The main objective of the emerging field of metabolomics is the analysis of all small molecule metabolites present in a particular living system in order to provide better understanding of dynamic processes occurring in living systems. This type of studies is of interest in various fields including systems biology, medicine and drug discovery. The main requirements for sample preparation methods used in global metabolomic studies are lack of selectivity, incorporation of a metabolism quenching step and good reproducibility. The efficiency of metabolism quenching and stability of analytes in selected biofluid or tissue dictate how accurately the analytical results represent true metabolome composition at the time of sampling. However, complete quenching of metabolism is not easily accomplished, so sample preparation can significantly affect metabolome's composition and the quality of acquired metabolomics data. In this research, the feasibility of the use of solid-phase microextraction (SPME) in direct extraction mode for global metabolomic studies of biological fluids based on liquid chromatography-mass spectrometry (LC-MS) was investigated for the first time. Initial research presented in this thesis focused on resolving several outstanding issues regarding the use of SPME for the analysis of biological fluids. SPME was not simultaneously capable to provide high-sample throughput and high degree of automation when coupled to LC-MS. This was successfully addressed through the development and evaluation of a new robotic station based on a 96-well plate format and an array of 96 SPME fibres. The parallel format of extraction and desorption allowed increased sample throughput of >1000 samples/day which represents the highest throughput of any SPME technique to date. This exceeds sample throughput requirements for a typical metabolomics study whereby ~100 samples/day are processed. SPME can also be used for direct in vivo sampling of flowing blood of an animal without the need to isolate a defined sample volume. This format of SPME is particularly attractive for metabolomic studies as it decreases the overall number of steps and also eliminates the need for metabolism quenching step because only small molecular weight species are extracted by the device, whereas large biological macromolecules such as proteins are not extracted by the coating. In current work, in vivo SPME sampling was successfully applied for sampling of mice for the first time. The proposed sampling procedure was fully validated against traditional terminal and serial sampling approaches for a pharmacokinetic study of carbamazepine and its metabolite. Excellent agreement of pharmacokinetic parameters such as systemic clearance, steady-state volume of distribution and terminal half-life was found for all three methods, with no statistically significant differences (p>0.05). The performance of new prototype commercial SPME devices based on hypodermic needle was also evaluated within the context of the study. The availability of such single-use devices with excellent inter-fibre reproducibility (<10% RSD) presents an important step forward in order to gain wider acceptance of in vivo SPME sampling. Finally, existing SPME coatings were not suitable for the simultaneous direct extraction of both hydrophilic and hydrophobic species, which is one of the requirements for a successful global metabolomics study. To address this issue, a systematic study of 40 types of commercially available sorbents was carried out using a metabolite standard test mixture spanning a wide molecular weight (80-777 Da) and polarity range (log P range of -5 to 7.4). The best performance for balanced extraction of species of varying polarity was achieved by (i) mixed-mode coating containing octadecyl or octyl group and benzenesulfonic acid ion exchange group, (ii) polar-enhanced polystyrene-divinylbenzene polymeric coatings and (iii) phenylboronic acid coatings. The second aspect of the research focused on the evaluation of SPME for a global metabolomics study of human plasma using two complementary LC-MS methods developed on benchtop Orbitrap MS system: reverse-phase method using pentafluorophenyl LC stationary phase and HILIC method using underivatized silica stationary phase. The parameters influencing overall method sensitivity such as voltages, mass ranges and ion inject times into C-trap were optimized to ensure best instrument performance for global metabolomic studies. Orbitrap system provided