Solid phase microextraction (SPME) has been widely used in a variety of sample matrices and proven to be a simple, fast and solvent-free sample preparation technique. A challenging limitation in the further development of this technique has been the insufficient sensitivity for some trace applications. This limitation lies mainly in the small volume of the extraction phase. According to the fundamentals of SPME, different strategies can be employed to achieve higher sensitivity for SPME sampling. These include cooling down the extraction phase, preparing a high capacity particle-loading extraction phase, as well as using a thin film with high surface area-to-volume ratio as the extraction phase. In this thesis, four sampling approaches were developed for high sensitivity sampling by employing cold fiber, thin film, cooling membrane and particle loading membrane as sampling tools. These proposed methods were applied to liquid, solid and particularly trace gas analysis. First, a fully automated cold fiber device that improves the sensitivity of the technique by cooling down the extraction phase was developed. This device was coupled to a GERSTEL® MultiPurpose Sampler (MPS 2), and applied to the analysis of volatiles and semi-volatiles in aqueous and solid matrices. The proposed device was thoroughly evaluated for its extraction performance, robustness, reproducibility and reliability by gas chromatograph/mass spectrometer (GC/MS). The evaluation of the automated cold fiber device was carried out using a group of compounds characterized by different volatilities and polarities. Extraction efficiency and analytical figures of merit were compared to commercial SPME fibers. In the analysis of aqueous standard samples, the automated cold fiber device showed a significant improvement in extraction efficiency when compared to commercial polydimethylsiloxane (PDMS) and non-cooled cold fiber. This was achieved due to the low temperature of the coating during sampling. Results from the cold fiber and commercial divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber analysis of solid sample matrices were obtained and compared. Results demonstrated that the temperature gap between the sample matrix and the coating significantly improved the distribution coefficient, and consequently, the extraction amount. The newly automated cold fiber device presents a platform for headspace analysis of volatiles and semi-volatiles for a large number of samples, with improved throughput and sensitivity. Thin film microextraction (TFME) improves the sensitivity by employing a membrane with a high surface area-to-volume ratio as the extraction phase. In Chapter 3, a simple non-invasive sample preparation method using TFME is proposed for sampling volatile skin emissions. Evaluation experiments were conducted to test the reproducibility of the sampling device, the effect of the membrane size, and the method for storage. Results supported the reproducibility of multi-membrane sampling, and demonstrated that sampling efficiency can be improved using a larger membrane. However, ability to control the sampling environment and time was proved to be critical in order to obtain reliable information; the in vivo skin emission sampling was also influenced by skin metabolism and environmental conditions. Next, the method of storage was fully investigated for the membrane device before and after sampling. This investigation of storage permitted the sampling and instrument analysis to be conducted at different locations. Finally, the developed skin sampling device was applied in the identification of dietary biomarkers after garlic and alcohol ingestion. In this experiment, the previously reported potential biomarkers dimethyl sulphone, allyl methyl sulfide and allyl mercaptan were detected after garlic intake, and ethanol was detected after the ingestion of alcohol. Experiments were also conducted in the analysis of volatile organic compounds (VOCs) from upper back, forearm and back thigh of the body on the same individual. Results showed that 27 compounds can be detected from all of the 3 locations. However, these compounds were quantitatively different. In addition, sampling of the upper back, where the density of sebaceous glands is relatively high, detected more compounds than the other regions. In Chapter 4, a novel sample preparation method that combines the advantages of cold fiber and thin film was developed to achieve the high extraction efficiency necessary for high sensitivity gas sampling. A cooling sampling device was developed for the thin film microextraction. Method development for this sampling approach included evaluation of membrane temperature effect, membrane size effect, air flow rate and humidity effect. Results showed that high sensitivity for equilibrium sampling can be achieved by either cooling down the membrane and/or using a large volume extraction phase. On the other hand, for pre-equilibrium extraction, in which the extracted amount was mainly determined by membrane surface area and diffusion coefficient, high sensitivity was obtained by thin membranes with a large surface area and/or high sampling flow rate. In addition, humidity evaluations showed no significant effect on extraction efficiency due to the absorption property of the liquid extraction phase. Next, the limit of detection (LOD) and reproducibility of the developed cooling membrane gas sampling method were evaluated. LOD with a membrane radius of 1 cm at room temperature sampling were 9.24 ng/L, 0.12 ng/L, 0.10 ng/L for limonene, cinnamaldehyde and 2-pentadecanone, respectively. Intra- and inter-membrane sampling reproducibility had a relative standard deviation (RSD%) lower than 8% and 13%, respectively. Results uniformly demonstrated that the proposed cooling membrane device could serve as a powerful tool for gas in trace analysis. In Chapter 5, a particle-loading membrane was developed to combine advantages of high distribution coefficient and high surface area geometry, and applied in trace gas sampling. Bar coating, a simple and easy preparation method was applied in the preparation of the DVB/PDMS membrane. Membrane morphology, particle ratio, membrane size and extraction efficiency were fully evaluated for the prepared membrane. Results show that the DVB particles are uniformly distributed in the PDMS base. The addition of a DVB particle enhanced the stiffness of the membrane to some extent, and improved the extraction capacity of the membrane. Extraction capacity for benzene was enhanced by a factor of 100 when the membrane DVB particle ratio increased from 0% to 30%. Additionally, the prepared DVB/PDMS membrane provided higher extraction efficiency than pure PDMS membrane and DVB/PDMS fiber, especially for highly volatile and polar compounds. The high reproducibility of the prepared DVB/PDMS membrane in air sampling demonstrated the advantage of the bar coating preparation method, and also permitted quantitative analysis. Last, the prepared particle-loading membrane was applied to semi-quantitative and quantitative analysis of indoor and outdoor air, respectively. Both the equilibrium calibration method and diffusion-based calibration method were proposed for the quantitative analysis. Results showed that the high capacity particle-loading membrane can be used for monitoring trace analytes such as perfume components and air pollutants.

