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In quantitative proteomics, many of the LC-MS based approaches employ stable isotopic labelling to provide relative quantitation of the proteome in different cell states. In a typical approach, peptides are first detected and identified by tandem MS scans prior to quantifying proteins. This provides the researcher with a large amount of data that are not useful for quantitation. It is desirable to improve the throughput of current approaches to make proteomics a more routine experiment with an enhanced capacity to detect differentially expressed proteins. This thesis reports the developments towards this goal, including an assessment of the viability of stable dimethyl labelling for comparative proteomic measurements and the evaluation of a dynamic algorithm called Parallel Isotopic Tag Screening (PITS) for the detection of isotopically labelled peptides for quantitative proteomics without the use of tandem MS scans.

In this work were synthesized ketamine isotopomers. Ketamine is used in human medicine and veterinary sectors. It has very broad spectrum of pharmacological effects: anesthetic, analgesic, hallucinogenic, bronchodilator, cardiovascular and antidepressive, which is currently in the research. At first was synthesized precursor of ketamine, N- desmethylketamine which was subsequently labeled the deuterium, tritium and carbon- 14. For the determination of purity and identity mass spectrometry and nuclear magnetic resonance spectroscopy were used. KEY WORDS synthesis of ketamine, ketamine, N-desmethylketamine, nor-ketamine, isotopically labeled ketamine, deuterium, tritium, carbon-14.

Estudou-se em casa-de-vegetação o efeito do volume de terra no desenvolvimento das plantas de feijão e milho, na absorção de fósforo pelas culturas e na determinação do P isotopicamente trocável (Valor L), em função do periódo de desenvolvimento dessas culturas e dois níveis de fertilidade do solo quanto ao fósforo. Avaliou-se também o efeito do P na planta proveniente da semente no cálculo do Valor L (L: valor L calculado sem descontar o P proveniente da semente e L (-Ps): valor L calculado descontando o P proveniente da semente). O estudo foi realizado com Latossolo Vermelho-amarelo em vasos plásticos contendo: 0,5; 1,0; 2,0; 4,0; 8,0; 12,0 e 18,0 L de terra; com e sem adubação de P (50 mg kg-1 terra de P) e colhendo as plantas em dois períodos: 30 e 50 dias após a germinação. O delineamento experimental foi inteiramente casualizado com 3 repetições no esquema fatorial 7x2x2. As variações nos períodos de colheita e níveis de fertilidade são representadas por: E1F1 (30 dias, sem adubação), E1F2 (30 dias, adubado), E2F1 (50 dias, sem adubação) e E2F2 (50 dias, adubado). A concentração de P nas plantas não foi um parâmetro adequado para estabelecer o estado nutricional das plantas e os vasos de 0,5 e 1,0 L tiveram desenvolvimento prejudicado e conseqüentemente no conteúdo de P, não sendo também adequados para determinação do valor L. Houve efeito do P na planta proveniente da semente no valor L para volumes de terra menor que 4,0 L para plantas de feijão e 8,0 L para plantas de milho. O volume de terra de 2,0 L é recomendado para estudos do valor L e L (-Ps) em plantas de feijão para as seguintes variáveis: E1F1, E1F2, E2F1 e em plantas de milho para E1F1 e E2F1. Para valor L e L (- Ps) em plantas de feijão para E2F2 e em plantas de milho para E1F2 e E2F2, recomenda-se o volume de terra de 4,0 L. A maior produção de massa seca e de conteúdo de P em plantas de feijão E1F2 foram obtidos com 12,0 L. Vasos com volume de terra de 13,0 L são os indicados para avaliação do desenvolvimento em plantas de milho E1F2 e E2F1. Com 14,0 L de terra obteve-se o maior conteúdo de P e massa seca em plantas de feijão E1F2 e conteúdo de P em plantas de milho E1F1. O volume de terra de 15,0 L é indicado para conteúdo de P em plantas de milho E1F2. Para a obtenção da máxima produção de massa seca em feijão em ambos os solos e épocas (E2F1 e E2F2) e milho no solo adubado e 50 dias (E2F2), assim como para conteúdo de P para feijão no E2F1 e E2F2 e milho E2F2, há necessidade de se usar vaso com volume de terra maior que 18,0 L. / The effect of soil volume on the growth of bean and corn crop, phosphorus uptake and isotopically exchangeable P (L value), as affected by the plant growing period and two soil P fertility level were studied in green house. The effect of seed derived P in the plant on the L value (L: L value calculated without discounting the seed derived P and L (- Ps): L value calculated discounting the seed derived P) was