a powerful platform for metabolomics because of its high resolution and mass accuracy, thus helping to distinguish between metabolites with same nominal mass. The acquisition speed of the instrument at the highest resolution setting was insufficient for use with ultrahigh performance liquid chromatography (UHPLC), so all methods were developed using conventional LC. However, overall metabolite coverage achieved in current study compared well or even exceeded metabolite coverage reported in literature on different LC-MS or UHPLC-MS platforms including time-of-flight, quadrupole time-of-flight and hybrid Orbitrap instruments. The performance of SPME was fully compared versus traditional methods for global metabolomics (plasma protein precipitation and ultrafiltration). The main findings of this systematic study show that SPME provides improved coverage of hydrophobic metabolites versus ultrafiltration and reduces ionization suppression effects observed with both plasma protein precipitation and ultrafiltration methods. Using SPME, <5% and <20% of peaks showed significant matrix effects in reverse phase and HILIC methods, respectively and the observed effects were mostly correlated to elution within retention time window of anticoagulant for the majority of metabolites showing this effect. This improves overall quality of collected metabolomics data and can also improve metabolite coverage. For example, the highest number of metabolite features (3320 features) was observed using SPME in combination with negative ESI reverse-phase LC method, while in positive ESI mode plasma protein precipitation with methanol/ethanol mixture provided the most comprehensive metabolite coverage (3245 features versus 1821 features observed for SPME). Method precision of SPME method was excellent as evaluated using median RSD (11-18% RSD) of all metabolites detected. A proof-of-concept in vivo SPME study was also performed on mice to study the effects of carbamazepine administration and shows that SPME can be used as successful sample preparation method for global metabolomic studies in combination with unsupervised statistical data analysis techniques. This study highlights important advantages of in vivo sampling approaches including the ability to capture short-lived and/or unstable metabolites, to achieve truer representation of the metabolome at the time of sampling than achievable by blood withdrawal methods and the ability to use smaller animal cohorts while obtaining highly-relevant data sets. The experimental results provide new and useful insight into the effects of different sample preparation methods on the collected metabolomics data, and establish both in vitro and in vivo SPME as a new tool for global LC-MS metabolomics analysis for the first time.

Evaluation of solid-phase microextraction as an alternative official method for analysis of polymers migration. The objective this study was to compaire the official methods with solid-phase microextraction (SPME)for the analysis of compounds migrating from cross-linked polyethylene into water. A medium polarity polydimethylsiloxane/divinylbenzene (PDMS/DVB)was proved most efficient for the SPME extraction. However, when applied to water samples in contact with polyethylene, SPME proved to be immensely more sensitive and have a greater extraction range than liquid-liquid extraction (LLE). It was proved the migration of Phenol, 2,4-bis(1,1-dimethylethyl). Concentration of this compound 0,6-0,15mg/l.It was proved the migration of Cyclohexadiene-1,4-dione, 2,6-bis(1,1-dimethylethyl).

Pasiūlyta nauja kietafazės mikroekstrakcijos sistema, kurioje nerūdijančio plieno strypelis dengtas anglies nanovamzdeliais, ištirtas jos terminis stabilumas ir atrankumas, nustatyta, kad sistema tinka lakių aromatinių angliavandenilių ekstrakcijai iš tirpalo ir iš viršerdvės. Ištirtos keturių skysčių-skysčių mikroekstrakcijos metodų - skysčių-skysčių mikroekstrakcijos kapiliare, mikroekstrakcijos užšaldomu tirpiklio lašu, dispersinės skysčių-skysčių mikroekstrakcijos ir dispersinės skysčių-skysčių mikroekstrakcijos užšaldant ekstraktą - galimybės ekstrahuoti lakius aromatinius angliavandenilius. Optimizuotos tirtų metodų ekstrakcijos sąlygos, nustatytos pagrindinės analizinės charakteristikos. Visų metodų rezultatų pasikartojamumas ir aptikimo ribos artimi. Išimtis – dispersinė skysčių-skysčių mikroekstrakcija, kuria gautos kiek didesnės aptikimo ribos. Greičiausi ekstrakcijos metodai - dispersinė skysčių-skysčių