination of antiproliferative potential of different Hypericum species extracts has shown additive effects in combination with bleomycin (radiomimetic), as well as high selectivity toward cervix and melanoma cancer cells. On the other hand, moderate antiproliferative potential and low selectivity during treatment of cancer cells only with the examined extracts was recorded. Significant share of in toto commercially available tea samples based on St. John&#39;s wort was noticed, while different subspecies of H. perforatum were identified as biological sources of the examined teas. Chemical profile and biological potential resemblance between a large number of investigated taxa and H. perforatum point to possibilities of their exploitation. However, the dependence of listed parameters from geographical origin of plant material emphasizes the importance of abiotic ecological factors for the quality of herbal drug. Chemical composition and morphological examination of monocomponent teas based on St. John&rsquo;s wort stress the importance of their quality control.</p>

<p>U ovoj doktorskoj disertaciji analizirano je bra&scaron;no strnih žita (p&scaron;enice, spelte, ječma,<br />raži, tritikalea, ovsa), prosolikog žita (kukuruza) i pseudocerealija (heljde i<br />amarantusa) primenom gasne hromatografije sa masenom spektrometrijom (GC-MS) u<br />kombinaciji sa multivarijantnom analizom, u cilju ispitivanja mogućnosti za<br />određivanje autentičnosti navedenih biljnih vrsta. U tu svrhu, iz bra&scaron;na navedenih<br />cerealija i pseudocerealija ekstrahovani su mali molekuli (lipidi i &scaron;ećeri). Lipidne<br />komponente bra&scaron;na ekstrahovane su heksanom. Nakon vi&scaron;estrukog obezma&scaron;ćivanja<br />istih uzoraka bra&scaron;na heksanom i su&scaron;enja, usledila je ekstrakcija &scaron;ećernih komponenata<br />96%-tnim etanolom. Dobijeni heksanski i etanolni ekstrakti derivatizovani su<br />odgovarajućim reagensima. Za derivatizacaju lipidnih komponenata kori&scaron;ćen je<br />rastvor TMSH (trimetilsulfonijum hidroksida, 0,2 M u metanolu). Time se masne<br />kiseline prevode u odgovarajuće metil-estre. Za derivatizaciju &scaron;ećernih komponenata<br />kori&scaron;ćen je etanolni rastvor natrijum-hidroksida i hidroksilamin-hidrohlorida u<br />kombinaciji sa BSTFA (bis-(trimetilsilil-trifluoroacetamidom). Na taj način su prosti<br />&scaron;ećeri prevedeni u odgovarujuće trimetilsilil-oksime. Ovako pripremljeni ekstrakti<br />uzoraka bra&scaron;na su analizirani na GC&ndash;MS uređaju.<br />Dobijeni hromatogrami međusobno pokazuju veliku sličnost, a naročito u okviru<br />uzoraka iste botaničke vrste. U obradi hromatograma i masenih spektara, kori&scaron;ćen je<br />MSD Productivity ChemStation program uz primenu Wiley 275 biblioteke masenih<br />spektara. Dobijeni podaci analizirani su na tri načina. Prvi način podrazumeva<br />kreiranje numeričkih matrica u modu ukupne jonske struje (TIC). Drugi postupak<br />podrazumeva kreiranje numeričkih matrica izolovanjem karakterističnih fragmentnih jona sa hromatograma (kod lipida 74 m/z, a kod &scaron;ećera 73 m/z u kombinaciji sa bar<br />jednim od sledećih jona: 204 m/z, 217 m/z, i 361 m/z). Kod trećeg načina obrade<br />podataka, analiza je urađena kreiranjem matrica primenom binarnog sistema (1/0), gde<br />&bdquo;1&ldquo; označava prisustvo određene komponente, a &bdquo;0&ldquo; označava njeno odsustvo u<br />posmatranom uzorku. Tako dobijeni podaci podvrgnuti su multivarijantnoj analizi<br />primenom statističkih programa &ndash; PAST i STATISTICA. U sva tri slučaja ispitivani<br />uzorci grupisani su u međusobno jasno odvojene grupe, prema odgovarajućoj biljnoj<br />vrsti. Uočeno je jasno razlikovanje pseudocerealija (heljde i amarantusa) i prosolikog<br />žita (kukuruza) od grupe strnih žita (p&scaron;enice, spelte, ječma, raži, tritikalea i ovsa).<br />Primenjenim metodama postiže se manje selektivna klasifikacija biljnih vrsta u okviru<br />strnih žita, sa izuzetkom uzoraka ovsa i spelte. Ovi rezultati pokazuju da je novim<br />navedenim postupcima moguće utvrditi autentičnost bra&scaron;na različitih botaničkih vrsta<br />cerealija i pseudocerealija.<br />Na isti način tretirani su i uzorci proizvedenog hleba (kore i sredine) u kojima je<br />p&scaron;enično bra&scaron;no supstituisano sa 0, 20, 40, 50, 60, 80 i 100% heljdinog bra&scaron;na.<br />Multivarijantnom analizom GC-MS podataka dobijaju se jasno odvojeni klasteri u<br />skladu sa porastom udela bra&scaron;na heljde u kori i sredini hleba. Najbolji rezultati<br />postignuti su koreliranjem sastava i sadržaja ugljenohidratnih komponenata u kori<br />hleba.<br />Krajnji ishod razvoja ovih novih originalnih metoda je mogućnost njihove praktične<br />primene u cilju rutinskih kontrola kvaliteta bra&scaron;na i gotovih pekarskih proizvoda u<br />laboratorijama, proizvodnim pogonima i inspekcijskim službama. Posebna pogodnost<br />primene predloženih metoda je to &scaron;to su one semi-kvalitativnog i semi-kvantitativnog<br />karaktera, pa se mogu izvoditi bez tačne identifikacije eluirajućih malih molekula</p> / <p>In this doctoral thesis various flour samples of small grains (wheat, spelt, barley, rye,<br />triticale, oats), corn and pseudocereals (buckwheat and amaranth) were analyzed using<br />gas chromatography - mass spectrometry (GC-MS) in combination with multivariate<br />analysis, in order to examine the possibilities for determining the authenticity of the<br />plant species listed. Small molecules (lipids and sugars) were extracted from flour<br />samples of the above-mentioned cereals and pseudocereals. The lipid components of<br />the flour were extracted with hexane. The extraction of sugar components from the<br />same flour samples was performed with 96% ethanol solution, after multiple defatting<br />with hexane and drying of the samples. The obtained hexane and ethanol extracts were<br />derivatized with appropriate reagents. For the derivatization of the lipid components, a<br />solution of TMSH (trimethylsulfonium hydroxide, 0.2 M in methanol) was used. Thus,<br />the fatty acids were converted into the corresponding methyl esters. For the<br />derivatization of the sugar components, an ethanol solution of sodium hydroxide and<br />hydroxylamine hydrochloride combined with BSTFA (bis- (trimethylsilyl<br />trifluoroacetamide) was used. Thus, the free sugars were converted into the<br />corresponding trimethylsilyl oximes. The derivatized extracts prepared in this manner<br />were analyzed on a GC -MS device.