Sample preparation is the most time-consuming and error-prone step in chemical analysis. Miniaturization and automation of the sample preparation equipment eliminating or reducing the use of hazardous organic solvents, online hyphenation of sample preparation with analytical instruments in a cost-effective way are important factors that need to be considered to design and implement innovative sample preparation techniques and strategies. Solid-phase microextraction (SPME) is a simple, environmentally benign technique well suited for hyphenation with analytical instruments. However, poor coating stability is a significant drawback of SPME employing conventionally prepared coatings. This shortcoming arises from the lack of chemical bonding between the sorbent coating and the substrate. Introduction of sol-gel coatings in SPME greatly improved thermal stability and solvent stability in SPME, by providing direct chemical bonding between substrate and the sol-gel coating. In traditional fiber format of SPME (where the sorbent coating is placed on the outer surface of an end-segment of the fiber) the coating remains vulnerable to mechanical damage. Capillary microextraction (CME), the capillary format of SPME (also known as in-tube SPME), allows to overcome this shortcoming by securing the sorbent coating on inner walls of the capillary. This dissertation focuses on the development and systematic investigation of novel silica- and germania-based perfluorinated and non-fluorinated sol-gel sorbents in the form of CME surface coatings: their preparation, material characterization, CME performance evaluation, preconcentration and recovery of various analytes including environmental pollutants. This research established that germania-based sol-gel sorbents are characterized by superior microextraction performance than analogous silica-based sorbents. This enhanced performance provided by germania-based sol-gel sorbents may be explained based on thermogravimetric analysis suggests that higher carbon loading on germania-based sol-gel sorbents. Germania-based phenyl- (Ph), phenethyl- (PhE), octyl- (C8), octadecyl- (C18) and cyclohexenylethyl- (ChE) ligand-containing sol-gel sorbents were prepared and various pollutants with aromatic rings (such as aromatic ketones, aldehydes and polycyclicaromatic hydrocarbons) were extracted and analyzed by CME-GC and CME-HPLC. It was observed that sol-gel sorbents containing aromatic ligands (PhE and Ph) provided superior microextraction performance for the analytes with aromatic ring(s) in their structure, than the sorbents with aliphatic ligands (C8 and C18). Investigation of sol-gel sorbents containing hydrophobic perfluorooctyl (PF-C8) and perfluorododecyl (PF-C12) ligands revealed that PF-C8 and PF-C12 sol-gel sorbents provided ~ 3 times higher microextraction efficiency (measure in terms of specific extraction, SE) than corresponding non-fluorinated counterparts, C8- and C12-, respectively. The synthesis and design of silica- and germania-based dual ligands sol-gel sorbents simultaneously providing superhydrophobicity and π-π interactions with analytes represent a significant accomplishment of this research. Such sorbents contained a PF-C12 and PhE ligands incorporated in sorbent chemical structure. In this case, perfluoro- group provided enhanced hydrophobic interaction and PhE group provided π-π interaction with the analytes. Combination of such interactions proved to be quite effective in the microextraction of alkylbenzenes and related compounds. Dual-ligand sol-gel sorbents with both equimolar and non-equimolar ligand concentrations were prepared. Experimentally it was established that sorbents with higher perfluorinated alkyl ligand concentrations had higher affinity for aliphatic hydrocarbons; however; when PhE concentration was higher, the dual-ligand sorbent showed enhanced affinity for aromatic compounds. The prepared sol-gel sorbents were characterized by less than 5% run-to-run RSD values, and also less than 5% capillary-to-capillary RSD values, which indicate that the sol-gel technique used in sorbent preparation was highly reproducible. The prepared sol-gel sorbents also showed that their performance does not deteriorate under aqueous saline matrix; therefore, it could be useful in the microextraction of pollutants from ocean water.

No description available.