mikroekstrakcija ir dispersinė skysčių-skysčių mikroekstrakcija užšaldant ekstraktą, ilgiausiai trunka mikroekstrakcija užšaldomu tirpiklio lašu. Švarių mėginių ekstrakcijai tinka visi tirti metodai, užterštiems mėginiams geriau tinka kietafazė mikroekstrakcija iš viršerdvės arba skysčių-skysčių mikroekstrakcija kapiliare. Paruoštos lakių aromatinių angliavandenilių mikroekstrakcijos metodikos pritaikytos vandens ir sniego mėginių analizei. / A new solid phase microextraction system composed of nanotubes coating fixed on a stainless steel support is suggested. Thermal stability and selectivity of the system was examined. It was determined that the system can be used for direct and headspace extraction of volatile aromatic hydrocarbons. Possibilities of four liquid phase microextraction techniques – hollow fibre liquid phase microextraction, liquid phase microextraction based on the solidification of a floating drop, dispersive liquid-liquid microextraction and dispersion-solidification liquid-liquid microextraction – for the extraction of volatile aromatic hydrocarbons were investigated. Extraction conditions of the investigated methods were optimized and the main analytical characteristics were determined. For all the methods detection limits and repeatability of the results are close. An exception is dispersive liquid-liquid microextraction with slightly higher detection limits. An extraction is especially fast using dispersive liquid-liquid microextraction and dispersion-solidification liquid-liquid microextraction. The most time-consuming method is liquid phase microextraction based on the solidification of a floating drop. All the methods are suitable for clean sample extraction. For the extraction from complex matrices the most suitable methods are headspace solid phase microextraction and hollow fibre liquid phase microextraction. The prepared microextraction techniques were applied for water and snow... [to full text]

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.

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.

Heating milk to high temperatures such as 140 ºC, as used in ultra high temperature (UHT) processing, causes physical and chemical changes in the milk. The production of a cooked flavour is a major change which reduces consumer acceptance of the UHT milk. It has been correlated with the formation of volatile sulphur compounds (VSCs) that result from milk proteins, principally the whey proteins β-lactoglobulin, containing the the sulphur amino acids cystine, cysteine and methionine. The VSCs in milk, whose concentrations are in the parts per billion to parts per million range, are highly reactive, easily oxidised, and sensitive to heat during thermal processing and analysis; this makes them a challenge to analyse. A sensitive method based on gas chromatography with pulsed flame photometric detection coupled with headspace sampling by solid phase microextraction (SPME/GC/PFPD) was developed to detect these compounds in commercial UHT milk and to investigate their production and disappearance during heating and storage. The SPME/GC/PFPD procedure was optimised using different extraction time (15 min, 30 min, & 60 min) – temperature (30 oC, 45 oC & 60 oC) combinations with CAR/PDMS fibre to obtain maximum sensitivity. A short extraction time (15 min) at low temperature (30 oC) was chosen to provide high sensitivity for detecting all VSCs in UHT milk without introducing artefactual VSCs. The extraction method and GC run time (16 min) make this method simple and fast. Nine VSCs were detected in commercial indirectly processed UHT milk, skim and whole. These are hydrogen sulphide (H2S), carbonyl sulphide (COS), methanethiol (MeSH), dimethyl sulphide (DMS), carbon disulphide (CS2), dimethyl disulphide (DMDS), dimethyl sulphoxide (DMSO), dimethyl sulphone (Me2SO2) and dimethyl trisulphide (DMTS). An additional unknown compound was detected but could not be identified by GC/MS because its concentration was below the detection limit of the MS detector. The concentrations of H2S, DMS and DMTS were higher than their threshold values indicating their importance in milk flavour, especially cooked flavour. Several attempts have been made to reduce the cooked flavour in UHT milk. In the current research, the use of hydrogen peroxide (H2O2) to oxidise the VSCs and thereby reduce cooked flavour was investigated. H2O2 is used as a milk preservative and is generally recognised as safe (GRAS) in USA. Several concentrations of H2O2 (0.001%, 0.005%, 0.01%, 0.02% & 0.03%) were added to milk to assess its effects on VSCs and on whey proteins denaturation in UHT milk. H2O2 effectively reduced the concentration of all VSCs, except DMDS which was increased, presumably by oxidation of MeSH. H2S was completely oxidised or reduced below its threshold value. Low concentrations of H2O2 (0.001% & 0.005%) had no effect on, or decreased, the extent of denaturation of β-lactoglobulin when added after or before processing, respectively. Some UHT plants use severe heating conditions, leading to high levels of denaturation of whey proteins, particularly β-Lg, the main source of the VSCs in milk. Correlations between heat severity, β-Lg denaturation and individual VSC generation were investigated in milk batch-heated at 80 oC and 90 oC, and UHT milk processed at 120-150 oC. In accordance with previous reports, β-Lg was more heat-sensitive than α-La. Only five VSCs were detected. The concentrations of H2S and MeSH correlated well with denaturation of β-Lg and α-La. DMS concentration correlated well with β-Lg in UHT milk but not in the batch-heated milk. CS2 did not show a good correlation with heat intensity and appeared to plateau out after a certain level of heating. Conversely, COS and MeSH seemed to require a certain minimum amount of heat before generation commenced; this corresponded to denaturation of β-Lg above 49% and 89% respectively at 80 oC. The higher concentrations of DMS and H2S in UHT milk compared with batch-heated samples having similar degrees of denaturation suggested other possible sources for their production and the importance of the heat severity in generating them. For example, at high heat intensity, S-methylmethionine and thiamine could be sources of DMS and H2S respectively. Furthermore, in whole milk as used in this work, milk fat globule membrane proteins are another source of VSCs. The outcome of this study will help UHT manufacturers to understand the production and disappearance of the VSCs in commercial UHT milk and how to adjust the processing conditions to avoid generation of cooked flavour. Additionally, the promising results of using low concentrations of H2O2 to oxidise the VSCs will provide the industry with another means of reducing cooked flavour. Before H2O2 use is implemented in UHT processing, future studies are required to evaluate all of its effects, including sporicidal effects. Overall, this study makes a contribution to finding a solution to the cooked flavour problem in UHT milk, thereby increasing market share of this milk in countries such as Australia, the UK and North America where cooked flavour is the main barrier to its consumer acceptance.

Ample research has been published regarding the effects of environmental decomposition on volatile organic profiles of tissue, however literature concerning the volatile organic profiles of thermally degraded tissue is limited in quantity and scope. The purpose of this study was to investigate the effects of temperature on the headspace volatile organic compounds produced by muscle, subcutaneous fat, skin, and punch biopsy samples. The majority of the compounds for each tissue type were alcohols and aldehydes. Compounds were extracted using solid phase microextraction and identified using gas chromatography/mass spectrometry. Compounds such as nonanal, 1-octen-3-ol, octanal, and hexanal were present in the volatile organic compound profile for many tissue types at a majority of the temperatures, particularly from 150°C to 300°C. 2-pentylfuran was the most abundant component in the profile of skin samples from 150°C to 300°C. The profile of fresh subcutaneous fat had numerous branched alkanes, while thermally degraded subcutaneous fat profiles were comprised mostly of aldehydes and alcohols. The profile of muscle was primarily composed of alcohols and aldehydes up to 300°C, whereas the most abundant compound at 350°C was trimethylpyrazine. There were consistent compounds identified among each tissue group. The abundance patterns of alcohols and aldehydes over increasing temperatures differed for each tissue type. Analysis of the data gathered in this study indicates that muscle, subcutaneous fat, and skin contribute characteristic compounds, such as alcohols and aldehydes, to the profile of the punch biopsy samples. The findings further suggest that temperature affects the volatile organic profile of tissue in terms of the compounds identified and the abundance trends of certain compounds.

