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.

Solid phase microextraction (SPME), needle trap device (NTD), and membrane extraction with a sorbent interface (MESI) are solvent-free sample preparation techniques that were developed to perform the rapid routine analysis of organic compounds (VOCs) in various environmental matrices by integrating sampling, extraction, preconcentration and sample introduction procedures into one step. A portable ion mobility spectrometry (IMS) analyzer has some advantages, such as small size, light weight, operability under ambient pressure, air as carrier gas, and sensitivity, all of which make IMS suitable for on-site monitoring for low concentration of analytes. The aforementioned sampling and preconcentration techniques were coupled with a portable IMS analyzer, as well as a thermal desorption unit that can accommodate SPME, NTD and MESI, which was modified and combined with IMS for on-site monitoring of volatile organic compounds (VOCs) from human breath and plant emissions. Experimental results demonstrated that low detection limits were achievable for gaseous analytes, (25 ng/L for acetone (SPME-IMS), 43 ng/mL (NTD-IMS) and 2.3 ng/mL (MESI-IMS) for α-pinene). These three analytical systems were applied for on-site rapid determination of acetone in human breath and α-pinene from plant emissions respectively. The salient features of these systems that make them suitable for on-site monitoring of volatile organic compounds in different sources are: small size, simple operation, fast and/or on-line sampling, rapid analysis.

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.

For the first time, octahydroxyl methylresorcinarene with four hexyl groups on the lower rim was utilized in the in-situ preparation of a silica-based sol-gel organic-inorganic hybrid coating for sample preconcentration by capillary microextraction (CME). Tetraethoxysilane (TEOS) was chosen as a sol gel precursor to create a cross-linked sol-gel network via acid-catalyzed hydrolytic polycondensation reactions. Sol-gel chemistry helped in the in situ preparation of resorcinarene-containing extraction phase in the form of a surface coating. It also provided an effective means to chemically bind the coating to the inner surface of fused silica capillary via condensation of the hydroxyl groups in the sol-gel network with the silanol groups on the fused silica capillary inner surface. These chemically bonded sol-gel coatings demonstrated excellent thermal stability (up to 350 oC). The sol-gel resorcinarene coatings successfully extracted traces of polycyclic aromatic hydrocarbons (PAHs), ketones, phenols, amines, and alcohols from aqueous samples providing parts per trillion level detection limits (0.828 - 46.01 ng/L) in GC using a Flame Ionization Detector (FID). CME was performed by passing the aqueous samples through the resorcinarene coated microextraction capillary (10 cm). The extracted analytes where then thermally desorbed into the GC column connected to the exit end of the sol-gel microextraction capillary via a press-fit quartz connector. Peak area relative standard deviation (RSD %), a measure of the extraction performance reproducibility for the coated capillary, was found in the range of (1.1 % - 8.3 %). The sol-gel resorcinarene sorbent was characterized by FTIR spectrum which indicated the presence of hydroxyl groups in the coating even after the sol-gel reactions were completed which explains the affinity of the resorcinarene sol-gel coating toward polar analytes. Scanning Electron Microscopy (SEM) images of the coating reveraled the porous morphology and thickness of 3.5 - 4.0 µm for the coating. The sol-gel resorcinarene coated capillary provided excellent extraction performance for wide range of analytes.

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).

Sample preparation is often a bottleneck in systems for chemical analysis. The aim of this work was to investigate and develop new techniques to address some of the shortcomings of current sample preparation methods. The goal has been to provide full automation, on-line coupling to detection systems, short sample preparation times and high-throughput. In this work a new technique for sample preparation that can be connected on-line to liquid chromatography (LC) and gas chromatography (GC) has been developed. Microextraction in packed syringe (MEPS) is a new solid-phase extraction (SPE) technique that is miniaturized and can be fully automated. In MEPS approximately 1 mg of sorbent material is inserted into a gas tight syringe (100-250 μL) as a plug. Sample preparation takes place on the packed bed. Evaluation of the technique was done by the determination of local anaesthetics in human plasma samples using MEPS on-line with LC and tandem mass spectrometry (MS-MS). MEPS connected to an autosampler was fully automated and clean-up of the samples took about one minute. In addition, in the case of plasma samples the same plug of sorbent could be used for about 100 extractions before it was discarded. A further aim of this work was to increase sample preparation throughput. To do that disposable pipette tips were packed with a plug of porous polymer monoliths as sample adsorbent and were then used in connection with 96-well plates and LC-MS-MS. The evaluation of the methods was done by the analysis of local anaesthetics lidocaine and ropivacaine, and anti-cancer drug roscovitine in plasma samples. When roscovitine and lidocaine in human plasma and water samples were used as model substances, a 96-plate was handled in about two minutes. Further, disposable pipette tips may be produced at low cost and because they are used only once, carry-over is eliminated.

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.

