This thesis deals with the reliability of methods for the speciation of mercury in environmental and biological samples. Problems with speciation methods that couple chromatography to atomic spectrometric detection and how to overcome the problems are discussed. Analytical techniques primarily studied and evaluated are high performance liquid chromatography-cold vapour-atomic absorption spectrometry (HPLC-CV-AAS), HPLC-inductively coupled plasma-mass spectrometry (HPLC-ICP-MS), capillary electrophoresis-ICP-MS (CE-ICP-MS) and gas chromatography-ICP-MS (GC-ICP-MS). Applying a multi-capillary approach increased the analyte amount injected into a CE-ICP-MS system and improved the overall sensitivity. A microconcentric nebulizer with a cyclone spray chamber was shown to improve the detection limits for mercury species 3-13 times in HPLC-ICP-MS and 11-19 times in CE-ICP-MS compared to a cross-flow nebulizer with a Scott spray chamber. To decrease the interference of water vapour in HPLC-CV-AAS a Nafion dryer tube was inserted between the CV-generation and the detector. Methyl mercury was however lost in the Nafion unless it was reduced to elemental mercury prior transport through the dryer tube. During sample pre-treatment, incomplete extraction, losses and transformation (alkylation, dealkylation, oxidation and reduction) of mercury species can lead to significant errors (underestimation and overestimation) in the determination of the concentrations. Methods to detect and determine the degree of transformation as well as correct for errors caused by transformation are presented in the thesis. The preferable method use species-specific enriched stable isotope standards in combination with MS detection and a matrix based calculation scheme. This approach is very powerful as both the concentrations of the species as well as the degrees of transformation can be determined within each individual sample.

A análise pelas técnicas hifenadas CLAE-DAD-EM e CLAE-DAD-EM/EM de extratos foliares de Lychnophora ericoides Mart. levou à identificação de 34 metabólitos secundários diretamente a partir dos extratos vegetais. Destes, 19 foram relatados pela primeira vez na subtribo Lychnophorinae. Uma metodologia para extração e análise destes metabólitos por CLAE-DAD foi desenvolvida, validada e utilizada para determinação de suas variações populacionais e temporais. Perfis metabólicos bem semelhantes, com diferenças quantitativas, mas não qualitativas, foram observados entre os indivíduos de uma mesma população. Entre as cinco populações analisadas, foi observado um metabolismo secundário bem mais diversificado, caracterizado pela presença de lactonas sesquiterpênicas, nas plantas da população de Ibiraci-MG. As concentrações mais elevadas de ácidos cafeoilquínicos foram encontradas no período da seca no cerrado (inverno) e as menores quando a planta se encontrava florida. Os teores de flavonóides foram maiores durante o período de chuvas (verão) e os de C-glicosilflavonas e lactonas sesquiterpênicas se mantiveram estáveis. Variações circadianas significativas de flavonóides e ácidos cafeoilquínicos também foram observadas nas duas populações nas quais estes fatores foram estudados. Foi verificado que o envelhecimento das folhas resulta em uma pronunciada redução no conteúdo de todos os metabólitos secundários e concentrações menores dos metabólitos foram observadas nas folhas dos ramos floridos em relação aos ramos sem flores de um mesmo indivíduo. Finalmente, conclui-se que as variações temporais e populacionais observadas em L. ericoides podem ser determinantes para a intensidade da atividade antiinflamatória e/ou analgésica desta planta, utilizada popularmente para estes propósitos. / The analyses of Lychnophora ericoides Mart. leaves extracts by HPLC-DAD-MS and HPLC-DAD-MS/MS resulted in the identification of 34 secondary metabolites directly from the plant extracts. Nineteen of these metabolites are being reported for the first time in the Lychnophorinae subtribe. A method for the extraction and analyses of these metabolites by CLAE-DAD was developed, validated and employed for the determination of their populational and temporal variations. Very similar metabolic profiles, exhibiting quantitative but not qualitative differences, were found among each population individuals. Among the five studied populations, a more diversified secondary metabolism, characterized by sesquiterpene lactones presence, was observed for the plants collected at Ibiraci-MG. The highest concentrations of caffeoylquinic acids were found in the cerrado dry season (winter), and the lowest when the plants were blooming. The flavoinoid contents were higher during the rainy season (summer), while C-glucosylflavones and sesquiterpene lactones levels remained stable. Significant circadian variations were also detected regarding flavonoids and caffeoylquinic acids in the two populations analyzed on this respect. It was also found a pronounced reduction in the contents of all the secondary metabolites as the leaves get old. Lower levels of all the metabolites were also found for the flowery branches when compared to the non flowery ones in the same individual. Finally, it can be concluded that the temporal and populational variations detected in L. ericoides leaves could highly influence the intensity of the anti-inflammatory and/or analgesic activities of this plant, used in popular medicines for these purposes.