<br />The obtained chromatograms were very similar, and especially within the samples of<br />the same botanical species. In the processing of the chromatograms and mass spectra,<br />MSD Productivity ChemStation program was used with the Wiley 275 Mass Spectra<br />Library. The obtained data were processed in three ways. The first method involves the<br />creation of numerical matrices in the total ion current mode (TIC). The second method<br />involves the creation of numerical matrices by isolating characteristic ion fragments from the TIC chromatograms (74 m/z for the lipid components, and 73 m/z in<br />combination with at least one of the following ions: 204 m/z, 217 m/z, and 361 m/z, for<br />the sugar components). In the third way of data processing, the analysis was done by<br />creating a matrix using a binary system (1/0), where &ldquo;1&rdquo; denotes the presence of a<br />particular component, and &ldquo;0&rdquo; denotes its absence in the observed sample. The data<br />thus obtained were subjected to multivariate analysis using statistical programs - PAST<br />and STATISTICA. In all three cases, the analyzed samples were grouped in clearly<br />separated groups according to the appropriate plant species. A clear distinction was<br />observed between pseudocereals (buckwheat and amaranth), corn and the group of<br />small grains (wheat, spelt, barley, rye, triticale, oats). The applied methods achieved a<br />less selective classification of plant species within the group of small grains, with the<br />exception of oats and spelt samples. These results show that it is possible to determine<br />the authenticity of flour of various botanical species of cereals and pseudocereals,<br />applying new methods described in this doctoral thesis.<br />The samples of produced bread (crust and crumbs) in which wheat flour was<br />substituted with 0, 20, 40, 50, 60, 80 and 100% of buckwheat flour were treated in the<br />same way. Multivariate analysis of GC-MS data provided clearly separated clusters in<br />accordance with the increase in the share of buckwheat flour in bread crusts and<br />crumbs. The best results were achieved by correlating the composition and content of<br />the carbohydrate components in bread crusts.<br />The ultimate outcome of the development of these new original methods is the<br />possibility of their practical application for the purpose of routine quality control of<br />flour and bakery products in laboratories, production facilities and inspection services.<br />A particular advantage of applying the proposed methods is that they are semiqualitative<br />and semi-quantitative. Therefore, they can be performed without the exact<br />identification of eluting small molecules (lipids and free sugars), nor their<br />quantification using analytical standards.</p>

<p>Ispitana&nbsp; je&nbsp; mogućnost ekstrakcije fumonizina iz kukuruza&nbsp;sa vodom i fosfatnim puferom, umesto sa sme&scaron;om&nbsp;acetonitril&ndash;metanol&ndash;voda.&nbsp; Ekstrakcione metode&nbsp;neorganskim&nbsp; rastvaraĉima&nbsp; su&nbsp; uspe&scaron;no&nbsp; primenjene za&nbsp;odreĊivanje&nbsp; fumonizina&nbsp; B₁, B_2&nbsp;i&nbsp; B_3&nbsp;metodom&nbsp; tečne&nbsp;hromatografije sa fluorescentnom detekcijom, kao i&nbsp;ukupnih fumonizina pomoću&nbsp; imunohemijske metode.&nbsp;Analiziran&nbsp; je&nbsp; veći broj&nbsp; uzoraka na&nbsp; prisustvo i sadržaj&nbsp;fumonizina.&nbsp; Ispitani su uzorci&nbsp; kukuruza (235), sakupljeni&nbsp;tokom vi&scaron;egodi&scaron;njeg perioda (2005.&nbsp; i 2009&minus;2013.&nbsp; godine)&nbsp; i&nbsp;p&scaron;enice (83) roda 2010. i 2012. godine, sa područja&nbsp; severne&nbsp;Srbije.&nbsp; Ispitana je kontaminiranost kukuruza sa područja&nbsp;<br />severne Srbije fumonizinima, drugim mikotoksinima i&nbsp;plesnima, kao i mogući uticaj klimatskih faktora na stepen&nbsp;kontaminacije.&nbsp; Rezultati kontaminiranosti useva&nbsp;fumonizinima&nbsp; za svaku godinu&nbsp; pojedinačno&nbsp; su povezivani&nbsp;sa vremenskim prilikama koje su&nbsp; pratile istu. Različita&nbsp;hrana na bazi žitarica je analizirana ELISA metodama.&nbsp;Razvijena je ELISA i metoda&nbsp; tečne hromatografije sa&nbsp;fluorescentnom detekcijom&nbsp; za određivanje fumonizina u tri&nbsp;vrste lekovitog bilja sa područja Srbije.&nbsp; Ispitana je&nbsp;mogućnost&nbsp; primene infracrvene spektroskopije sa&nbsp;Furijeovim transformom&nbsp; za &nbsp;odredivanje&nbsp; fumonizina.&nbsp;Razvijen je&nbsp; ekspertni sistem za re&scaron;avanje problema izbora&nbsp;<br />optimalnog postupka određivanja fumonizina u kukuruzu.</p> / <p>Possibility of fumonisin extraction from maize using water and phosphate buffer instead of acetonitrile-methanol-water mixture was examined. The methods of extraction without organic solvents have successfully been applied for the determination of fumonisins&nbsp; B1, B2 and&nbsp; B3 using&nbsp; liquid chromatography-fluorescence detection&nbsp; method,&nbsp; as well as total fumonisins using enzyme-linked immunosorbent&nbsp; assay&nbsp; (ELISA).&nbsp; Large number of samples was analyzed for the presence and content of fumonisins. Maize samples (total 235) collected throughout several-year period (2005 and 2009-2013) and wheat samples (total 83) from 2010 and 2012 harvest originating from the territory of northern Serbia were analyzed. Contamination of maize originating from the territory of northern Serbia with&nbsp;fumonisins and other mycotoxins and moulds was examined, as well as the potential impact of climatic factors on contamination level.&nbsp; The results on the fumonisin-contamination of cereals obtained for each individual year are related with the climatic&nbsp; conditions characteristic for the relevant year. A variety of cereal-based food was analyzed using ELISA methods. Improved&nbsp; liquid chromatography-fluorescence&nbsp; detection method and ELISA for the determination of fumonisins in three &nbsp;medicinal plant&nbsp; species from the territory of Serbia were developed.&nbsp; Possible application of&nbsp; Fourier transform&nbsp; infrared spectroscopy&nbsp; (FTIR) for quantification of fumonisin was investigated.&nbsp; An expert system&nbsp; to solve the problem of selecting an optimal&nbsp;method for&nbsp; determination of fumonisins in&nbsp; maize has been developed.</p>

<p>Određivanje koncentracije etanola u telesnim tečnostima, pre svega u krvi, neophodan je uslov da bi se ustanovio uticaj alkoholemije na psihomotorne sposobnosti. Poznavanje stabilnosti lekova, droga i metabolita u biolo&scaron;kim uzorcima je od ključne važnosti kada se ukaže potreba za ponovljenom analizom i evaluacijom rezultata u sudskom postupku. Osnovni ciljevi ovog rada su da se uz pomoć HS-GC metode (hedspejs gasna hromatografija) ustanovi da li postoji statistički značajna promena koncentracije etanola u uzorcima krvi dobijenih od živih osoba i u biolo&scaron;kim uzorcima uzorcima sa autopsijskog materijala. Na osnovu rezultata potrebno je bilo utvrditi u kojem tipu uzorka uzetog sa le&scaron;nog materijala postoji najmanja promena koncentracije tokom perioda čuvanja uzorka. Istraživanje je bilo otvoreno, randomizirano i prospektivnog tipa. Biolo&scaron;ki uzorci krvi krvi živih osoba i le&scaron;nog materijala (krv, mokraća i staklasto telo) uzimani su metodom slučajnog izbora, u rasponu alkoholemije od 0,1 mg/ml do 5 mg/ml. Nakon inicijalne dvostruke analize, jedan biolo&scaron;ki uzorak čuvan je u trajanju od 180 dana, dok je drugi otvaran i analiziran nakon 60, 120 i 180 dana. Ukupan broj analiza alkoholemije u krvi živih osoba iznosio je 500. Ukupan broj analiza koncentracije etanola u krvi, mokraći i staklastom telu sa le&scaron;eva iznosio je 360. Etanol je u uzorcima krvi živih osoba, kao i u biolo&scaron;kim uzorcima sa autopsijskog materijala određivan metodom HS GC. Tokom čuvanja biolo&scaron;kih uzoraka u periodu od &scaron;est meseci ustanovljeno je da je do&scaron;lo do značajnog smanjenja koncentracije etanola u svim analiziranim uzorcima, nezavisno od njegovog porekla. Promena koncentracije etanola tokom čuvanja u zavisnosti je od tkivne vrste uzorka, inicijalne alkoholemije, dužine čuvanja, integriteta vijala i čepova, temperature, odnosa tečne i gasne faze, prisustva konzervansa i potencijalnog intermitentnog otvaranja radi analiza.