Parabens are effective antibacterial and anti-fungal agents and are used extensively as preservatives in cosmetics, food and pharmaceutical products. Because of the presence of parabens in the environment and their negative effects on human health, there is an increasing interest in their trace analysis. Since the concentration of parabens in the environment are rather low and cosmetics present rather complex matrices for the analysis, it is necessary to apply a preconcentration or isolation step prior to the chromatographic analysis. The aim of this work was to develop liquid-phase microextraction methods – single drop microextraction, hollow fibre liquid phase microextraction and dispersive liquid-liquid microextraction – for parabens and to apply the methods for determination of parabens in water samples and cosmetic products. Extraction conditions for single drop microextraction, hollow fibre liquid phase microextraction and dispersive liquid-liquid microextraction were optimized and the quality parameters of the suggested methods were calculated. The influence of derivatization on the paraben extraction efficiency was evaluated. Prepared microextraction methods were applied for the determination of parabens in real samples. / Parabenai pasižymi antibakterinėmis bei priešgrybelinėmis savybėmis ir plačiai naudojami kosmetikos, maisto, farmacijos pramonėje kaip konservantai, apsaugantys produktus nuo ankstyvo gedimo ir prailginantys jų galiojimo laiką. Padidėjus įtarimams, jog prasiskverbę per odą parabenai gali sukelti alergines reakcijas, ardyti endokrininę sistemą, skatinti vėžinių ląstelių gamybą, buvo pradėta kontroliuoti jų kiekį aplinkoje, maisto bei kosmetikos produktuose. Tačiau parabenų koncentracijos dažnai yra per mažos, o mėginiai per daug sudėtingi, kad juos būtų galima analizuoti nesukoncentravus ir neizoliavus nuo trukdančios matricos. Šioje daktaro disertacijoje apibendrintų mokslinių tyrimų tikslas – sukurti parabenų skysčių-skysčių mikroekstrakcijos metodus – mikroekstrakciją tirpiklio lašu, skystafazę mikroekstrakciją kapiliare ir dispersinę skysčių-skysčių mikroekstrakciją – bei pritaikyti juos parabenų nustatymui vandenyje bei kosmetikos produktuose. Optimizuotos mikroekstrakcijos tirpiklio lašu, skystafazės mikroekstrakcijos kapiliare ir dispersinės skysčių-skysčių mikroekstrakcijos ekstrakcijos sąlygos ir nustatytos pagrindinės analizinės charakteristikos. Ištirta parabenų derivatizacijos įtaka parabenų dujų chromatografinio nustatymo efektyvumui. Paruošti parabenų mikroekstrakcijos metodai pritaikyti vandens ir kosmetikos mėginių analizei.

Parabenai pasižymi antibakterinėmis bei priešgrybelinėmis savybėmis ir plačiai naudojami kosmetikos, maisto, farmacijos pramonėje kaip konservantai, apsaugantys produktus nuo ankstyvo gedimo ir prailginantys jų galiojimo laiką. Padidėjus įtarimams, jog prasiskverbę per odą parabenai gali sukelti alergines reakcijas, ardyti endokrininę sistemą, skatinti vėžinių ląstelių gamybą, buvo pradėta kontroliuoti jų kiekį aplinkoje, maisto bei kosmetikos produktuose. Tačiau parabenų koncentracijos dažnai yra per mažos, o mėginiai per daug sudėtingi, kad juos būtų galima analizuoti nesukoncentravus ir neizoliavus nuo trukdančios matricos. Šioje daktaro disertacijoje apibendrintų mokslinių tyrimų tikslas – sukurti parabenų skysčių-skysčių mikroekstrakcijos metodus – mikroekstrakciją tirpiklio lašu, skystafazę mikroekstrakciją kapiliare ir dispersinę skysčių-skysčių mikroekstrakciją – bei pritaikyti juos parabenų nustatymui vandenyje bei kosmetikos produktuose. Optimizuotos mikroekstrakcijos tirpiklio lašu, skystafazės mikroekstrakcijos kapiliare ir dispersinės skysčių-skysčių mikroekstrakcijos ekstrakcijos sąlygos ir nustatytos pagrindinės analizinės charakteristikos. Ištirta parabenų derivatizacijos įtaka parabenų dujų chromatografinio nustatymo efektyvumui. Paruošti parabenų mikroekstrakcijos metodai pritaikyti vandens ir kosmetikos mėginių analizei. / Parabens are effective antibacterial and anti-fungal agents and are used extensively as preservatives in cosmetics, food and pharmaceutical products. Because of the presence of parabens in the environment and their negative effects on human health, there is an increasing interest in their trace analysis. Since the concentration of parabens in the environment are rather low and cosmetics present rather complex matrices for the analysis, it is necessary to apply a preconcentration or isolation step prior to the chromatographic analysis. The aim of this work was to develop liquid-phase microextraction methods – single drop microextraction, hollow fibre liquid phase microextraction and dispersive liquid-liquid microextraction – for parabens and to apply the methods for determination of parabens in water samples and cosmetic products. Extraction conditions for single drop microextraction, hollow fibre liquid phase microextraction and dispersive liquid-liquid microextraction were optimized and the quality parameters of the suggested methods were calculated. The influence of derivatization on the paraben extraction efficiency was evaluated. Prepared microextraction methods were applied for the determination of parabens in real samples.