also evalueted. The study was carried out in plastic pots containing: 0.5; 1.0, 2.0, 4.0, 8.0, 12.0 and 18.0 L of Red Yellow Latosol, with and without P addition (50 mg kg-1 soil P) and harvesting the plants in two periods: 30 and 50 days after seeding. The experimental design was completely randomized with 3 replications in 7x2x2 factorial scheme. The variations in harvest period and soil P fertility level are presented as: E1F1 (30 days, without P application), E1F2 (30 days, with P), E2F1 (50 days, without P) and E2F2 (50 days, with P). The plant P concentration was not an adequate parameter for establishing the plant nutritional state. The plants of 0.5 and 1.0 L pots had their growth limited, which consequently affected the P content, therefore also not being adequate for the L value determination. There was effect of seed derived P on L value for soil volumes smaller than 4.0 L for bean plants, and 8.0 L for corn plants. The 2.0 L soil volume is recommended for L and L (-Ps) values studies with bean plants to the following variables: E1F1, E1F2, E2F1 and with corn plants to E1F1 and E2F1. For L and L (-Ps) values in beans plants to E2F2 and with corn plants to E1F2 and E2F2, the 4.0 L soil volume is recommended. The highest dry matter weight and P content in E1F2 bean plants were obtained with 12.0 L soil volume. Pots with 13.0 L soil are indicated for growth evaluation of E1F2 and E2F1 corn plants. The highest P content and dry matter weigh of E1F2 bean plants and E1F1 corn plants P content were obtained with 14.0 L soil volume. The 15.0 L soil volume is suggested for the P content study with E1F2 corn plants. For obtaining maximum dry matter yield of bean plants in both soils and growth periods (E2F1 and E2F2) and of corn in P fertilized soil harvesting at 50 days (E2F2), as well as for P content of E2F1 and E2F2 bean plants and E2F2 corn plants, pots with larger soil volume than 18.0 L should be used.

Estudou-se em casa-de-vegetação o efeito do volume de terra no desenvolvimento das plantas de feijão e milho, na absorção de fósforo pelas culturas e na determinação do P isotopicamente trocável (Valor L), em função do periódo de desenvolvimento dessas culturas e dois níveis de fertilidade do solo quanto ao fósforo. Avaliou-se também o efeito do P na planta proveniente da semente no cálculo do Valor L (L: valor L calculado sem descontar o P proveniente da semente e L (-Ps): valor L calculado descontando o P proveniente da semente). O estudo foi realizado com Latossolo Vermelho-amarelo em vasos plásticos contendo: 0,5; 1,0; 2,0; 4,0; 8,0; 12,0 e 18,0 L de terra; com e sem adubação de P (50 mg kg-1 terra de P) e colhendo as plantas em dois períodos: 30 e 50 dias após a germinação. O delineamento experimental foi inteiramente casualizado com 3 repetições no esquema fatorial 7x2x2. As variações nos períodos de colheita e níveis de fertilidade são representadas por: E1F1 (30 dias, sem adubação), E1F2 (30 dias, adubado), E2F1 (50 dias, sem adubação) e E2F2 (50 dias, adubado). A concentração de P nas plantas não foi um parâmetro adequado para estabelecer o estado nutricional das plantas e os vasos de 0,5 e 1,0 L tiveram desenvolvimento prejudicado e conseqüentemente no conteúdo de P, não sendo também adequados para determinação do valor L. Houve efeito do P na planta proveniente da semente no valor L para volumes de terra menor que 4,0 L para plantas de feijão e 8,0 L para plantas de milho. O volume de terra de 2,0 L é recomendado para estudos do valor L e L (-Ps) em plantas de feijão para as seguintes variáveis: E1F1, E1F2, E2F1 e em plantas de milho para E1F1 e E2F1. Para valor L e L (- Ps) em plantas de feijão para E2F2 e em plantas de milho para E1F2 e E2F2, recomenda-se o volume de terra de 4,0 L. A maior produção de massa seca e de conteúdo de P em plantas de feijão E1F2 foram obtidos com 12,0 L. Vasos com volume de terra de 13,0 L são os indicados para avaliação do desenvolvimento em plantas de milho E1F2 e E2F1. Com 14,0 L de terra obteve-se o maior conteúdo de P e massa seca em plantas de feijão E1F2 e conteúdo de P em plantas de milho E1F1. O volume de terra de 15,0 L é indicado para conteúdo de P em plantas de milho E1F2. Para a obtenção da máxima produção de massa seca em feijão em ambos os solos e épocas (E2F1 e E2F2) e milho no solo adubado e 50 dias (E2F2), assim como para conteúdo de P para feijão no E2F1 e E2F2 e milho E2F2, há necessidade de se usar vaso com volume de terra maior que 18,0 L. / The effect of soil volume on the growth of bean and corn crop, phosphorus uptake and isotopically exchangeable P (L value), as affected by