Submitted by Ana Hilda Fonseca (anahilda@ufba.br) on 2016-08-31T15:22:10Z No. of bitstreams: 1 VERSÃO FINAL DA DISSERTAÇÃO DE MESTRADO - RAFAEL DOURADO PIMENTA FONTES.pdf: 2834377 bytes, checksum: 4df31943280d22c5475b7e744e8d6683 (MD5) / Approved for entry into archive by Vanessa Reis (vanessa.jamile@ufba.br) on 2016-09-02T16:18:17Z (GMT) No. of bitstreams: 1 VERSÃO FINAL DA DISSERTAÇÃO DE MESTRADO - RAFAEL DOURADO PIMENTA FONTES.pdf: 2834377 bytes, checksum: 4df31943280d22c5475b7e744e8d6683 (MD5) / Made available in DSpace on 2016-09-02T16:18:17Z (GMT). No. of bitstreams: 1 VERSÃO FINAL DA DISSERTAÇÃO DE MESTRADO - RAFAEL DOURADO PIMENTA FONTES.pdf: 2834377 bytes, checksum: 4df31943280d22c5475b7e744e8d6683 (MD5) / CNPq e FAPESB / Na atualidade, o consumo de organismos marinhos (principalmente peixes e camarões) tem aumentado cada vez mais, devido à sua constituição proteica. Nos últimos anos, com a elevada demanda de pescados, intensificou-se também o cultivo (aquicultura), principalmente de camarão, para suprir o mercado consumidor. A aceitação de alimentos de origem marinha pelo consumidor está diretamente relacionada ao odor e sabor que devem ser atrativos e agradáveis. As principais substâncias químicas identificadas com as responsáveis pelo “flavor” dos alimentos marinhos são os bromofenóis simples 2-bromofenol (2-BF), 4-bromofenol (4-BF), 2,4-dibromofenol (2,4-DBF), 2,6-dibromofenol (2,6-DBF) e 2,4,6-tribromofenol (2,4,6-TBF). A microextração com gota única (SDME) é uma técnica que apresenta muitas vantagens, quando comparada com as técnicas clássicas, pois permite o isolamento e pré-concentração dos analitos em um passo único, seguida de introdução da amostra em sistema de análise por CG-EM. Esta técnica vem ganhando destaque por não ser exaustiva, utilizar uma quantidade muito pequena de solvente (estando de acordo com os preceitos da química verde), requer um curto tempo de análise, tem elevada sensibilidade e baixo custo. Nesse trabalho foi desenvolvida uma nova metodologia analítica baseada em SDME e CG-EM para determinação de bromofenóis em abdômen e ração de camarão cultivado (carcinicultura) (Lithopenaeus vannamei) e abdômen de camarão pescado (Xiphopenaeus kroyeri, camarão sete barbas) em diferentes estações do ano. A otimização da técnica por CG-EM permitiu boa separação dos bromofenóis simples em apenas 15 minutos. Esta nova metodologia foi validada em função da linearidade das curvas analíticas, limite de detecção e quantificação, precisão e recuperação para cada um dos cinco analitos estudados. A recuperação e precisão variaram, respectivamente, de 50,8 a 103% e de 2,27 a 18,8%. Os limites de detecção e quantificação variaram, respectivamente, de 0,200 a 0,499 ng mL-1 e de 0,500 a 1,000 ng mL-1. Não foi perceptível uma relação regular ou linear entre a sazonalidade e a concentração dos bromofenóis nos camarões pescados. Tanto na primavera quanto no verão a maior concentração detectada foi do 2,4-DBF, enquanto no outono e inverno foram o 4-BF e o 2-BF respectivamente. Já no abdômen do camarão de cativeiro e na ração deste a maior concentração foi do 4-BF. A menor concentração na ração, no abdômen do camarão cultivado e na estação verão corresponde ao 2-BF, na primavera e no outono ao 2,6-DBF, enquanto no inverno ao 4-BF. A metodologia desenvolvida, além de apresentar baixos limites de detecção e quantificação, envolve menor tempo de análise, menor consumo de energia e solventes, sendo assim compatível com os preceitos da Química Verde. / At present, the consumption of marine organisms (mainly fish and shrimp) has increased even more, due to its constitution protein. In recent years, with the high demand of fish, also accelerated cultivation (aquaculture), especially shrimp, to supply the consumer market. Acceptance of marine foods by consumers is directly related to odor and flavor that should be attractive and pleasant. The main chemicals identified as responsible for the "flavor" of marine foods are simple bromophenol 2-bromophenol (2-BF), 4-bromophenol (4-BF), 2,4-dibromophenol (2,4-DBF) 2,6-dibromophenol (2,6-DBF) and 2,4,6-tribromophenol (2,4,6-TBF). A single droplet with microextraction (SDME) is a technique that has many advantages compared with conventional techniques, since it allows the isolation and preconcentration of analytes in a single step, followed by introduction of the sample analysis system GC-MS. This technique has been gaining attention for not being exhaustive, use a very small amount of solvent (which is consistent with the principles of green chemistry), requires a short analysis time, has high sensitivity and low cost. In this work we developed a new analytical methodology based SDME and GC-MS to determine bromophenols in abdomen and feed farmed shrimp (Lithopenaeus vannamei) shrimp fished and abdomen (Xiphopenaeus kroyeri, seven shrimp whiskers) in different seasons year. Optimization of GC-MS technique allowed good separation of bromophenols simple in just 15 minutes. This new methodology was validated on the basis of the calibration curves linearity, limit of detection and quantification accuracy and recovery for each of the five analytes studied. The recovery and precision ranging, respectively, from 50,8 to 103% and from 2,27 to 18,8%. The limits of detection and quantification varied, respectively, from 0,200 to 0,499 ng ml-1 and 0,500 to 1,000 ng ml-1. It was not noticeable or a regular relationship between the linear and the concentration of bromophenol seasonality in shrimp fished. Both in spring and in summer the highest concentration detected was 2,4-DBF, while in autumn and winter were the 4-BF and 2-BF respectively. Already in the abdomen of shrimp feed in captivity and this was the highest concentration of 4-BF. The lowest concentration in the feed, abdomen farmed shrimp and summer season match 2-BF, in spring and autumn to 2,6-DBF, while in winter the 4-BF. The methodology developed, and have low limits of detection and quantification, involves less analysis time, lower power consumption and solvents and is therefore compatible with the principles of Green Chemistry

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.