</p> / <p>Determination of ethanol concentration in body fluids, especially blood, is a necessary objective to establish the influence of alcohol on psychomotor skills. Knowing the stability of medicines, drugs and metabolites in biological samples is of crucial importance when there is a need for repeated analysis and result evaluation in court. The main objectives of this work were to determine whether there was a statistically significant change in ethanol concentration in blood samples obtained from living subjects and from autopsy material, by using HS-GC method (headspace gas chromatography). Based on the results it was necessary to determine which type of sample collected from autopsy showed the lowest change in concentration during the storage period. The study was open, randomized and prospective. Biological samples of living person&#39;s blood and autopsy biological samples (blood, urine and the vitreous humor) were taken at random, in the level range between 0.1 mg/ml and 5 mg/ml. After an initial duplicate analysis, one biological sample was stored for a period of 180 days, while the other was opened and analyzed after 60, 120 and 180 days. Total number of analysis of living person&#39;s blood samples was 500. The total number of analysis of autopsy biological samples was 360. All concentrations were determined by HS-GC method. During the storage, results showed that there has been a significant decrease in the concentration of ethanol in all of the analyzed samples, regardless of its origin. The level of this change was dependent on the type of tissue sample, initial alcohol concentration, duration of storage, integrity of the vials and stoppers, temperature, ratio of liquid and gas phases, presence of preservatives and intermittent opening for analysis.</p>

<p>Sprovedena studija po prvi put izve&scaron;tava o prisustvu patulina, sekundarnog metabolita određenih vrsta plesni, u proizvodima od jabuka, kao i proceni rizika usled unosa patulina od strane odojčadi, dece, adolescenata i odrasle populacije u Republici Srbiji. Ukupno 356 uzoraka sokova i ka&scaron;ica za odojčad i malu decu (48 i 66, redom), sokova za decu (mala pakovanja sa cevčicom, 100) i sokova u porodičnom pakovanju (142), sakupljeno je sa trži&scaron;ta tokom tri godine i analizirano primenom tečne hromatografije sa ultraljubičastom detekcijom, metodom koja je predhodno validirana. Prisustvo patulina je detektovano u 44% i 17% uzoraka sokova i ka&scaron;ica za odojčad i malu decu, redom, u količinama nižim od zakonskog ograničenja od 10 &mu;g/kg (maksimalno 8,3 i 7,7 &mu;g/kg, redom). Udeo kontaminiranih uzoraka među sokovima za decu iznosio je 43%, sa najvi&scaron;om koncentracijom patulina od 30,2 &mu;g/kg ispod maksimalno dozvoljenog nivoa od 50 &mu;g/kg. Patulin je detektovan u 51% sokova u porodičnom pakovanju, sa 0,7% uzoraka iznad zakonske granice od 50 &mu;g/kg (prosečna koncentracija 4,3 &mu;g/kg). Sokovi od jabuka su pokazali značajno vi&scaron;i udeo kontaminiranih uzoraka (74% u odnosu na 28%), kao i vi&scaron;i prosečni sadržaj patulina (6,4 u odnosu na 2,1 &mu;g/kg) u poređenju sa sokovima od me&scaron;anog voća. Procena rizika usled unosa patulina od strane odojčadi, dece, adolescenata i odrasle populacije u Republici Srbiji, sprovedena primenom determinističkog i probabilističkog pristupa, uključujući rezultate biodostupnosti patulina, pokazala je da je odnos između procenjenog dnevnog unosa i toksikolo&scaron;ke referentne doze za patulin od 0,4 &mu;g/kg telesne mase, koji se označava kao &ldquo;hazard quotient&rdquo;, znatno ispod 1, &scaron;to ukazuje na tolerantan nivo izloženosti i nepostojanje razloga za zabrinutost za zdravlje populacije. Dodatno, analiza ostataka pesticida i toksičnih metala potvrdila je bezbednost proizvoda od jabuka na trži&scaron;tu. Međutim, identifikacija vi&scaron;estrukih ostataka pesticida je razlog za aktivan pristup i pažljivo planiranje i sprovođenje monitoringa bezbednosti hrane, posebno hrane za odojčad i malu decu, kao najosetljivije populacione grupe.</p> / <p>This study reports for the first time the occurrence of patulin, a secondary metabolite of certain fungi, in apple-based food, as well as risk assessment related to patulin intake by infants, children, adolescents and adults in Serbia. In total, 356 samples of infant fruit juices (48), infant pur&eacute;e (66), juices for children (small package with straw, 100), and juices in family package (142) were collected from the market over three years (2013&ndash;15) and analysed using validated method based on liquid chromatography with ultraviolet detection. Patulin was found in 44% of infant juices and 17% of infant pur&eacute;e, with all values below the legal limit of 10 &mu;g/kg (maximum 8.3 and 7.7 &mu;g/kg, respectively). The proportion of contaminated samples among fruit juices for children was 43%, with the highest patulin concentration at 30.2 &mu;g/kg, not exceeding the maximum allowed level of 50 &mu;g/kg. Patulin was found in 51% of juices in family package, with 0.7% of the samples in excess of the legal limit of 50 &mu;g/kg (mean 4.3 &mu;g/kg). Apple juices showed significantly higher percentage of contaminated samples (74% versus 28%), as well as higher mean patulin content (6.4 versus 2.1 &mu;g/kg) when compared with the multifruit ones. Risk assessment of patulin intake by Serbian infants, children, adolescents and adults, conducted by deterministic and probabilistic approaches and including the bioaccessibility results, revealed a ratio between exposure and toxicological reference dose for patulin of 0,4 &mu;g/kg body weigth, called hazard quotients, well below 1, indicating a tolerable exposure level and no health concern. Furthermore, analysis of pesticide residues and toxic metals confirmed safety of apple products on the market. However, identification of multiple pesticide residues is a reason for an active attitude and carefully planned and conducted monitoring of food safety, expecialy in the case of food for infants and young children, as they are the most susceptible population group.</p>

