<p>U okviru doktorske disertacije razvijena je brza i selektivna metoda za<br />određivanje i praćenje ostataka herbicida na bazi sulfonilurea (nikosulfuron,<br />oksasulfuron, tribenuron metil, tritosulfuron, triasulfuron, rimsulfuron i<br />prosulfuron) u zemlji&scaron;tu primenom visoko pritisne tečne hromatografije sa<br />UV detektor sa nizom fotoosetljivih dioda (HPLC-UV-DAD). Razdvajanje<br />sulfonilurea je izvedeno uz primenu Zorbax Eclipse XDB-C18 kolone. U cilju<br />definisanja odgovarajućih uslova analize, ispitan je i definisan uticaj<br />najznačajnijih eksperimentalnih faktora. Usvojeni su sledeći optimalni uslovi<br />gradijentne elucije: mobilna faza acetonitril (A) i 0,1% sirćetna kiselina (B) t<br />= 0 min, 52% A; t = 2-2,5 min, 47% A; t = 2,5-5 min, 52% A, pri konstatnom<br />protoku od 1 mL min-1 i temperaturi kolone od 250C. Na osnovu odziva<br />analitičkog signala, njegove reproduktivnosti i linearnosti, talasna dužina od<br />240 nm je usvojena kao odgovarajuća. Utvrđeno je da je uticaj matriksa bio<br />izražen te je stoga za kvantitativno određivanje sadržaja SU u uzorcima<br />zemlji&scaron;ta primenom HPLC-UV-DAD metode kori&scaron;ćena MMC kalibraciona<br />kriva. Pri analizi ekstrakata nekontaminiranog zemlji&scaron;ta ostvarena je granica<br />kvantitativnog određivanja za nikosulfuron, oksasulfuron, tribenuron metil,<br />tritosulfuron, triasulfuron, rimsulfuron i prosulfuron od 3,16 &mu;g kg-1, 3,40 &mu;g<br />kg-1, 3,76 &mu;g kg-1, 4,13 &mu;g kg-1, 3,60 &mu;g kg-1, 3,04 &mu;g kg-1 i 2,97 &mu;g kg-1,<br />redom. Za potrebe osetljivijih, selektivnijih i tačnijih određivanja SU u<br />zemlji&scaron;tu, definisana je metoda uz primenu tečne hromatografije sa<br />tandemskom masenom spektrometrijom (HPLC-MS-MS). HPLC-MS-MS je<br />kori&scaron;ćena kao referntna tehnika za proveru rezultata koji su dobijeni<br />primenom HPLC uz UV-DAD detektor. Definisan je efikasan postupak za<br />pripremu uzoraka zemlji&scaron;ta u cilju hromatografskog određivanja SU.<br />Najefikasnija ekstrakcija ostvarena je primenom sme&scaron;e dihlormetanacetonitril<br />(2:1, v/v), zaki&scaron;eljene sirćetnom kiselinom (0,8%, v/v), uz dodatak<br />uree (0,3 g/ 10g zemlji&scaron;ta). Postupak mikrotalasne estrakcije u zatvorenom<br />sistemu omogućio je najbolje uslove za ekstrakciju SU. U cilju izdvajanja SU,<br />iz matriksa ekstrakata zemlji&scaron;ta, primenjena je ekstrakcija na čvrstoj fazi (SPE) sa silika gelom kao adsorbensom. Definisana metoda za određivanje<br />ispitivanih SU, kao i razvijeni postupak pripreme uzoraka, uspe&scaron;no je<br />primenjen u analizi realnih uzoraka zemlji&scaron;ta. Veoma dobre vrednosti testa<br />povrata ostvarene pri analizi primenom HPLC-UV-DAD metode, potvrdile su<br />pre svega ispravnost postupka pripreme uzoraka, a delom i kompletne<br />metode. Tačnost razvijene metode za određivanje SU u zemlji&scaron;tu potvrđena je<br />paralelnim analizama uzoraka zemlji&scaron;ta kori&scaron;ćenjem HPLC-MS-MS.<br />Primenom Studentovog t-testa (upareni), utvrđeno je da se rezultati paralelnih<br />analiza statistički značajno ne razlikuju (P=95%). Istraživanja u okviru<br />disertacije obuhvatila su i praćenje disipacije nikosulfurona, rimsulfurona,<br />oksasulfurona i prosufurona u različitim komercijalnim dozama primene<br />tokom 50 dana u polju na tri različite dubine (0-15 cm, 15-30 cm i 30-45 cm),<br />kao i laboratorijskim uslovima na dve temperature (250C i 300C). Vreme<br />polurazgradnje (DT50) ispitivanih SU (nikosulfuron, oksasulfuron i<br />prosulfuron) definisano Mitttag-Lefler-ovom funkcijom u ogledima u<br />laboratorijskim uslovima na 250C iznosilo je 0,65 dana (50 g ha-1 a.k.), 0,64<br />dana (160 g ha-1 a.k.) i 0,66 dana (30 g ha-1 a.k.), redom, a na 300C za<br />nikosulfuron 0,20 dana (50 g ha-1 a.k.) i prosulfuron 0,49 dana (50 g ha-1 a.k.).<br />DT50 za ispitivane SU u polju, takođe definisano Mitttag-Lefler-ovom<br />funkcijom, iznosilo je 72 minuta (0,05 dana), 0,23 dana, 0,12 dana i 0,15<br />dana, za nikosulfuron, rimsulfuron, oksasulfuron i prosulfuron, redom.<br />Značajno veća disipacija koja je utvrđena istraživanjima u okviru ove<br />disertacije najverovatnije je uzrokovana uticajem abiotičkih i biotičkih<br />procesa, kao i faktora spolja&scaron;nje sredine.</p> / <p>In this thesis a fast and selective method for determination and monitoring<br />of residues sulfonylurea herbicides (SU) (nicosulfuron, oxasulfuron,<br />tribenuron methyl, tritosulfuron, triasulfuron, rimsulfuron and prosulfuron)<br />in soil, by applying high-performance liquid chromatography with UV<br />detector with diode array diodes (HPLC-UV-DAD) was developed. The<br />separation of the sulfonylurea was performed by use of a Zorbax Eclipse<br />XDB-C18 column. In order to define appropriate conditions of analysis,<br />the influence of the most significant the experimental factors was<br />investigated and defined. The following optimal conditions for gradient<br />elution: mobile phase acetonitrile (A) and 0.1% acetic acid (B) t = 0 min,<br />52% A; t = 2-2.5 min, 47% A; t = 2.5-5 min, 52% A were adopted at a<br />constant flow rate of 1 mL min-1 and column temperature of 250C. On<br />basis of the response of the analytical signal, its reproducibility and<br />linearity, the wavelength of 240 nm was adopted as an appropriate. The<br />influence of the matrix was expressed and therefore for the quantitative<br />determination of investigated SU in soil samples application of HPLCUV-<br />DAD methods was used MMC calibration curve. In the analysis of<br />extracts of nocontaminated soil limits of quantitation for nicosulfuron,<br />oxasulfuron, tribenuron methyl, tritosulfuron, triasulfuron, rimsulfuron and<br />prosulfuron was achieved 3.16 &mu;g kg-1, 3.40 &mu;g kg-1, 3.76 &mu;g kg-1, 4.13 &mu;g<br />kg-1, 3.60 &mu;g kg-1, 3.04 &mu;g kg-1 and 2.97 &mu;g kg-1, respectively. The method<br />by using liquid chromatography-tandem mass spectrometry (HPLC-MSMS)<br />was defined in order to find more sensitive, more selective and more<br />accurate way to determine concentration SU in soil. HPLC-MS-MS was<br />used as the reference technique for checking results that have been<br />obtained by HPLC with UV-DAD detector. An efficient procedure<br />preparation of soil samples for the purpose chromatographic determination<br />of SU was defined. Using a mixture of dichloromethane-acetonitrile (2: 1,<br />v / v), acidified with acetic acid (0.8%, v / v), with the addition of urea (0.3<br />g / 10 g soil) was applied as the most effective way for extraction SU.<br />Microwave extraction process in a closed system allowed the best<br />conditions for the extraction of SU. In order to extract SU from matrix<br />exctracts soil the solid phase the extraction (SPE) with silica gel as an<br />adsorbent was used. The defined method for determination of investigated SU, as well as the developed procedure of sample preparation, was<br />successfully applied in the analysis of real samples of soil. The correctness<br />of procedure of sample preparation was confirmed with very good values<br />of recovery test which was applied in the analysis by using HPLC-UVDAD.<br />The accuracy of the developed method for determination of SU was<br />confirmed by coupled analysis soil samples using HPLC-MS-MS. The<br />results of parallel analysis does not differ significantly at 95% confidence<br />level. It was obtained using Student t-test (paired). The method developed<br />in this thesis was used for monitoring the dissipation of nicosulfuron,<br />rimsulfuron, oxasulfuron prosufuron, in different commercial doses of<br />application during 50 days in field at three different depths (0-15 cm, 15-<br />30 cm and 30-45 cm) and for laboratory conditions at two temperatures<br />(25&ordm;C and 30&ordm;C). The half-life time (DT50) of investigated SU were<br />calculated by use of Mitttag-Lefler function in experiments under<br />laboratory conditions at 25&ordm;C. The values of DT50 for nicosulfuron,<br />prosufuron and oxasulfuron read: 0.65 days (50 g ha-1 a.k.), 0.64 days (160<br />g ha-1 a.k.) and 0.66 days (30 g ha-1 a.k.), respectively. In the same type of<br />experiments at 30&ordm;C we get DT50 for nicosulfuron equals 0.20 days (50 g<br />ha-1 a.k.) and for prosulfuron equals 0.49 days (50 g ha-1 a.k.). The values<br />of DT50 on experiments in field were obtain also by use of Mitttag-Lefler&#39;s<br />function, and half-life times for nicosulfuron, rimsulfuron, oxasulfuron and<br />prosufuron from the commercially recommended doses, equal 72 minutes<br />(0.05 days), 0.23 days, 0.12 days and 0.15 days, respectively. Significantly<br />greater dissipation which was determined in this thesis was probably<br />caused by the influence of abiotic and biotic processes as well as<br />environmental factors.</p>