L’objectif de cette thèse est d’étudier le rôle de la fermentation malolactique (FML) sur l’arôme fruité des vins rouges. Les bactéries lactiques (BL) modifient la composition du vin mais il n’existe pas de consensus concernant spécifiquement cette famille aromatique. Contrairement aux idées empiriques sur la FML, ce travail a démontré l’absence à court terme d’un " masque lactique ", cependant l’apparition d’une telle interaction olfactive pourrait être plus tardive. Par contre, il est montré l’existence d’un masque proche de la note de réduction, de type fumé/grillé, dont la caractérisation n’a pas été effectuée dans cette étude.Le suivi des principaux marqueurs fruités du vin (70 molécules) a été rendu possible par le développement des méthodes d’analyse chromatographique en phase gazeuse couplée à la microextraction sur phase solide (esters, C13-norisoprénoïdes, lactones, thiols). En particulier, une " base de données esters " (32 composés) a rendu plus robuste l’ensemble des variations constatées au cours du développement des BL. En effet, les modifications des teneurs en esters sont démontrées comme un processus majeur de la balance de la note fruitée au cours de la FML. Cette fermentation permet à court terme, aussi bien la synthèse que l’hydrolyse des esters grâce aux activités estérases et, à plus long terme, la formation tardive d'esters éthyliques d'acides branchés issus du catabolisme de certains acides aminés. La spécificité des estérases vis-à-vis de la nature et de la longueur de la chaîne carbonée des esters est mise en évidence, ainsi que l'importance de la disponibilité des substrats, liée en partie à l'activité des levures.L’étude de l’influence des souches de BL et de la co-inoculation levures/bactéries a permis de confirmer le rôle clé des interactions entre les microorganismes, ainsi que l’importance de la composition de la matrice vin. / The aim of this thesis is to study the role of malolactic fermentation (MLF) on the fruity aroma of red wines. Lactic acid bacteria (LAB) modify wine composition but there is no consensus concerning this aromatic group specifically. In opposition to empirical ideas on MLF, this work has demonstrated the absence, in short-term, of a “lactic-mask” although this kind of olfactory interaction may still occur in a later stage of wine development. Nevertheless, the existence of a smoked/toasted reduction-like mask note was proved. Its characterization has not been done in this work. The survey of the main fruity markers of wine (70 compounds) was made possible by the development of several gas chromatography coupled with solid-phase microextraction analytical methods (esters, C13-norisoprenoids, lactones, thiols). In particular, the creation of an “ester database” (32 compounds) has improved the detection of variations during LAB development in terms of analysis robustness. In fact, changes on esters contents are proved to be responsible for a major part of fruity notes evolution during MLF. Initially, this fermentation allows both synthesis and hydrolysis of esters as a consequence of esterase activity. Moreover, it promotes late-production of ethylic esters through the catabolism of certain aminoacids. Esterases specificity towards nature and size of the esters carbon chain is pointed out along with substrates availability, partially related to yeast activity.The study of the influence of both LAB strains and yeast/bacteria co-inoculation has confirmed microorganisms interactions and wine matrix composition to be of the great importance.

A análise estereosseletiva tem um lugar de destaque em várias áreas e, dentre elas, a farmacêutica, pois diversos fármacos quirais são comercializados como misturas racêmicas. A análise estereosseletiva empregando a cromatografia líquida de alta eficiência e a eletroforese capilar é prática e bastante eficaz para aplicações que envolvem a determinação de enantiômeros ao nível de traços em matrizes complexas, como por exemplo, em estudos de disposição cinética. Entretanto, devido à complexidade das matrizes biológicas, há necessidade da preparação da amostra antes de sua análise. Entre as diversas técnicas existentes, as mais utilizadas são a extração líquido-líquido e a extração em fase sólida. Contudo, essas técnicas apresentam algumas desvantagens,sendo a principal delas o uso de grandes quantidades de solventes. Portanto, técnicas que requerem pouco ou nenhum consumo de solventes orgânicos são bastante desejáveis. Entre essas técnicas destacam-se a microextração em fase sólida (SPME) e a microextração em fase líquida (LPME). Essas técnicas relativamente recentes têm como vantagens a utilização de quantidades mínimas de solventes orgânicos, o alto poder de concentração, a remoção dos interferentes da matriz biológica e a simplicidade de automação. Nesse trabalho propusemos a utilização da SPME e LPME como técnicas de preparação de amostras de urina, visando o desenvolvimento de métodos estereosseletivos com detectabilidade e seletividade adequadas para aplicação em estudos posteriores de disposição cinética. A SPME foi empregada para análise estereosseletiva do ibuprofeno e de seus principais metabólitos, carboxiibuprofeno e 2-hidroxiibuprofeno e da hidroxicloroquina e seus principais metabólitos, enquanto que a LPME