Os vinhos espumantes elaborados pelo método Tradicional são elaborados, comumente, a partir das uvas Chardonnay, Pinot Noir, Chadonnay, Riesling, Viognier, Trebbiano e Pinot Noir e os componentes voláteis destes espumantes já têm merecido a atenção de diversos estudos científicos. Entretanto, vinhos espumantes produzidos a partir de outros varietais de uvas ainda não foram alvo de pesquisas científicas. A segunda fermentação ocorre dentro da garrafa e acaba por conferir uma maior complexidade aromática ao espumante produzido pelo método Tradicional, devido ao contato do vinho com as leveduras em meio redutor, por um determinado período de tempo. Isto acontece devido aos produtos secundários do metabolismo das leveduras, durante a conversão de açúcares em etanol e dióxido de carbono. Esta conversão depende dos nutrientes adicionados, chamados adjuvantes de fermentação, bem como da espécie de levedura utilizada, visto que cada levedura possui um metabolismo diferente para a utilização dos nutrientes e açúcares presentes no vinho base. Assim, objetivou-se, neste trabalho, caracterizar os componentes voláteis de vinhos espumantes de uma vinícola gaúcha, que emprega um assemblage inovador, empregando uvas Chadonnay, Riesling, Viognier, Trebbiano e Pinot Noir. Do assemblage deste vinho base utilizou-se, para segunda fermentação, duas espécies de leveduras comerciais: Saccharomyces cerevisiae e Saccharomyces bayanus. Para cada levedura utilizada na fermentação do vinho base, oito diferentes adjuvantes de fermentação foram empregados. A determinação dos compostos voláteis se deu através da técnica de microextração em fase sólida no modo headspace (HS-SPME) e cromatografia gasosa acoplada a detector de espectrometria demassa quadrupolar (GC/MS). Ao total, 25 compostos foram tentativamente identificados nos vinhos em estudo, sendo os compostos majoritários citados a seguir, com sua possível contribuição para o aroma destes vinhos: octanoato de etila (aroma de fruta), álcool isoamílico (aroma de banana), ácido octanoico (aroma de pimentão) e álcool feniletílico (aroma floral). O fenetil fenilacetato, um dos compostos minoritários tentativamente identificado em alguns dos vinhos, até então não reportado em vinho espumante, é associado a aroma frutado. Não foi possível distinguir subgrupos entre os 16 vinhos em estudo, provenientes de diferentes condições na segunda fermentação, quando as áreas cromatográficas dos compostos voláteis destes 16 vinhos foram submetidas a análise de cluster. Isto implica em que, nas condições experimentais deste estudo, não foi possível distinguir os voláteis dos vinhos fermentados (2ª fermentação) com S. cereviseae e os fermentados com S. bayanus. A mesma análise de cluster mostrou a subdivisão dos compostos voláteis dos 16 vinhos em dois grupos, os quais se distinguiram, provavelmente, devido aos diferentes adjuvantes nutricionais empregados: fosfato e Thiazote. Desta forma, através de análise qualitativa por HS-SPME-GC/MS, foi possível verificar a homogeneidade do perfil volátil dos 16 vinhos espumantes, obtidos a partir de diferentes adjuvantes de fermentação e duas espécies distintas de leveduras Saccharomyces sp, além de comparar os componentes voláteis presentes nestes espumantes com aqueles reportados na literatura para outros vinhos espumantes. / Sparkling wines elaborated by Traditional Method are usually produced by the grapes Chardonnay, Pinot Noir and Riesling, in which the volatile compounds of these sparkling wines have been calling attention to scientific studies. However, sparkling wines produced by other varietal grapes have not been a target of scientific research yet. The second fermentation occurs inside the bottle, in which confer a greater aromatic complexity to the sparkling wine produced by Traditional Method, due to the contact of it with lees in a reducing medium during a certain period of time. This happens due to secondary products of yeast metabolism, during the conversion of sugar in ethanol and carbon dioxide. This conversion depends on the nutrients added, called fermentation adjuvants, as the yeast used, since each one has a different metabolism for using this nutrients and sugars presented in the base wine. Thus, this work aims to characterize the volatile compounds of a south Brazilian winery, which use an innovative assemblage, using the grapes