Beyond the expected DBP exposure from drinking water, dermal from bathing, and inhalation, occurrence in food and beverage items can provide an additional occurrence pathway. Synthetic dyes are often added to beverages for aesthetic purposes and many are in the form of a reactive azo (-N=N-) dye or triarylmethane dye, both with a characteristic aromatic ring. The presence of dyes in beverages that are reconstituted with disinfected tap water pose the risk of reactions with the residual chlorine in the drinking water resulting in decolorization, and of greater concern, disinfection byproduct formation. Additionally, oral over-the-counter (OTC) medications contain chemical constituents that when reconstituted with tap water present a risk of DBP formation. Several studies were performed to evaluate the kinetic decay rates of the dyes and drugs in disinfectants, and the effects of water quality conditions on DBP formation. Commercial beverage products and OTC medications were evaluated for the DBPs that were detected in the free chlorine-treated precursor samples. The dye and drugs precursors followed second order kinetics, with the fastest rates for brilliant blue and phenylephrine in chlorinated water. The effects of water properties on precursor degradation and DBP formation was complex due to the influence of characteristics of precursor molecules. The cytotoxic and anti-estrogenic responses were measured in the dye and drug precursors and their respective beverages and OTC medications, to determine potential links. Mio Energy showed estrogenic character and Alka Seltzer induced an anti-estrogenic and cytotoxic response, however there were no clear linkages between the beverage/ medication and their respective dye and drug precursors.