<p>Doktorsku disertaciju čine tri celine kojima je zajednički element ispitivanje fenola, u pogledu njihovog sadržaja i dostupnosti u dijetetskim suplementima na bazi grožđa i japanskog troskota, ispitivanja uticaja suplementacije kod eksperimentalnih životinja, odnosno mogućnosti ekstrakcije fenolnih jedinjenja iz komine grožđa. Osnovni ciljevi prvog dela bili su ispitivanje bezbednosti i kvaliteta 14 suplemenata na bazi grožđa i japanskog troskota u pogledu ujednačenosti mase, sadržaja i brzine rastvorljivosti. U drugom delu je radi provere terapijske delotvornosti suplementa koji sadrži čist resveratrol ispitan njegov uticaj na glikemiju i lipidni status eksperimentalnih životinja. Treći deo je obuhvatio analizu pet različitih komina grožđa i razvoj metode ekstrakcije fenola iz odabrane komine kako bi se dobili ekstrakti sa &scaron;to je moguće većim prinosom, koristeći rastvarače koji su bezbedni po ljudsko zdravlje. Rezultati su pokazali da dva od četrnaest suplemenata nisu ispunila zahteve farmakopeje za lekove u pogledu ujednačenosti mase i sadržaja aktivne komponente, dok ni jedan suplement nije ispunio zahteve u pogledu brzine rastvorljivosti aktivne supstance, zbog čega se postavlja pitanje delotvornosti ispitanih suplemenata. Ni kod jednog suplementa nisu kvantifikovani ostaci pesticida ni toksični metali, čime je potvrđena njihova bezbednost. Farmakodinamska ispitivanja na pacovima pokazala su antioksidantna, anti-dijabetska i hipolipidemijska svojstva dijetetskog suplementa na bazi resveratrola, čime je potvrđena njegova delotvornost. Za potvrdu delotvornosti suplementa na ljudima neophodno je sprovesti dobro dizajnirano kliničko ispitivanje na dovoljnom broju ispitanika. Analiza pet različitih komina grožđa pokazala je da ovaj nusprodukt proizvodnje vina sadrži različite fenole, od kojih je samo katehin bio prisutan u značajnoj količini. Kvalitativan i kvantitativan sastav komine značajno varira zavisno od sorte i berbe grožđa, kao i od primenjenog tehnolo&scaron;kog procesa proizvodnje vina. Imajući u vidu sveobuhvatne kriterijume u pogledu efikasnosti ekstrakcije, zaključeno je da su 55% etanol, odnos uzorak / rastvarač 1:40, pH 4,5, T 55&deg;C i vreme od 30 min optimalni eksperimentalni uslovi za ekstrakciju fenola iz komine grožđa. U zavisnosti od osnovnog cilja procesa ekstrakcije ovi parametri se mogu lako modifikovati. Vi&scaron;estruke su mogućnosti za iskori&scaron;ćenje komine grožđa, a jedan od njih bi mogla biti i da se koristi kao sirovina za izradu dijetetskih suplemenata.</p> / <p>The dissertation consists of three parts with a common element &ndash; testing phenols in terms of content and availability in grape and Polygonum cuspidatum-based dietary supplements, testing the supplement&rsquo;s impact on experimental animals and the possibility to extract phenolic compounds from grape pomace. The primary objectives of the first part were testing safety and quality of fourteen grape and Polygonum cuspidatum-based supplements in terms of uniformity of mass, content and dissolution profile. In the second part, to test the therapeutic efficacy of a supplement containing pure resveratrol, its effect on the glycemia and lipid profile of experimental animals was examined. The third part covers the analysis of five different grape pomaces and the development of a phenol extraction method from the selected grape pomace to obtain extracts with the highest possible yield, using solvents that are safe for human health. The results showed that two out of fourteen supplements have not met the Pharmacopoeia requirements in terms of uniformity of mass and active substance content, while no supplement has met requirements in terms of dissolution test of the active substance, which raises the question of the effectiveness of the tested supplements. No pesticide or heavy metal residues were quantified in any of the supplements which confirms their safety. Pharmacodynamic testing on rats has shown antioxidant, antidiabetic and hypolipidemic properties of the resveratrol-based dietary supplement which confirms its efficacy. In order to confirm the supplement efficacy on people, it is necessary to conduct a well-designed clinical trial on a sufficient number of human participants. The analysis of five different grape pomaces revealed that this byproduct of vine production contains different phenols, and only catechin was present in significant amounts. The qualitative and quantitative composition of grape pomace varies significantly depending on grape variety and harvesting as well as on the technological process that was applied in vine production. Considering the comprehensive criteria in terms of extraction efficacy, it was concluded that optimal experimental conditions for the extraction of phenol from grape pomace include 55% ethanol, sample/solvent ratio 1:40, pH 4.5, T 55&deg;C for 30 minutes. Depending on the primary objective in the extraction process, these parameters can be easily modified. There are multiple possibilities for utilizing grape pomace and one of them could be as raw material in production of dietary supplements.</p>

<p>Emerging substances of concern (EmS) are wide groups of chemicals frequently used, that are not included in legislation nor mandatory monitored like priority and hazardous priority substances. Group of substances overlapping in priority and emerging substances list provided by legislation are Endocrine Disruptive Compounds (EDCs), referred to as emerging xenobiotics (EXs). The modules of emerging xenobiotics detection and identification presents an interactive, complex and dynamic process shown in the research. Analytical method, LLE followed by GC-MS was successfully adapted and applied to screening of emerging and priority substances in wastewater. The screening and target analyses results were evaluated via risk assessment (ERA) and detected substances chemical properties were statistically evaluated (PCA, HCA an PC) for the purpose of correlation of chemical properties and predicted treatment possibilities from wastewater.</p> / <p>Емергентне супстанце (ЕмС) припадају широј групи хемикалија које се фреквентно користе, а нису обухваћене законски прописаном мониторингу, као приоритетне и хазардне приоритетне супстанце. Емергентни ксенобиотици (ЕК), а посебно супстанце које ометају ендокрини систем (ЕД), су група једињења које припадају и приоритетним и емергентним супстанцама. Модули одређивања ксенобиотика су интерактиван, сложен и динамичан процес, подложан променама, детаљно описан у тези. Аналитичка метода, ТТЕ праћене ГЦ-МС, успешно је прилагођена за скрининг идентификацију емергентних и приоритетних супстанци у отпадној води. За евалуацију добијених резултата коришћене су статистичке методе (ПЦА, ХЦА и ПК) у сврху корелације хемијских карактеристика са предвиђеним третманима отпадних вода, као и процена ризика за животну средину.</p> / <p>Emergentne supstance (EmS) pripadaju široj grupi hemikalija koje se frekventno koriste, a nisu obuhvaćene zakonski propisanom monitoringu, kao prioritetne i hazardne prioritetne supstance. Emergentni ksenobiotici (EK), a posebno supstance koje ometaju endokrini sistem (ED), su grupa jedinjenja koje pripadaju i prioritetnim i emergentnim supstancama. Moduli određivanja ksenobiotika su interaktivan, složen i dinamičan proces, podložan promenama, detaljno opisan u tezi. Analitička metoda, TTE praćene GC-MS, uspešno je prilagođena za skrining identifikaciju emergentnih i prioritetnih supstanci u otpadnoj vodi. Za evaluaciju dobijenih rezultata korišćene su statističke metode (PCA, HCA i PK) u svrhu korelacije hemijskih karakteristika sa predviđenim tretmanima otpadnih voda, kao i procena rizika za životnu sredinu.</p>