Chardonnay, Riesling, Viognier, Trebbiano and Pinot Noir. To the base wine, two different commercial yeasts were added: Saccharomyces cerevisiae and Saccharomyces bayanus to the performance of second fermentation. To each yeast used for fermenting the base wine, eight different fermentation adjuvants were used. The determination of volatile compounds were performed by Headspace solid-phase Microextraction (HS-SPME) and gas chromatography coupled to a mass quadrupole spectrometry (GC/MS). In total, 25 compounds were tentatively identified in the studied sparkling wines, being the majority listed as it follows, with their possible contribution to these sparkling wines aroma: ethyl octanoate (fruity), isoamyl alcohol (banana), octanoic acid (green pepper), and phenethyl alcohol (flower). Phenethyl phenylacetate, one of the minority compounds tentatively identified in some of the sparkling wines, is associated with fruity aroma. It was not possible to distinguish subgroups from different conditions during the second fermentation, when submitting the chromatographic areas of volatile compounds to cluster analysis. It implies that, under the experimental conditions of these study, it was not possible to differ the volatile compounds of the fermented (2nd fermentation) with S. cerevisiae and those which were fermented with S. bayanus. The same cluster analysis showed a subdivision of volatile compounds of the 16 wines in two groups, in which were probably distinguished due to the different nutritional adjuvants used: phosphate and Thiazote. Thus, throughout qualitative analysis by HS-SPME-GC/MS, it was possible to verify the homogeneity of volatile profile of the 16 sparkling wines, obtained by different fermentation adjuvants and two different yeast species of Saccharomyces sp, besides the comparison of volatile compounds presented in these sparkling wines with those others reported in the literature.

A depressão, tida como a doença do final do século XX, acarreta diversos distúrbios físicos, mentais e emocionais. Amitriptilina,. imipramina, nortriptilina e desipramina são antidepressivos tricíclicos (ADT) largamente usados no tratamento de desordens depressivas. Os ADT são fármacos que apresentam alta variabilidade interindividual na farmacocinética e resposta clínica não facilmente ou não imediatamente mensurável. Assim faz-se necessário o desenvolvimento e validação de métodos analíticos capazes de determinar com a devida confiabilidade suas concentrações em amostras de plasma. Os métodos desenvolvidos possuem uma etapa de extração para posterior separação e quantificação. Os métodos de extração avaliados foram a extração líquido-líquido (LLE) e a microextração em fase sólida (SPME). As técnicas usadas para separação e quantificação foram cromatografia líquida (LC) e eletroforese capilar (CE). Dois métodos foram desenvolvidos e validados: LLE/LC e LLE/CE. Usando uma fibra com recobrimento PDMS 100 mm, um método SPME/LC (modo de dessorção off-line) foi desenvolvido e os parâmetros relativos a SPME foram otimizados fazendo uso de planejamento fatorial (23) e simplex. Usando as mesmas condições otimizadas para a fibra PDMS testou-se uma fibra PDMS/DVB 65 mm. Avaliou-se a CE para a determinação dos ADT em formulações farmacêuticas e o comportamento eletroforético destes compostos em diferentes meio (água, metanol e acetonitrila) foi avaliado. De acordo com os parâmetros avaliados (linearidade, precisão (intra e inter ensaios), recuperação, limites de quantificação (LOQ) e detecção) ambos os métodos (LLE/LC e LLE/CE) mostraram-se aplicáveis para a determinação dos ADT em níveis plasmáticos. O método SPME/LC (off-line) que fez uso da fibra com recobrimento PDMS/DVB apresentou LOQ suficientemente baixos para determinar os ADT em concentrações plasmáticas, apesar de consumir um tempo maior para execução, quando comparado aos métodos LLE/LE e LLE/CE. A validação e aplicação de um método fazendo uso de CE para a análise dos ADT em formulações farmacêuticas mostra a aplicabilidade desta técnica para análises de rotina. Determinou-se por CE o pKa* dos ADT em água, metanol e ACN. A ordem dos valores obtidos em meio aquoso e metanólico é inversa em relação a ordem obtida em ACN. Assim, pôde-se correlacionar a carga das espécies em cada condição de análise com a ordem de migração dos ADT em cada meio. / Depressive disorders