This study had a twofold purpose. First, the usefulness of the simplex algorithm as a short method of optimization in high performance liquid chromatographic separations was investigated. The second was to test a modified simplex method. The volume fractions of mobile phase components were chosen as the variable factors in the optimization process. Four test cases were performed which included separation of cholesterol esters, naphthalene and its derivatives, polycyclic aromatic compounds, and the thiane compounds. The standard for accepting an optimum was based on the baseline separation of two adjacent peaks and the analysis time. In addition to successful separations, the correlation between the separation and the chemical characteristics of mobile phase compositions was calculated and this could then be used for further modification of simplex search strategy.

This research project sought to explore, compare and develop chemometric methods with the goal of resolving chromatographically overlapped peaks though the use of spectral information gained from the four-way data sets associated with comprehensive two-dimensional liquid chromatography with diode array detection (LC ´ LC-DAD). A chemometric method combining iterative key set factor analysis (IKSFA) and multivariate curve resolution-alternating least squares (MCR-ALS) was developed. In the section of urine data analyzed, over 50 peaks were found, with 18 visually observable and 32 additional compounds found only after application of the chemometric method. Upon successful chemometric resolution of chromatographically overlapped peaks, accurate and precise quantification was then necessary. Of the compared methods for quantification, the manual baseline method was determined to offer the best precisions. Of the 50 found peaks from the urine analysis, 34 were successfully quantified using the manual baseline method with percent relative standard deviations ranging from 0.09 to 16. The accuracy of quantification was then investigated by the analysis of wastewater treatment plant effluent (WWTPE) samples. The chemometrically determined concentration of the unknown phenytoin sample was found to not exhibit a significant difference from the result obtained by the LC-MS/MS reference method, and the precision of the IKSFA-ALS method was better than that of the precision of the LC-MS/MS analysis. Chromatographic factors (data complexity, large dynamic range, retention time shifting, chromatographic and spectral peak overlap and background removal, were all found to affect the quantification results. The last part of this work focused on rapid screening methods that were capable of locating peaks between samples that exhibited significant differences in concentration. The aim here was to reduce the amount of data required to be resolved and quantified to only those peaks that were of interest. This would then reduce the time required to analyze large, complex samples by eliminating the need to first quantify all peaks in a given sample for many different samples. Both the similarity index (SI) method and the Fisher ratio (FR) method were found to fulfill this requirement in a rapid means of screening fifteen wine samples.