Tebuconazole belongs to widely utilized plant fungicides, mostly in vineyards. Its traces are present in plant tissues, wine as well as in soil. The aim of the study was to determine stoichiometries and stabilities of metal (Cu, Zn, Cd, Ca and Ag) complexes with tebuconazole (1) by means of electrospray ionization mass spectrommetry (ESI-MS). It was found that the complexes of tebuconazole with copper ions are particularly stable ([Cu(1)]+ has appearance energy 224 kJ·mol-1 , corresponding with the dissociation of the water molecule from the tebuconazole molecule), while the stability of another tebuconazole complexes is lower, about 150 kJ·mol-1 . In real environmental samples, the detection of 1 itself is hampered by its spreading into various species. The method involved here is based upon a hyphenation of electrochemistry (amperometry) and electrospray-ionization mass spectrometry (ESI-MS). It uses the fact that Cu creates very stable complexes with 1. We demonstrate that at appropriate potentials (at higher than 500 mV) Cu cations can be generated in an electrochemical cell and subsequently form complexes with 1 present in the sample. The resulting complex [Cu(1)]+ is detected with ESI-MS. The intensity of detected complexes depends on the concentration of 1 and the oxidation level of the...

De nombreux métaux tels que, par exemple le Zn, le Fe, le Cu ou le Ni jouent un rôle essentiel dans la croissance et le développement normal des plantes car ils sont impliqués dans différents processus physiologiques pouvant être cependant perturbés par une carence ou un excès en métaux. Par conséquent, afin de réguler l'absorption des métaux, leur translocation et leur accumulation, les plantes ont développé des mécanismes divers, comme la production de métabolites de faible poids moléculaire pouvant se lier aux métaux. La connaissance de ces complexes métalliques et des processus qu'ils subissent dans les plantes peut être utilisée dans des études environnementales, nutritionnelles et toxicologiques. Cependant, cette connaissance était très limitée à cause de l'absence de méthodologie appliquée avec succès pour étudier la spéciation des traces de métaux dans les végétaux. Par conséquent, le but de cette étude fut le développement d'une méthodologie d'analyse des espèces métalliques dans des échantillons de plantes. La nouvelle approche est basée sur l'utilisation conjointe du (i) couplage HPLC – ICP MS permettant la détection de nombreuses espèces, souvent peu concentrées, contenant des métaux avec (ii) une identification en parallèle par couplage HPLC – electrospray Orbitrap MS/MS. Elle a permis l'identification (i) d’environ 60 espèces métalliques, la plupart jamais observées précédemment dans différents fluides (xylème, endosperme liquide) de Pisum sativum (petit pois) et (ii) de plusieurs complexes mixtes fer - aluminium - citrate chez Plantago almogravensis. La méthodologie développée a également été appliquée à (iii) la spéciation du sélénium dans Brassica nigra permettant l'identification de plus de 30 composés de faible poids moléculaire contenant du sélénium. / Numerous metals, such as, e.g. Zn, Fe, Cu or Ni play an essential role in normal plant growth and development as they are involved in different physiological processes that may be, however, disrupted by metal deficiency or excess. Therefore, to regulate metal uptake, translocation and accumulation plants have developed diverse mechanisms including the production of low molecular mass metal binding metabolites. The knowledge of these forms of metals and the processes they undergo in plants can be used in environmental, nutrition and toxicological studies. However, this knowledge was very limited as there was a lack of methodology that could be successfully applied to investigate trace metal speciation in plants. Therefore, the aim of this study was the development of the methodology for the analysis of metal species in complex plant samples. The novel approach is based on the use of combination of (i) HPLC – ICP MS coupling allowing the detection of numerous, often low concentrated, metal-containing species with (ii) a parallel identification using HPLC – electrospray Orbitrap MS/MS coupling. The developed approach allowed the identification of (i) ca. 60 different, mainly previously unreported metal species in saps of Pisum Sativum (green pea) and (ii) several mixed iron – aluminum – citrate complexes in Plantago almogravensis. This developed methodology was also applied to (iii) investigate selenium speciation in Brassica nigra allowing the identification of more than 30 selenium-containing low molecular mass compounds.