foi usada para a análise estereosseletiva da hidroxicloroquina e seus metabólitos. Inicialmente, foi realizada a otimização da separação dos fármacos e metabólitos em diversas colunas quirais,a otimização da separação da hidroxicloroquina e seus metabólitos por eletroforese capilar e,em seguida, a otimização dos procedimentos de extração e a validação dos métodos desenvolvidos. Utilizando a coluna Chiralpak AD-RH® e fase móvel composta por solução de ácido fosfórico 1 mol L-1 pH 3 : metanol (75 : 25, v/v), foi validado um método para análise enantiosseletiva do ibuprofeno em urina. A SPME foi empregada para extração do ibuprofeno das amostras de urina utilizando a fibra PDMS-DVB 60 6;m. O método mostrou ser linear na faixa de concentração de 0,25 a 25 μg mL–1 para cada enantiômero. Para a análise do 2-hidroxiibuprofeno e carboxiibuprofeno, foi utilizada a coluna Chiralpak AS® e fase móvel composta por hexano: isopropanol (94 : 6, v/v) + 0,05% de ácido trifluoracético. A extração desses metabólitos foi feita empregando a fibra de CW-TPR 50 µm. O método mostrou ser linear na faixa de concentração de 5 a 50 µg mL -1. Já para a análise da hidroxicloroquina e seus principais metabólitos, DHCQ e DCQ, foi utilizada a coluna Chiralcel OD-H® e fase móvel composta por hexano : etanol : metanol (96 : 2 : 2, v/v/v) + 0,2% de dietilamina. A extração desses metabólitos foi feita empregando a fibra de PDMSDVB 60 μm. O método mostrou ser linear na faixa de concentração de 50 a 1000 ng mL -1para HCQ e 42 - 416 ng mL-1 para os metabólitos. A microextração em fase líquida foi avaliada na análise da hidroxicloroquina e seus metabólitos, BDCQ, DHCQ e DCQ e separação por eletroforese capilar. Para tanto foi utilizado um capilar de sílica fundida nãorecoberto com um comprimento efetivo de 42 cm, e 30 mmol L-1 de HP-b-CD + 1% de CD-b- sulfatada dissolvida em tampão tris(hidroxiaminometano) 100 mmol L-1 pH 9 como tampão de análise. O método mostrou ser linear na faixa de concentração de 10 - 1000 ng mL-1 para HCQ e 21-333 ng mL-1 para os metabólitos. Obteve-se precisão com coeficientes de variação inferiores a 15% e exatidão com erros relativos menores que 15% para todos os métodos desenvolvidos. Após validação, os métodos foram empregados na determinação da quantidade excretada acumulada do do ibuprofeno, da hidroxicloroquina e de seus metabólitos após administração de de rac-ibuprofeno e rac-hidroxicloroquina a voluntários sadios. Em suma, as duas técnicas foram eficientes na extração dos fármacos e metabólitos estudados. A SPME mostrou ser uma técnica de mais fácil manuseio, porém com baixos valores de recuperação. Por outro lado, a LPME apresentou valores de recuperação maiores, porém o manuseio do sistema de extração foi mais difícil, necessitando de um tempo maior para o domínio da técnica. / The stereoselective analysis has been standing out in several areas, and it is mainly present in the pharmaceutical industry, since many drugs are marketed as racemic mixtures. The stereoselective analysis employing high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) is very useful for the determination of enantiomers at very low concentrations, as the ones found in biological matrices, for instance, in pharmacokinetic studies. The first step in the analysis of drugs in biological fluids is the extraction procedure. The most common extraction procedures employed are liquid-liquid extraction and solidphase extraction. These techniques show several drawbacks, such as the high amount of organic solvent consumed. So, based on this fact, techniques that consume small amounts of organic solvents are desirable. Among these techniques, solid-phase microextraction (SPME) and liquid-phase microextraction (LPME) have been stood out. The main advantages of these techniques are the small amount of organic solvent consumed and its high capacity in drug concentration. In this work, our purpose was to employ the SPME and the LPME as sample preparation techniques to develop stereoselective methods to be further applied in pharmacokinetic studies. SPME was employed for the stereoselective analysis of ibuprofen, hydroxychloroquine and their major metabolites.On the other hand,LPME was employed only for the enantioselective analysis of hydroxychloroquine and its metabolites. The first step was the separation optimization of the drugs and their metabolites by HPLC using several chiral columns, and the separation optimization of hydroxychloroquine and its metabolites by CE. Next, the extraction optimizations and method validations were performed. The enantioselective analysis of ibuprofen in human urine was carried out in the Chiralpak AD-RH® column, using methanol-pH 3.0 phosphoric acid solution (75 : 25 v/v) as mobile phase. The method was linear over the 0.5 - 25 µg mL-1 concentration range for both enantiomers. The fiber used in this method was PDMS-DVB 60 µm.The analysis of 2-hydroxyibuprofen and carboxyibuprofen was performed on a Chiralpak AS® column using hexane:isopropanol (95 : 5 v/v) plus 0.05% trifluoroacetic acid as the mobile phase. The method was linear over the 5 - 50 µg mL-1 concentration range. To perform the extractions, a CW-TPR 50 µm coated fiber was employed. The analysis of hydroxychloroquine and its major metabolites (DCQ and DHCQ) was done on a Chiralcel OD-H® column using hexane-methanol-ethanol (96 : 2 : 2, v/v/v) plus 0.2% diethylamine as mobile phase. The extraction was performed using a PDMS-DVB 60 µm coated fiber. The method was linear over the range of 50 - 1000 ng mL-1 for HCQ enantiomers and over the range of 42 - 416 ng mL-1 for DCQ and DHCQ enantiomers. LPME and CE were applied for the chiral determination of hydroxychloroquine and its metabolites (DCQ, DHCQ, BDCQ) in human urine. The electrophoretic separations were carried out in 100 mmol L-1tris(hydroxymethyl)aminomethane buffer (pH adjusted to 9.0 with phosphoric acid) containing 1% (w/v) S-b-CD and 30 mg mL-1 HP-?