<p>&Scaron;tavelj (Rumex crispus, Polygonaceae) je vi&scaron;egodi&scaron;nja zeljasta biljka, koja predstavlja bogat izvor fenolnih komponenti. Iako se smatra invazivnim korovom, mlado li&scaron;će &scaron;tavelja je jestivo i često se koristi kao salata. Dalje, upotreba plodova &scaron;tavelja opisana je u srpskoj i turskoj narodnoj medicini u lečenju gastrointestinalnih tegoba. Cilj ovog rada bio je procena in vitro i in vivo antioksidantne/prooksidantne i citotoksične aktivnosti, i određivanje eventualnog in vitro antiinflamatornog efekta vodenog ekstrakta ploda Rumex crispus. Ukupan sadržaj flavonoida određen je spektrofotometrijskom metodom. Kvalifikacija i kvantifikacija flavonoida potvrđena je visokoefikasnom tečnom hromatografijom (HPLC). Antioksidantna aktivnost vodenog ekstrakta ploda &scaron;tavelja procenjena je na osnovu in vitro testova: Ferric-reducing antioxidant power (FRAP), sposobnosti ekstrakta da neutrali&scaron;e slobodne radikale NO&bull;, OH&bull; i DPPH&bull; i uticaja na lipidnu peroksidaciju u lipozomima. Citotoksičnost ispitivanog ekstrakta je određena in vitro na tumorskim ćelijskim linijama: humani karcinom cerviksa (HeLa), adenokarcinom (HT-29) i adenokarcinom dojke (MCF7). Takođe, moguća in vivo hepatoprotektivna i antioksidantna svojstva ekstrakta određena su kod oksidativnog stresa izazvanog CCl4 kod eksperimentalnih životinja. Pored toga, proverena je hipoteza u kojoj testiran ekstrakt pokazuje in vivo antiproliferativnu aktivnost kod Ehrlich-ovih (EAC) i Hepatoma AS30D ćelija, merenjem zapremine ascitesa, procenta vijabilnih ćelija i nivoa nekoliko antioksidantnih enzima. Optimizovan in vitro test za određivanje potencijala inhibicije ciklooksigenaze-1 (COX-1) i 12-lipooksigenaze (12-LOX) preduzet je u svrhu procene antiinflamatornog efekta vodenog ekstrakta ploda R. crispus. HPLC analiza otkrila je da je mikvelianin najdominantniji flavonoidni konstituent ekstrakta. Testirani ekstrakt pokazao je potencijalnu antioksidantnu aktivnost rezultujući velikom moći u neutralizaciji slobodnih radikala, i sposobno&scaron;ću da smanji lipidnu peroksidaciju u lipozomima. Rezultati su ukazali na tkivno-selektivnu citotoksičnost ekstrakta ploda R. crispus in vitro. Najizraženija antitumorska aktivnost primećena je prema HeLa i MCF7 ćelijskim linijama. Podaci sugeri&scaron;u da bi se ispitivani ekstrakt mogao smatrati potencijalnim in vivo hepatoprotektivnim i antioksidantnim agensom, sprečavajući oksidativna o&scaron;tećenja jetre. S druge strane, pomenuti ekstrakt može pokazati in vivo prooksidantna svojstva, uzrokujući oksidativni stres u maligno transformisanim EAC i AS30D ćelijama i smanjujući zapreminu ascitesa i udeo vijabilnih ćelija, u poređenju sa kontrolnom grupom. Promene u aktivnosti antioksidantnih enzima su verovatno posledica indukovanog oksidativnog stresa u EAC i AS30D ćelijama, naročito kod pretretiranih životinja. Vodeni ekstrakt ploda &scaron;tavelja pokazao je COX-1, kao i 12-LOX inhibitornu aktivnost, navodeći da bi ispitivani ekstrakt mogao biti antiinflamatorni agens. Vodeni ekstrakt ploda R. crispus ima potencijalnu antioksidantnu, citotoksičnu i antiinflamatornu aktivnost. Ispoljavanje prooksidantnih svojstava predstavlja mogući mehanizam antiproliferativnog efekta ekstrakta.</p> / <p>Curly dock (Rumex crispus, Polygonaceae) is a wild perennial herbaceous plant, which products are described as a rich source of phenolic compounds. Apart from being considered a seriously invasive weed, young leaves of curly dock are edible and often used as salad. Furthermore, the use of its fruits has been described in Serbian and Turkish traditional medicine against stomach complaints. The objectives of this study were to evaluate in vitro and in vivo antioxidant/prooxidant and cytotoxic activities, and to determine an eventual in vitro anti-inflammatory effect of the aqueous extract of Rumex crispus fruits. Total flavonoid content was determined by spectrophotometric method. Qualification and quantification of flavonoids were confirmed using High performance liquid chromatography (HPLC). The aqueous extract of curly dock fruits was evaluated for its antioxidant activity by in vitro assays for Ferric-reducing antioxidant power (FRAP), NO&bull;, OH&bull; and DPPH&bull;-free radical scavenging activities and the influence on lipid peroxidation in liposomes. The cytotoxicity of tested extract was examined in vitro in human cervix carcinoma (HeLa), colon adenocarcinoma (HT-29) and breast adenocarcinoma (MCF7). Also, the potential in vivo hepatoprotective and antioxidant properties of investigated extract were determined on CCl4-induced oxidative stress in experimental animals. Furthermore, the hypothesis that the examined extract might show in vivo antiproliferative activity in Ehrlich carcinoma (EAC) and Hepatoma AS30D cells was tested by measuring volume of ascites, percentage of viable cells and level of several antioxidant enzymes. The optimized in vitro test for determination of cyclooxygenase-1 (COX-1) and 12-lipoxygenase (12-LOX) inhibition potency was undertaken in order to estimate an anti-inflammatory effect of aqueous extract of R. crispus fruits. HPLC analysis revealed miquelianin as the most abundant flavonoid constituent of the extract. The tested extract might have an antioxidant activity resulting in scavenging of free radicals and ability to decrease lipid peroxidation in liposomes. The results could indicate tissue-selective cytotoxicity of R. crispus fruit extract in vitro. The most prominent antitumor activity was observed towards HeLa and MCF7 cell lines. The data suggested that investigated extract may be considered as potential in vivo hepatoprotective and antioxidant agent due to prevention of the liver injuries induced by oxidative damage. On the other hand, mentioned extract could exhibit in vivo prooxidant property, causing the oxidative stress in malignant transformed EAC and AS30D cells and reducing volume of ascites and percentage of viable cells, in comparison with control group. Changes in activities of antioxidant enzymes might be the results of induced oxidative stress in EAC and AS30D cells, especially in the pretreated animals. The aqueous extract of curly dock fruits showed COX-1, as well as 12-LOX inhibitory activity, suggesting that tested extract might be an anti-inflammatory agent. It could be concluded that aqueous fruit extract of R. crispus might have antioxidant, cytotoxic and anti-inflammatory activities. The prooxidant properties of examined extract could be the mechanism of potential antiproliferative effect of extract.</p>