imply in a variety of physical, mental and emotional disturbances. Amitriptyline, imipramine, nortriptyline e desipramine are tricyclic antidepressants (TAD) largely used in depression treatment. The TADs are drugs that show high inter individual variability in pharmacokinetics, low clinical response or even they are not straightforwardly measured. Therefore, it is necessary the development and validation of analytical methods for TAD determination in plasma samples. The developed methods have an extraction step for subsequently separation and quantification. The extraction methods appraised were liquid-liquid extraction (LLE) and solid phase microextraction (SPME). The analytical techniques used for separation and further quantification were liquid chromatography (LC) and capillary electrophoresis (CE). Two methods were developed and validated: LLE/LC and LLE/CE. Using a PDMS coating (100 mm), a SPME/LC method (with off-line dessorption) was developed and the experimental conditions relative to SPME were optimized using a factorial planning (23) and a simplex methodology. The same optimized conditions (for PDMS) were used for a PDMS/DVB (65 mm) coating. According to the appraised parameters such as linearity, precision (intra and inter assays), recovery and limits of quantification and detection, the methods LLE/LC and LLE/CE showed suitable for TAD determination in plasma samples regarding the therapeutic plasmatic concentration. The SPME/LC method (off-line desorption) with PDMS/DVB coating has also shown LOQ lower than the minimum therapeutic plasmatic concentration, implying that may be subject to validation. This method may be used for TAD analysis even though the total analysis time was greater those from LLE/LC and LLE/CE methods. CE was also applied for TAD determination in pharmaceutical formulations and the electrophoretic behavior such compounds in different separation media, especially non-aqueous solvents, was studied. The validation and application of a method using CE for TAD determination in pharmaceutical formulations has shown the applicability of this technique for routine analysis. The migration order in aqueous and methanolic media is opposite to obtained in acetonitrile, and the pKa* of the TAD were determined in different media (aqueous, methanol and acetonitrile) by CE. With the calculated values, it was possible to correlate the species total charge to the migration order in each medium.

Os vinhos espumantes elaborados pelo método Tradicional são elaborados, comumente, a partir das uvas Chardonnay, Pinot Noir, Chadonnay, Riesling, Viognier, Trebbiano e Pinot Noir e os componentes voláteis destes espumantes já têm merecido a atenção de diversos estudos científicos. Entretanto, vinhos espumantes produzidos a partir de outros varietais de uvas ainda não foram alvo de pesquisas científicas. A segunda fermentação ocorre dentro da garrafa e acaba por conferir uma maior complexidade aromática ao espumante produzido pelo método Tradicional, devido ao contato do vinho com as leveduras em meio redutor, por um determinado período de tempo. Isto acontece devido aos produtos secundários do metabolismo das leveduras, durante a conversão de açúcares em etanol e dióxido de carbono. Esta conversão depende dos nutrientes adicionados, chamados adjuvantes de fermentação, bem como da espécie de levedura utilizada, visto que cada levedura possui um metabolismo diferente para a utilização dos nutrientes e açúcares presentes no vinho base. Assim, objetivou-se, neste trabalho, caracterizar os componentes voláteis de vinhos espumantes de uma vinícola gaúcha, que emprega um assemblage inovador, empregando uvas Chadonnay, Riesling, Viognier, Trebbiano e Pinot Noir. Do assemblage deste vinho base utilizou-se, para segunda fermentação, duas espécies de leveduras comerciais: Saccharomyces cerevisiae e Saccharomyces bayanus. Para cada levedura utilizada na fermentação do vinho base, oito diferentes adjuvantes de fermentação foram empregados. A determinação dos compostos voláteis se deu através da técnica de microextração em fase sólida no modo headspace (HS-SPME) e cromatografia gasosa acoplada a detector de espectrometria demassa quadrupolar (GC/MS). Ao total, 25 compostos foram tentativamente identificados nos vinhos em estudo, sendo os compostos majoritários citados a seguir, com sua possível contribuição