In the first part of this work, LC-MS data were used to calculate the in-vitro intrinsic clearances (CLint) for the metabolism of p-methoxyrnethamphetamine (PMMA) and fluoxetine by the CYP2D6 enzyme using a steady-state (SS) approach and a new general enzyme (GE) screening method. For PMMA, the SS experiment resulted in a CLint of 2.7 ± 0.2 µL pmol 2D6-1min-1 and the GE experiment resulted in a CLint of 3.0 ± 0.6 µL pmol 2D6-1min-1. For fluoxetine, the SS experiment resulted in a CLint of 0.33 ± 0.17 µL pmol 2D6-1min-1 and the GE experiment resulted in a CLint of 0.188 ± 0.013 µL pmol 2D6-1min-1. The inhibition of PMMA metabolism by fluoxetine was also demonstrated.In the second part of the work, target factor analysis was used as part of a library search algorithm for the identification of drugs in LC-DAD chromatograms. The ability to resolve highly overlapped peaks using the spectral data afforded by the DAD is what distinguished this method from conventional library searching methods. A validation data set of 70 chromatograms was used to calculate the sensitivity (correct identification of positives) and specificity (correct identification of negatives) of the method, which were 92% and 94% respectively.Finally, the last part of the work shows the development of data analysis methods for four-way data generated by two-dimensional liquid chromatography separations with DAD. Maize seedlings were analyzed, specifically focusing on indole-3-acetic acid (IAA) and related compounds. Window target testing factor analysis was used to identify the spectral groups represented by the standards in the mutant and wild-type chromatograms. Two curve resolution algorithms were applied to resolve overlapped components in the data and to demonstrate the quantitative potential of these methods. A total of 95 peaks were resolved. Of those peaks, 45 were found in both the mutant and wild-type maize, 16 peaks were unique to the mutants, 13 peaks were unique to the wild-types, and the remaining peaks were standards. Several IAA conjugates were quantified in the maize samples at levels of 0.3 - 2 µg/g plant material.

Phenylephrine (PE) is the most commonly used over-the-counter nasal decongestant. The problem associated with phenylephrine is that it undergoes extensive first pass metabolism in the intestinal gut wall leading to its poor and variable oral bioavailability. This research project aims at developing strategies in order to increase the oral bioavailability of PE by co-administration of GRAS compounds. A HILIC assay method was developed to detect the parent drug, phenylephrine (PE) and its sulfate metabolite (PES).The enzyme kinetic studies were done with phenolic dietary or GRAS compounds using LS180 human intestinal cell model, recombinant SULT enzymes and human intestinal cytosol (HIC). From the screening studies done, one inhibitor was selected in order to study the mechanism of inhibition. In conclusion the studies done in vitro provided a basis in order to predict in vivo intrinsic clearance through the sulfation pathway.

4 Abstract Estrogens are considered to belong to chemicals that negatively affect the endocrine system, even if present at very low concentrations. They are discharged into environment as a result of an increasing application of drugs etc. This work is focused on the separation and quantification of five estrogens, namely estrone (E1), 17β-estradiol (βE2), 17α-estradiol (αE2), 17α-ethynylestradiol (EE2) and estriol (E3) in natural water samples by HPLC-UV method. The chromatographic system consisted of a C18 stationary phase (SunFire® C18, 150 x 4.6 mm, octadecyl bounded to silica gel, particle size 5 µm) and binary mobile phase of acetonitrile/water in various ratios in isocratic separation mode. The effect of acetonitrile content in the mobile phase and flow rate of the mobile phase on retention and separation parameters was tested. Under the optimized separation conditions (acetonitrile/water 40/60 (v/v), 1.3 ml/min), all the compounds were baseline resolved and eluted within 15 min. These experimental conditions were applied to the calibration measurements which were carried out within the concentration range from 0.001 to 1 mg/ml. Limits of detection (LOD) and limits of quantification (LOQ) for the individual estrogens and their mixture (standards dissolved in methanol) were determinated. The detection...