<p>Insekticidi novije generacije, neonikotinoidi, odlikuju se specifičnim načinom &nbsp;delovanja na&nbsp;nervni sistem insekata. Radi dobijanja &scaron;to brže i kvalitetnije informacije o izloženosti životne sredine&nbsp;ovim insekticidima i količinama njihovih ostataka u hrani potrebno je raspolagati odgovarajućim&nbsp;instrumentalnim metodama za njihovo određivanje. Razvijene su i optimizovane analitičke metode&nbsp;zasnovane na tečnoj hromatografiji za određivanje sedam odabranih neonikotinoida (dinotefurana,&nbsp;nitenpirama, tiametoksama, klotianidina, imidakloprida, acetamiprida &nbsp;i tiakloprida) u medu i likeru od&nbsp;meda. Ispitivana je mogućnost određivanja klotianidina pomoću tečne hromatografije visoke efikasnosti&nbsp;sa detektrorom od niza dioda (HPLC-DAD) primenom kombinacije tečno-tečne i ekstrakcije na čvrstoj&nbsp;fazi iz uzoraka meda. Na osnovu preliminarnih rezultata može se zaključiti da kori&scaron;ćenje &nbsp;faznih-čvrsto&nbsp;kolona u kombinaciji sa tečno-tečnom ekstrakcijom dihlormetanom rezultira prihvatljivim prinosom&nbsp;klotianidina u uzorcima meda pri koncentraciji od oko 0,5 &micro;g g^-1&nbsp;klotianidina. Radi dobijanja većih&nbsp;prinosa odabrana je disperzna tečno-tečna mikroekstrakcija (DLLME) kao tehnika pripreme uzoraka&nbsp;meda. Testirana je upotreba acetonitrila kao disperznog sredstva. Pored hloroforma, kori&scaron;ćen je i&nbsp;dihlormetan kao drugo ekstrakciono sredstvo, kako bi se &nbsp;uporedila efikasnost ekstrakcije. Zabeleženi su&nbsp;prinosi klotianidina od 69,7 i 68,3% &nbsp;u zavisnosti da li je kori&scaron;ćen hloroform, odnosno DHM kao rastvor&nbsp;za ekstrakciju. Može se zaključiti da je prinos ekstrakcije bio povoljniji pri odnosu 0,5 mL ACN i 2,0&nbsp;mL DHM. Prinosi su se kretali od 68,4% do 92,1%, &scaron;to je ukazalo da su parametri DLLME ekstrakcije optimalni. Kako bi se detaljnije ispitali ključni parametri DLLME tehnike, kori&scaron;ćena je metodologija povr&scaron;ine odziva (RSM), kao i detekcija na osetljivijem kuplovanom masenom detektoru (MS/MS). Optimizovani su HPLC-MS/MS &nbsp;parametri kako bi se obezbedilo zadovoljavajuće hromatografsko &nbsp;razdvajanje i niske granice detekcije (GD, 0,5&ndash;1,0 &mu;g kg^-1) i određivanja (GO, 1,0&ndash;2,5 &mu;g kg^-1) ispitivanih neonikotinoida u medu. Upotrebom centralno kompozitnog dizajna konstruisani su kvadratni modeli ispitivanih faktora: zapremine ekstrakcionog (DHM, 1,0&ndash;3,0 mL) i disperznog (ACN, 0,0&ndash;1,0 mL) sredstva, izračunati statistički parametri i optimizovan proces DLLME upotrebom <em>Derringer</em>-ove funkcije poželjnih odgovora. Upotrebom MMC i SC krivih u opsegu GO&ndash;100,0 &mu;g kg^-1&nbsp;ispitan je uticaj matriksa pri čemu zaključeno je da je najveći uticaj matriksa bio na odziv analitičkog signala nitenpirama, dinotefurana i klotianidina. Ispitani su prinosi odabranih neonikotinoida (R, 74,3&ndash;113,9%), kao i preciznost metode u uslovima ponovljivosti (RSD, 2,74&ndash; 11,8%) i intermedijerne reproduktivnosti (RSD, 6,64&ndash;16,2%). Brza (retenciona vremena 1,5&ndash;9,9 min) i osetljiva metoda, koja tro&scaron;i malu količinu rastvarača, primenjena je za ispitivanje 15 realnih uzoraka meda različitog cvetnog porekla. Rezultati su pokazali da ispitivani med nije sadržao ostatke ispitivanih neonikotinoida u koncentracijama iznad GD. Dalje istraživanje je bilo usmereno ka&nbsp; razvijanju i optimizaciji HPLC-DAD analitičke metode upotrebom DLLME i QuEChERS tehnika za &nbsp;pripremu uzoraka za određivanje 7 neonikotinoida u uzorcima meda. U ovom delu istraživanja optimizovani su i hromatografski parametri, upotrebom RSM sa Box-Behnken-ovim dizajnom i Derringer-ovom funkcijom poželjnih odgovora. Od &nbsp;ispitivanih neonikotinoida dinotefuran i imidakloprid su bili u najvećoj meri izloženi uticaju matriksa, bez obzira na proceduru pripreme uzoraka. Može se istaći da je uticaj matriksa na analitički signal dinotefurana bio izraženiji u slučaju MS/MS, apostrofirajući manju robusnost ove metode određivanja. Prinosi neonikotinoida su &nbsp;bili (R, 73,1&ndash;118,3%), preciznost u uslovima ponovljivosti (RSD, 3,28&ndash;10,40%) i intermedijerne reproduktivnosti (RSD, 6,45&ndash;17,70%), a granice detekcije (GD, 1,5&ndash;2,5 &micro;g kg^-1) i određivanja (GO, 5,0&ndash;10,0 &micro;g kg^-1). Metoda je primenjena za ispitivanje 7 neonikotinoida u 104 uzorkameda različitog cvetnog porekla sa &nbsp;teritorije Autonomne Pokrajine Vojvodine. Detektovano je prisustvo tiakloprida, imidakloprida i tiametoksama u količinama koje su bile ispod MDK RS i EU. Analizirani su uzorci likera od meda - medice. Upoređivane su dve tehnike pripreme uzoraka, DLLME i QuEChERS i primenjeni optimizovani hromatografski &nbsp;uslovi i MS/MS parametri. U slučaju nitenpirama, dinotefurana i tiametoksama uticaj matriksa bio je najizraženiji. Metoda je validovana određivanjem prinosa neonikotinoida (R, 69,2&ndash;113,4%), preciznosti u uslovima ponovljivosti (RSD, 3,21&ndash;12,81%) i intermedijerne reproduktivnosti (RSD, 9,11&ndash;16,63%), kao i granice detekcije (GD, 0,5&ndash;2,5 &micro;g kg^-1) i određivanja (GO, 1,0&ndash;10,0 &micro;g kg^-1). Analizom 10 komercijalno dostupnih likera od meda otkriveno je&nbsp;prisustvo klotianidina i tiakloprida, evčzokinot&scaron; z&nbsp; na neophodnost daljeg kontrolisanja ovog proizvoda na&nbsp;prisustvo neonikotinoida. Ispitana je mogućnost uklanjanja odabranih neonikotinoida (dinotefurana,&nbsp;klotianidina i tiakloprida) iz vodene sredine (reke Dunav). Ispitivanje efikasnosti 6 različitih vrsta&nbsp;uklanjanja odabranih neonikotinoida (u prisustvu prirodne insolacije u laboratorijskim uslovima, sa&nbsp;dodatkom H2O2, sa dodatkom MWCNT, sa dodatkom MWCN+H&nbsp;₂O₂, sa dodatkom Fe-MWCNT, sa dodatkom Fe-MWCNT+H₂O₂) vr&scaron;eno je upotrebom prethodno razvijene HPLC-MS/MS metode. Krive uklanjanja odabranih neonikotinoida, pokazale su da tokom 60 minuta pri prirodnoj insolaciji&nbsp; u laboratorijskim uslovima koncentracija smanjenje oko 25%. Analitički signal dinotefurana dobijen u prisustvu H₂O_2&nbsp;pod istim uslovima ukazuje na uklanjanje ciljnog analita od oko 40%, tiakloprida od oko 70%, a klotianidina u potpunosti. Testirana je adsorpcija ciljnog analita na vi&scaron;ezidnim ugljeničnim nanocevima (MWCNT). Ovim postupkom može da se ukloni oko 30% dinotefurana, oko 50% klotianidina i 60% tiakloprida. U kombinaciji sa H₂O_2&nbsp;, MWCNT pokazuju bolju sposobnost uklanjanja za 15&ndash;50% u zavisnosti od ispitivanog neonikotinoida. Upotreba Fe-MWCNT i njihova kombinacija sa H₂O₂ otvorila je mogućnost za dalja &nbsp;ispitivanja mehanizma uklanjanja. Ustanovljeno je nastajanje intermedijera kojima odgovaraju m/z od 117,5 i 140,6 u slučaju razgradnje dinotefurana u sistemima sa H₂O₂, MWCNT+H₂O₂, Fe-MWCNT+H₂O_2&nbsp;i klotianidina u sistemu Fe-MWCNT+H₂O₂.