-CD, with a constant voltage of +18 kV. The method was linear over the concentration range of 10-1000 ng mL -1 for each HCQ stereoisomer and 21-333 ng mL -1 for each metabolite stereoisomer. Within-day and between-day assay precision and accuracy for all described methods were lower than 15%. The developed methods were applied for the determination of the cumulative urinary excretion of ibuprofen metabolites and hydroxychloroquine and its metabolites after oral administration of racibuprofen and rac-hydroxychloroquine to health volunteers. Comparing the techniques, both SPME and LPME were efficient to extract the drugs and their metabolites from human urine. iv SPME showed to be an easier technique to handle, however, the drug amount recovered by this technique was too small. On the contrary, LPME was a more difficulty technique to be handled, but better recovery values were obtained with this technique.

A new technique for sample preparation and trace analysis of organic pollutants in water using mixed-phase thin film (MPTF) devices, combined with direct thermal desorption, cold trapping, gas chromatography-mass spectrometry (GC-MS) is presented for the first time. Two novel analytical devices, Carboxen/polydimethylsiloxane (CAR/PDMS) and polydimethylsiloxane/divinylbenzene (PDMS/DVB) TF samplers were fabricated using spin coating technique and glass wool fabric mesh as substrate. The samplers were easily tailored in size and shape by cutting tools. Good durability and flat-shape stability were observed during extractions and stirring in water. The latter characteristic obviates the need for an extra framed holder for rapid thin film microextraction (TFME) and makes the samplers more robust and user-friendly. The analytical performance of the MPTF devices was satisfactorily illustrated and compared with those of solid phase microextraction (SPME) fibers and PDMS thin film membrane using water samples spiked with seven N–nitrosamines (NAs), known as disinfection by-products (DBPs) in drinking water. Marked enhancement of extraction efficiencies (typically more than one order of magnitude) for the N-nitrosamines, including the hydrophilic ones, was obtained with the MPTF devices under generally pre-equilibrium conditions, compared to the SPME fibers and PDMS thin film membrane. The analytical results obtained in this study, including linearity, repeatability and detection levels at low ng/L for the tested compounds, indicate that the new thin film devices are promising for rapid sampling and sample preparation of trace levels of polar organic pollutants in water with sensitivities higher than SPME fibers and with a wide application range typical of mixed-phase coatings. The user-friendly format and robustness of the novel devices are also advantageous for on-site applications, which is the ultimate use of thin film samplers. Moreover, the thin film fabrication approach developed in this study offers the possibility of making other novel samplers with PDMS or different absorptive polymers such as polyacrylate (PA) and polyethylene glycol (PEG) as particle-free, or as particle-loaded thin films with a variety of adsorptive solid particles. In another development in the course of this research, the performance and accuracy of the SPME fiber approach for sample preparation of selected DBPs were demonstrated and compared with the conventional liquid-liquid extraction (LLE) method by real drinking water samples analysis in collaboration with Health Canada. Four regulated trihalomethanes (THMs) and seven other DBPs known as priority by-products, including four haloacetonitriles, two haloketones and chloropicrin, were analyzed in real samples during two separate comparative studies. In each study, duplicate samples from several water treatment and distribution systems in Canada, collected and stabilized under the same protocol, were analyzed in parallel by two independent labs; in the University of Waterloo by an optimized headspace SPME-GC-MS and in Health Canada by a LLE-GC-ECD (electron capture detection) method equivalent to EPA 551.1. The values for the concentration of the analytes in the samples obtained by the two methods were in good agreement with each other in majority of the cases indicating that SPME affords the promise of a dependable sample preparation technique for rapid DBPs analysis. In particular, it was shown that the SPME fiber approach combined with GC-MS is a fast reliable alternative to the LLE-GC-ECD (EPA 551.1) method for analysis of the regulated THMs in the concentration ranges that are typical and relevant for drinking water samples.