para o aroma destes vinhos: octanoato de etila (aroma de fruta), álcool isoamílico (aroma de banana), ácido octanoico (aroma de pimentão) e álcool feniletílico (aroma floral). O fenetil fenilacetato, um dos compostos minoritários tentativamente identificado em alguns dos vinhos, até então não reportado em vinho espumante, é associado a aroma frutado. Não foi possível distinguir subgrupos entre os 16 vinhos em estudo, provenientes de diferentes condições na segunda fermentação, quando as áreas cromatográficas dos compostos voláteis destes 16 vinhos foram submetidas a análise de cluster. Isto implica em que, nas condições experimentais deste estudo, não foi possível distinguir os voláteis dos vinhos fermentados (2ª fermentação) com S. cereviseae e os fermentados com S. bayanus. A mesma análise de cluster mostrou a subdivisão dos compostos voláteis dos 16 vinhos em dois grupos, os quais se distinguiram, provavelmente, devido aos diferentes adjuvantes nutricionais empregados: fosfato e Thiazote. Desta forma, através de análise qualitativa por HS-SPME-GC/MS, foi possível verificar a homogeneidade do perfil volátil dos 16 vinhos espumantes, obtidos a partir de diferentes adjuvantes de fermentação e duas espécies distintas de leveduras Saccharomyces sp, além de comparar os componentes voláteis presentes nestes espumantes com aqueles reportados na literatura para outros vinhos espumantes. / Sparkling wines elaborated by Traditional Method are usually produced by the grapes Chardonnay, Pinot Noir and Riesling, in which the volatile compounds of these sparkling wines have been calling attention to scientific studies. However, sparkling wines produced by other varietal grapes have not been a target of scientific research yet. The second fermentation occurs inside the bottle, in which confer a greater aromatic complexity to the sparkling wine produced by Traditional Method, due to the contact of it with lees in a reducing medium during a certain period of time. This happens due to secondary products of yeast metabolism, during the conversion of sugar in ethanol and carbon dioxide. This conversion depends on the nutrients added, called fermentation adjuvants, as the yeast used, since each one has a different metabolism for using this nutrients and sugars presented in the base wine. Thus, this work aims to characterize the volatile compounds of a south Brazilian winery, which use an innovative assemblage, using the grapes Chardonnay, Riesling, Viognier, Trebbiano and Pinot Noir. To the base wine, two different commercial yeasts were added: Saccharomyces cerevisiae and Saccharomyces bayanus to the performance of second fermentation. To each yeast used for fermenting the base wine, eight different fermentation adjuvants were used. The determination of volatile compounds were performed by Headspace solid-phase Microextraction (HS-SPME) and gas chromatography coupled to a mass quadrupole spectrometry (GC/MS). In total, 25 compounds were tentatively identified in the studied sparkling wines, being the majority listed as it follows, with their possible contribution to these sparkling wines aroma: ethyl octanoate (fruity), isoamyl alcohol (banana), octanoic acid (green pepper), and phenethyl alcohol (flower). Phenethyl phenylacetate, one of the minority compounds tentatively identified in some of the sparkling wines, is associated with fruity aroma. It was not possible to distinguish subgroups from different conditions during the second fermentation, when submitting the chromatographic areas of volatile compounds to cluster analysis. It implies that, under the experimental conditions of these study, it was not possible to differ the volatile compounds of the fermented (2nd fermentation) with S. cerevisiae and those which were fermented with S. bayanus. The same cluster analysis showed a subdivision of volatile compounds of the 16 wines in two groups, in which were probably distinguished due to the different nutritional adjuvants used: phosphate and Thiazote. Thus, throughout qualitative analysis by HS-SPME-GC/MS, it was possible to verify the homogeneity of volatile profile of the 16 sparkling wines, obtained by different fermentation adjuvants and two different yeast species of Saccharomyces sp, besides the comparison of volatile compounds presented in these sparkling wines with those others reported in the literature.