</p> / <p>Neonicotinoid insecticides, as one of the fastest growing new generation of insecticides, have&nbsp;contributed to a significant reduction of toxicity for the environment; therefore, monitoring and&nbsp;determination of trace levels of the neonicotinoids in honey are necessary and demands highly efficient,&nbsp;selective and sensitive analytical techniques. The objective of the present work was to develop a rapid,&nbsp;sensitive, optimized and accurate analytical method based on liquid chromatography for determining&nbsp;seven neonicotinoid insecticides, dinotefuran, nitenpyram, thiamethoxam, clothianidin, imidacloprid,&nbsp;acetamiprid and thiacloprid in honey and honey liqueur samples. The possibility for determination of&nbsp;clothianidin in honey samples was investigated by HPLC with a diode array detector (HPLC-DAD).&nbsp;Based on preliminary results, it can be concluded that the use of a solid-phase column in combination&nbsp;with a liquid-liquid extraction with dichloromethane results in an acceptable recovery of clothianidin in&nbsp;the samples with a clothianidin concentration of about 0.5 &micro;g g^-1. After obtaining low recovery of&nbsp;clothianidin, dispersed liquid-liquid microextraction (DLLME) was selected as a technique for the&nbsp;preparation of honey samples.. The adequacy of acetonitrile as a dispersing agent was investigated.&nbsp;Besides the chloroform, a dichloromethane was used as a second extracting agent , in order to compare&nbsp;the relative efficiency of the extraction solvents. It can be concluded that the extraction recovery (68.4&ndash;92.1%) was more favorable with the use of 0.5 mL ACN and 2.0 mL DHM. Furthermore, LC-MS/MS&nbsp;parameters were optimized to unequivocally provide good chromatographic separation, low detection&nbsp;(LOD, 0.5&ndash;1.0 &mu;g L^&minus;1) and quantification (LOQ, 1.0&ndash;2.5 &mu;g L^&minus;1) limits for acetamiprid, clothianidin,&nbsp;thiamethoxam, imidacloprid, dinotefuran, thiacloprid and nitenpyram in honey samples. Using different&nbsp;<br />types (chloroform, dichloromethane) and volumes of extraction (1.0&ndash;3.0 mL) and dispersive&nbsp;(acetonitrile; 0.0&ndash;1.0 mL) solvent and by mathematical modeling it was possible to establish the optimal&nbsp;sample preparation procedure. Matrix-matched calibration and blank honey sample spiked in the&nbsp;<span style="font-size: 12px;">concentration range of LOQ&ndash;100.0 &mu;g kg</span>^{<span style="font-size: 12px;">&minus;1&nbsp;</span>}<span style="font-size: 12px;">were used to compensate the matrix effect and to fulfill the&nbsp;</span><span style="font-size: 12px;">requirements of SANCO/12495/2011 for the accuracy (R 74.3&ndash;113.9%) and precision (expressed in&nbsp;</span><span style="font-size: 12px;">terms of repeatability (RSD 2.74&ndash;11.8%) and within-laboratory reproducibility (RSDs &nbsp;6.64&ndash;16.2%)) of&nbsp;</span><span style="font-size: 12px;">the proposed method. The rapid (retention times 1.5&ndash;9.9 min), sensitive and low solvent consumption&nbsp;</span><span style="font-size: 12px;">procedure described in this work provides reliable, simultaneous, and quantitative method applicable for&nbsp;</span><span style="font-size: 12px;">the routine laboratory analysis of seven neonicotinoid residues in 15 real honey samples. Neonicotinoid &nbsp;</span><span style="font-size: 12px;">residues were not detected in any of the investigated samples. The objective of next study was to&nbsp;</span><span style="font-size: 12px;">develop and optimize HPLC-DAD analytical method with dispersive liquid-liquid microextraction&nbsp;</span><span style="font-size: 12px;">(DLLME) and QuEChERS sample preparation procedures for the simultaneously analysis of seven&nbsp;</span><span style="font-size: 12px;">neonicotinoids in honey samples. The liquid chromatographic conditions were optimized by response&nbsp;</span><span style="font-size: 12px;">surface methodology with <em>Box-Behnken</em> design and the global <em>Derringer</em>&acute;s desirability. The optimized&nbsp;</span><span style="font-size: 12px;">method was &nbsp;validated to fulfill the requirements of SANCO/12495/2011 standard for both sample&nbsp;</span><span style="font-size: 12px;">pretreatment procedures providing results for accuracy (R, 73.1&ndash;118.3%), repeatability (RSD, 3.28&ndash;</span><span style="font-size: 12px;">10.40%) and within-laboratory reproducibility (RSD, 6.45&ndash;17.70%), limits of detection (LOD, 1.5&ndash;2.5&nbsp;</span><span style="font-size: 12px;">g&micro; kg</span>^{<span style="font-size: 12px;">-1</span>}<span style="font-size: 12px;">) and quantification (LOQ, 5.0&ndash;10.0 &micro;g kg</span>^{<span style="font-size: 12px;">-1</span>}<span style="font-size: 12px;">). For the first time, more than 100 honey samples&nbsp;</span><span style="font-size: 12px;">collected from all 7 counties of Autonomous Province of Vojvodina were analyzed. The presence of&nbsp;</span><span style="font-size: 12px;">thiacloprid, imidacloprid and thiametoxam was discovered in a small number of samples. The objective&nbsp;</span><span style="font-size: 12px;">of next study was to develop an optimized LC-MS/MS analytical method with DLLME and QuEChERS&nbsp;</span><span style="font-size: 12px;">procedures for analysis of 7 neonicotinoids in honey liqueur. The method was validated to fulfill the&nbsp;</span><span style="font-size: 12px;">requirements of SANCO/12495/2011 for both sample pretreatment procedures providing results for&nbsp;</span><span style="font-size: 12px;">accuracy (R, 69.2&ndash;113.4% for DLLME; 71.8&ndash;94.9% for QuEChERS), precision (RSD expressed in&nbsp;</span><span style="font-size: 12px;">terms of repeatability (3.21&ndash;10.20% for DLLME; 4.19&ndash;12.81% for QuEChERS) and within-laboratory&nbsp;</span><span style="font-size: 12px;">reproducibility (9.11&ndash;16.63% for DLLME; 11.32&ndash;16.40% for QuEChERS)), limits of detection (LOD,&nbsp;</span><span style="font-size: 12px;">0.5&ndash;1.5 g&micro; L</span>^{<span style="font-size: 12px;">-1&nbsp;</span>}<span style="font-size: 12px;">for DLLME; 1.0&ndash;2.5 g&micro; L</span>^{<span style="font-size: 12px;">-1&nbsp;</span>}<span style="font-size: 12px;">for QuEChERS) and quantification (LOQ, 1.0&ndash;5.0 g&micro; L</span>^{<span style="font-size: 12px;">-1&nbsp;</span>}<span style="font-size: 12px;">for&nbsp;</span><span style="font-size: 12px;">DLLME; 2.5&ndash;10.0 &micro;g L</span>^{<span style="font-size: 12px;">-1&nbsp;</span>}<span style="font-size: 12px;">for QuEChERS). Analysis of real honey liqueur samples obtained from local&nbsp;</span><span style="font-size: 12px;">markets showed the presence of clothianidin or thiacloprid in four of the analyzed samples, therefore&nbsp;</span><span style="font-size: 12px;">implicating the necessity of ongoing control of this type of traditional product. &nbsp;Removal of selected&nbsp;</span><span style="font-size: 12px;">neonicitinoid insecticides - dinotefuran, clothianidin and thiacloprid using MWCNT and H</span><span style="font-size: 12px;">₂O_2&nbsp;</span><span style="font-size: 12px;">from&nbsp;</span><span style="font-size: 12px;">Danube water matrix was investigated. &nbsp;Efficiency of different systems for neonicotinoids removal&nbsp;</span><span