An environmentally friendly, rapid and cost effective analytical procedure based on dispersive liquid-liquid microextraction was developed for the determination of six tetracyclines (TCs) in meat destined for human consumption. Meat extracts were analyzed for TCs using a sensitive and selective analytical technique, liquid chromatography tandem mass spectrometry. Various influencing factors on the extraction, separation and determination of TCs such as pH of mobile phases, type and volume of disperser solvent, type and volume of extraction solvent and sample pH were optimized. Validation parameters such as calibration function, limit of detection (LOD), limit of quantification (LOQ), detection capability (CCα), decision limit (CCβ), accuracy and precision were established according to EU commission decision 2002/657/EC. Linearity in the range of 25-200 μg kg-1 was obtained with regression coefficients ranging from 0.9991 to 0.9998. Recoveries of spiked blank muscle samples at three levels (i.e. 50, 100 and 150 μg kg-1) ranged from 80 to 101% and reproducibility was between 2 and 7%. The LODs and LOQs ranged from 2.22 to 3.59 μg kg-1 and from 7.38 to 11.49 μg kg-1 respectively. The CCα ranged from 105 to 111 μg kg-1 while CCβ ranged from 107 to 122 μg kg-1. The proposed method compared well with the dispersive solid phase extraction method and was successfully applied to the determination of TCs in meat samples. Some of the thirty bovine muscle samples obtained from local abattoirs and butcheries were found to contain two tetracycline antibiotics residues (chlortetracycline and oxytetracycline) with oxytetracycline being the most commonly detected. The concentration levels of the TC residues detected in the eleven bovine muscle samples were between 12.4 and 68.9 μg kg-1, levels that are lower than the European Union set maximum residue level (MRL) of 100 μg kg-1 hence the meat was fit for human consumption. / Chemistry / M. Sc. (Chemistry)

A análise estereosseletiva tem um lugar de destaque em várias áreas e, dentre elas, a farmacêutica, pois diversos fármacos quirais são comercializados como misturas racêmicas. A análise estereosseletiva empregando a cromatografia líquida de alta eficiência e a eletroforese capilar é prática e bastante eficaz para aplicações que envolvem a determinação de enantiômeros ao nível de traços em matrizes complexas, como por exemplo, em estudos de disposição cinética. Entretanto, devido à complexidade das matrizes biológicas, há necessidade da preparação da amostra antes de sua análise. Entre as diversas técnicas existentes, as mais utilizadas são a extração líquido-líquido e a extração em fase sólida. Contudo, essas técnicas apresentam algumas desvantagens,sendo a principal delas o uso de grandes quantidades de solventes. Portanto, técnicas que requerem pouco ou nenhum consumo de solventes orgânicos são bastante desejáveis. Entre essas técnicas destacam-se a microextração em fase sólida (SPME) e a microextração em fase líquida (LPME). Essas técnicas relativamente recentes têm como vantagens a utilização de quantidades mínimas de solventes orgânicos, o alto poder de concentração, a remoção dos interferentes da matriz biológica e a simplicidade de automação. Nesse trabalho propusemos a utilização da SPME e LPME como técnicas de preparação de amostras de urina, visando o desenvolvimento de métodos estereosseletivos com detectabilidade e seletividade adequadas para aplicação em estudos posteriores de disposição cinética. A SPME foi empregada para análise estereosseletiva do ibuprofeno e de seus principais metabólitos, carboxiibuprofeno e 2-hidroxiibuprofeno e da hidroxicloroquina e seus principais metabólitos, enquanto que a LPME foi usada para a análise estereosseletiva da hidroxicloroquina e seus metabólitos. Inicialmente, foi realizada a otimização da separação dos fármacos e metabólitos em diversas colunas quirais,a otimização da separação da hidroxicloroquina e seus metabólitos por eletroforese capilar e,em seguida, a otimização dos procedimentos de extração e a validação dos métodos desenvolvidos. Utilizando a coluna Chiralpak AD-RH® e fase móvel composta por solução de ácido fosfórico 1 mol L-1 pH 3 : metanol (75 : 25, v/v), foi validado um método para análise enantiosseletiva do ibuprofeno em urina. A SPME foi empregada para extração do ibuprofeno das amostras de urina utilizando a fibra PDMS-DVB 60 6;m. O método mostrou ser linear na faixa de concentração de 0,25 a 25 μg mL–1 para cada enantiômero. Para a análise do 2-hidroxiibuprofeno e carboxiibuprofeno, foi utilizada a coluna Chiralpak AS® e fase móvel composta por hexano: isopropanol (94 : 6, v/v) + 0,05% de ácido trifluoracético. A extração desses metabólitos foi feita empregando a fibra de CW-TPR 50 µm. O método mostrou ser linear na faixa de concentração de 5 a 50 µg mL -1. Já para a análise da hidroxicloroquina e seus principais metabólitos, DHCQ e DCQ, foi utilizada a coluna Chiralcel OD-H® e fase móvel composta por hexano : etanol : metanol (96 : 2 : 2, v/v/v) + 0,2% de dietilamina. A extração desses metabólitos foi feita empregando a fibra de PDMSDVB 60 μm. O método mostrou ser linear na faixa de concentração de 50 a 1000 ng mL -1para HCQ e 42 - 416 ng mL-1 para os metabólitos. A microextração em fase líquida