Nitrilases are well known for their unique property to effectively convert nitriles into corresponding carboxylic acids and ammonia. They can also form amides as by-products. In contrast to nitrile hydratases they do not require cofactors or prosthetic groups. The research in this work is focused on nitrilase from filamentous fungus Arthroderma benhamiae and cyanide hydratase from Aspergillus niger K10. Genes of these enzymes were expressed using pET-30a(+) plasmid in the bacterium Escherichia coli strain BL21-Gold (DE3). The products obtained were purified by a series of ion exchange chromatography and gel filtration and subsequently characterized with respect to oligomeric state of the protein and its usability for protein crystallography. To obtain information regarding the structural arrangement of the individual proteins, electrophoretic separation in polyacrylamide gel, gel filtration, analytical ultracentrifugation, mass spectrometry, dynamic light scattering and drop coating deposition Raman spectroscopy were used. Keywords: nitrilase, cyanide hydratase, Aspergillus niger, Arthroderma benhamiae, liquid chromatography (In Czech)

Charles University in Prague Pharmaceutical Faculty in Hradec Králové Department of Pharmaceutical Botany and Ecology Candidate: Miloslava Ďuráčová Consultant: RNDr. Jitka Vytlačilová, Ph.D. Title of thesis: HPLC-MS determination of active biological compounds in Elbe river Biologically active compounds have a various ways to enter environment. They can occur as pesticides, cosmetics and pharmaceutics and their metabolites. Such compounds are classified as environmental contaminants. There is a increased environmental concentration connected with increasing consumption of biologically active compounds. There is a urgent need to follow the effect on the different parts of ecosystem and levels of biologically active compounds. This work was prepared during the cooperation with Povodí Labe state enterprise. A novel analytical method was described in the experimental part of this thesis. I was employed to evaluate biologically compounds levels in the surface water samples. This method is now routinely used. 10 out of 19 evaluated compounds reached concentrations higher than LOD. Acetaminophene (559 ng/l, Králický stream), gabapentine (457 ng/l Elbe, LB Schmilka), cotinine (139 ng/l, Králický potok) were the biologically active compounds with the highest found concentrations. Keywords: HPLC-MS analysis,...

In recent years, endocrine disruption of corticosteroid signaling pathways in wildlife and humans by environmental chemicals have attracted increasing attention. The integrated potential of chemicals in the aquatic environment that disrupt corticosteroid actions have been evaluated using in vitro glucocorticoid receptor (GR) mediated bioassays. Exogenous natural and synthetic corticosteroids (CSs), which are widely used in human and animal therapeutic applications, were demonstrated to be the most important GR agonists, that can potentially cause adverse effects, especially on aquatic organisms. To date, only a few studies have investigated the occurrence and behavior of GR agonists in the aquatic environment and their removal in conventional wastewater treatment plants. Furthermore, there are hardly any data reported on the removal of GR agonists by advanced water and wastewater treatment, especially those synthetic CSs with high potency. To further understand the fate of GR agonists in water and wastewater treatment processes, a sensitive and robust LC-MS/MS method was successfully developed for analyzing a wide range of GR agonists in various environmental waters. The occurrence of GR agonists in surface water and groundwater was monitored along the Lower Santa Cruz River (SCR). Several GR agonists were detected, and a trend of degradation was observed downstream the two WWTP outfalls for both surface water and groundwater. The fate of GR agonists in a local wastewater treatment plant (WWTP) was investigated, and up to 14 GR agonists were detected at different stages. Highly potent synthetic CSs, including clobetasol propionate (CBP), fluticasone propionate (FTP), fluocinolone acetonide (FCA), and triamcinolone acetonide (TCA), were poorly removed in WWTP. Negative removal of some CSs was observed in primary treatment, which may due to the deconjugation of CS conjugates. Removal of GR agonists in secondary effluent during various advanced water treatment processes, including UV, ozonation, MF, RO and chlorination, were studied. UV and RO appeared to be the most efficient treatment process for the attenuation of GR agonists, followed by ozone, while chlorination had little effects on GR agonists in water. Bench-scale experiments were then carried out to investigate the removal of GR agonists by ultraviolet based advanced oxidation processes (UV/AOPs), and powder activated carbon (PAC). UV/chlorine and UV/H2O2 were demonstrated to be effective in removal GR agonists in wastewater, and UV photolysis would be the predominant mechanism in UV/AOP processes. Four types of PACs were tested for removing GR agonists in wastewater effluent, and Cabot HDB carbon was suggested, while Calgon PWA carbon was not recommended due to its low removal efficiency.