the plant growing period and two soil P fertility level were studied in green house. The effect of seed derived P in the plant on the L value (L: L value calculated without discounting the seed derived P and L (- Ps): L value calculated discounting the seed derived P) was also evalueted. The study was carried out in plastic pots containing: 0.5; 1.0, 2.0, 4.0, 8.0, 12.0 and 18.0 L of Red Yellow Latosol, with and without P addition (50 mg kg-1 soil P) and harvesting the plants in two periods: 30 and 50 days after seeding. The experimental design was completely randomized with 3 replications in 7x2x2 factorial scheme. The variations in harvest period and soil P fertility level are presented as: E1F1 (30 days, without P application), E1F2 (30 days, with P), E2F1 (50 days, without P) and E2F2 (50 days, with P). The plant P concentration was not an adequate parameter for establishing the plant nutritional state. The plants of 0.5 and 1.0 L pots had their growth limited, which consequently affected the P content, therefore also not being adequate for the L value determination. There was effect of seed derived P on L value for soil volumes smaller than 4.0 L for bean plants, and 8.0 L for corn plants. The 2.0 L soil volume is recommended for L and L (-Ps) values studies with bean plants to the following variables: E1F1, E1F2, E2F1 and with corn plants to E1F1 and E2F1. For L and L (-Ps) values in beans plants to E2F2 and with corn plants to E1F2 and E2F2, the 4.0 L soil volume is recommended. The highest dry matter weight and P content in E1F2 bean plants were obtained with 12.0 L soil volume. Pots with 13.0 L soil are indicated for growth evaluation of E1F2 and E2F1 corn plants. The highest P content and dry matter weigh of E1F2 bean plants and E1F1 corn plants P content were obtained with 14.0 L soil volume. The 15.0 L soil volume is suggested for the P content study with E1F2 corn plants. For obtaining maximum dry matter yield of bean plants in both soils and growth periods (E2F1 and E2F2) and of corn in P fertilized soil harvesting at 50 days (E2F2), as well as for P content of E2F1 and E2F2 bean plants and E2F2 corn plants, pots with larger soil volume than 18.0 L should be used.

Solid-phase microextraction (SPME) coupled with gas chromatography/mass spectrometry (GC/MS) is routinely used to analyze polycyclic aromatic hydrocarbons (PAHs) in water. A common SPME-GC/MS approach quantifies target analytes using isotopically labeled standards (IISs); one IIS is needed for each target analyte. This approach is challenging, even prohibitive since IISs are often expensive; moreover, they are generally not available for each analyte of interest. This study developed a novel SPME-GC/MS approach for the quantification of PAHs in water. The new method, which employs only a small number of IISs, uses relative response factor (RRF) (i.e., analyte corresponding to IIS) to quantify PAHs in water. Possible matrix dependency of RRFs values was examined using water that was modified concerning different physical-chemical characteristics (i.e., ionic strength, pH, suspended solids, humic acid, and biological organic carbon represented by hemoglobin). The results revealed that RRFs are not noticeably affected by changing ionic strength and pH; the other three parameters did affect the RRFs. However, the results also showed that the effect is minimal when the solution is dilute (i.e., low concentrations of suspended solids, humic acid or hemoglobin). Relatively stable RRFs for dilute water solutions indicates that this approach can be used for routine quantification of water that does not contain prohibitive amounts of suspended solids, humic acid, and biological organic matter. The developed method was employed to quantify trace levels of PAHs in three different types of water, namely river water, well water, and bottled water. PAH levels in every kind of water were less than 100 ng/L level (i.e., 0.1 ppb). Analyses of spiked water samples containing 2 ng PAHs revealed correlations between calculated RRFs and the physical-chemical properties of the PAHs investigated (i.e., vapor pressure, boiling point, octanol/water partition coefficient, octanol/air partition coefficient, GC retention time). This implies that RRFs for PAHs not examined in this study can be predicted. Overall, the results presented herein constitute a meaningful contribution to the development of SPME-GC/MS methods for quantitative analysis of PAHs and other chemicals in dilute aqueous solutions. Moreover, the development of methods that alleviate the need for IISs corresponding to each target analyte.