foi avaliada na análise da hidroxicloroquina e seus metabólitos, BDCQ, DHCQ e DCQ e separação por eletroforese capilar. Para tanto foi utilizado um capilar de sílica fundida nãorecoberto com um comprimento efetivo de 42 cm, e 30 mmol L-1 de HP-b-CD + 1% de CD-b- sulfatada dissolvida em tampão tris(hidroxiaminometano) 100 mmol L-1 pH 9 como tampão de análise. O método mostrou ser linear na faixa de concentração de 10 - 1000 ng mL-1 para HCQ e 21-333 ng mL-1 para os metabólitos. Obteve-se precisão com coeficientes de variação inferiores a 15% e exatidão com erros relativos menores que 15% para todos os métodos desenvolvidos. Após validação, os métodos foram empregados na determinação da quantidade excretada acumulada do do ibuprofeno, da hidroxicloroquina e de seus metabólitos após administração de de rac-ibuprofeno e rac-hidroxicloroquina a voluntários sadios. Em suma, as duas técnicas foram eficientes na extração dos fármacos e metabólitos estudados. A SPME mostrou ser uma técnica de mais fácil manuseio, porém com baixos valores de recuperação. Por outro lado, a LPME apresentou valores de recuperação maiores, porém o manuseio do sistema de extração foi mais difícil, necessitando de um tempo maior para o domínio da técnica. / The stereoselective analysis has been standing out in several areas, and it is mainly present in the pharmaceutical industry, since many drugs are marketed as racemic mixtures. The stereoselective analysis employing high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) is very useful for the determination of enantiomers at very low concentrations, as the ones found in biological matrices, for instance, in pharmacokinetic studies. The first step in the analysis of drugs in biological fluids is the extraction procedure. The most common extraction procedures employed are liquid-liquid extraction and solidphase extraction. These techniques show several drawbacks, such as the high amount of organic solvent consumed. So, based on this fact, techniques that consume small amounts of organic solvents are desirable. Among these techniques, solid-phase microextraction (SPME) and liquid-phase microextraction (LPME) have been stood out. The main advantages of these techniques are the small amount of organic solvent consumed and its high capacity in drug concentration. In this work, our purpose was to employ the SPME and the LPME as sample preparation techniques to develop stereoselective methods to be further applied in pharmacokinetic studies. SPME was employed for the stereoselective analysis of ibuprofen, hydroxychloroquine and their major metabolites.On the other hand,LPME was employed only for the enantioselective analysis of hydroxychloroquine and its metabolites. The first step was the separation optimization of the drugs and their metabolites by HPLC using several chiral columns, and the separation optimization of hydroxychloroquine and its metabolites by CE. Next, the extraction optimizations and method validations were performed. The enantioselective analysis of ibuprofen in human urine was carried out in the Chiralpak AD-RH® column, using methanol-pH 3.0 phosphoric acid solution (75 : 25 v/v) as mobile phase. The method was linear over the 0.5 - 25 µg mL-1 concentration range for both enantiomers. The fiber used in this method was PDMS-DVB 60 µm.The analysis of 2-hydroxyibuprofen and carboxyibuprofen was performed on a Chiralpak AS® column using hexane:isopropanol (95 : 5 v/v) plus 0.05% trifluoroacetic acid as the mobile phase. The method was linear over the 5 - 50 µg mL-1 concentration range. To perform the extractions, a CW-TPR 50 µm coated fiber was employed. The analysis of hydroxychloroquine and its major metabolites (DCQ and DHCQ) was done on a Chiralcel OD-H® column using hexane-methanol-ethanol (96 : 2 : 2, v/v/v) plus 0.2% diethylamine as mobile phase. The extraction was performed using a PDMS-DVB 60 µm coated fiber. The method was linear over the range of 50 - 1000 ng mL-1 for HCQ enantiomers and over the range of 42 - 416 ng mL-1 for DCQ and DHCQ enantiomers. LPME and CE were applied for the chiral determination of hydroxychloroquine and its metabolites (DCQ, DHCQ, BDCQ) in human urine. The electrophoretic separations were carried out in 100 mmol L-1tris(hydroxymethyl)aminomethane buffer (pH adjusted to 9.0 with phosphoric acid) containing 1% (w/v) S-b-CD and 30 mg mL-1 HP-?-CD, with a constant voltage of +18 kV. The method was linear over the concentration range of 10-1000 ng mL -1 for each HCQ stereoisomer and 21-333 ng mL -1 for each metabolite stereoisomer. Within-day and between-day assay precision and accuracy for all described methods were lower than 15%. The developed methods were applied for the determination of the cumulative urinary excretion of ibuprofen metabolites and hydroxychloroquine and its metabolites after oral administration of racibuprofen and rac-hydroxychloroquine to health volunteers. Comparing the techniques, both SPME and LPME were efficient to extract the drugs and their metabolites from human urine. iv SPME showed to be an easier technique to handle, however, the drug amount recovered by this technique was too small. On the contrary, LPME was a more difficulty technique to be handled, but better recovery values were obtained with this technique.