style="font-size: 12px;">(under natural insolation in laboratory, with H</span><span style="font-size: 12px;">₂O₂, with MWCNT, with MWCNT+ H</span><span style="font-size: 12px;">₂O₂, with Fe-MWCNT, with Fe-MWCNT+H₂O₂) was evaluated with developed LC-MS/MS method. Analysis of&nbsp;</span><span style="font-size: 12px;">degradation rates revealed loss of 25% of the initial neonicotinoid concentration under natural insolation in&nbsp;</span><span style="font-size: 12px;">the laboratory conditions during 60 min. Addition of chemical agent H₂O_2&nbsp;promoted loss of 40% of the&nbsp;</span><span style="font-size: 12px;">initial dinotefuran, 70% of thiacloprid concentration and total removal of clothianidin under same&nbsp;</span><span style="font-size: 12px;">conditions. With the addition &nbsp;of MWCNT concentration of dinotefuran, clothianidin and thiacloprid&nbsp;</span><span style="font-size: 12px;">decayed for 30, 50 and 60%, respectively. Iron modification of MWCNT in combination with H</span><span style="font-size: 12px;">₂O_2&nbsp;</span><span style="font-size: 12px;">increased the removal rate of selected neonicotinoid for 15&ndash;50%. Presence of intermediates was&nbsp;</span><span style="font-size: 12px;">discovered in systems of dinotefuran with H₂O₂, MWCNT+H</span><span style="font-size: 12px;">₂O₂, e-MWCNT+H₂O_2&nbsp;</span><span style="font-size: 12px;">and of&nbsp;</span><span style="font-size: 12px;">clothianidin in systems with Fe-MWCNT+H₂O_2&nbsp;</span><span style="font-size: 12px;">with m/z of 117,5 and 140,6.&nbsp;</span></p>

<p>Ricin je prirodni protein, toksin koji spada među najpristupačnije i najsmrtonosnije otrove. Nalazi se u biljci Ricinus (Ricinus communis), sa najvećim sadržajem u semenu (1-5 %). Ricin se smatra potencijalnim bioterorističkim oružjem i prema riziku za ljudsko zdravlje svrstan je u B kategoriju biolo&scaron;kog oružja. U novije vreme kori&scaron;ćen je za konstruisanje imunotoksina protiv tumorskih ćelija u terapiji maligniteta. Dokumentovana su mnoga trovanja ricinom, kako zadesna, tako i samoubilačka i ubilačka. U tu svrhu koristilo se intaktno seme ricinusa ili ekstrahovani ricin. Osim ricina, u semenu ricinusa je prisutan toksični alkaloid ricinin u količini 0.3-0.8 %. Ricinus je jedini poznati prirodni izvor ricinina, koji se ko-ekstrahuje sa ricinom iz semena biljke. Ricinin se jednostavno detektuje u kliničkim uzoracima metodom tečne hromatografije i masene spektrometrije i, s obzirom na komplikovanu identifikaciju ricina u biolo&scaron;kim uzorcima, smatra se biomarkerom za intoksikaciju ricinusom, odnosno ricinom. Osnovni ciljevi ovog istraživanja su da se uz pomoć HS-GC metode i patohistolo&scaron;kom&nbsp; analizom dokaže prisustvo ricinina u krvi laboratorijskih pacova u odnosu na vremenski interval koji je protekao od oralne aplikacije suspenzije do vremena žtvovanja, da se odredi distribucija i koncentracija ricinina u organima laboratorijskih pacova u različitim vremenima žrtvovanja, kao i da se utvrdi da li postoji značajna razlika u razvoju patomorfolo&scaron;kih promena na organima laboratorijskih pacova u različitim vremenima žrtvovanja. Istraživanje je bilo otvoreno, randomizirano i prospektivnog tipa. Laboratorijski pacovi su u istom vremenu oralno tretirani suspenzijom koja je sadržaja subletalnu koncentraciju ricina. Nakon žrtvovanja u precizno definisanim vremenskim intervalima uzeti su uzorci krvi i unutra&scaron;njih organa radi daljih analiza. Odgovarajući uzorci su analizirani metodom HC-GS u cilju određivanja koncentracije i distribucije ricinina, kao pouzdanog markera trovanja ricinom, u krvi i unutra&scaron;njim organima. Takođe je izvr&scaron;ena patohistolo&scaron;ka analiza uzoraka tkiva unutra&scaron;njih organa u cilju utvrđivanja promena izazvanim delovanjem ricina u odnosu na vreme proteklo od aplikacije suspenzije. Dobijeni rezultati su obrađeni odgovarajućim statističkim metodama. Rezultati istraživanja omogućavaju standardizaciju postupaka odabira reprezentativnih uzoraka prilikom sumnje na trovanje ricinusom i metode dokazivanja akutnog trovanja. Na taj način može se pouzdano i efikasno dokazati trovanje ricinusom.</p> / <p>Ricin is a naturally occurring protein, a toxin which belongs to the category of the most accessible and the most lethal poisons. It is obtained from the castor oil plant ( Ricinus communis), whose seeds contain its highest content (1-5%). Ricin is also thought to be a potential weapon of bioterrorism and taking into account the risk for human health, it is classified as a biological weapon category B. Lately it has been used for the construction of the immunotoxins against tumor cells in the therapy of malignant diseases. Numerous poisonings using ricin have been documented, not only accidental poisoning, but also in case of suicides and homicides. In those cases, intact ricin seeds or extracted ricin were used. Apart from ricin, castor oil plants also contain a toxic alkaloid ricinine (0.3-0.8%). Castor oil plants are the only known natural source of ricinine, which is co-extracted with ricin from the seeds of this plant. Ricinine is simply detected in clinical samples by using the method of liquid chromatography and mass spectrometry. Taking into account a complicated identification of ricin in biological samples, it is considered to be a biomarker for the intoxication by castor oil plant, or ricin itself. The main aim of this research is to use the HS-GC method and pathohistological analysis in proving the existence of ricinine in the blood of experimental rats in relation to the time interval between the oral application of solution of castor seeds in water and the time of sacrificing, to determine the distribution and concentration of ricinine in the organs of experimental rats, as well as to establish whether there was a significant difference in the development of pathomorphological changes on the organs of experimental rats at various points of sacrificing. The research was open, randomised and prospective. Experimental rats were simultaneously orally tested by the solution which contained sublethal concentration of ricin. After sacrificing, blood samples were taken from inner organs in specifically defined intervals of time and used for further analysis. The appropriate samples were analysed by HC-GS method in order to determine the concentration and distribution of ricinine as a reliable marker of ricin poisoning in blood and inner organs. Also, pathohistological analysis of the samples of inner organ tissues was made with the purpose of establishing the changes caused by the effects of ricinine in relation to time which passed from the application of the solution. The obtained results were processed by appropriate statistical methods. The results of this research allow for the standardisation of the actions in selecting the representative samples in case there is a possibility of ricin poisoning and the method of proving the acute poisoning. Following these steps, ricin poisoning can be proved in a reliable and an efficient way.</p>