The detection and quantification of hormones are important to assess the reproductive and stress status of experimental models and for the diagnosis of diseases in human and veterinary clinics. The peptide secretoneurin (SN) has been proposed as a new sex hormone, but effective quantification methods are challenging. Traditional methods require the use of antibodies with either radioactive or non-radioactive tracers. There are difficulties with these methods in terms of sensitivity, specificity, and inter-laboratory repeatability. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) can circumvent many of these challenges. Another source of variation is the extraction of lipophilic steroidal compounds, which is incompatible with the extraction of hydrophilic peptide hormones. I have developed efficient extraction and sensitive detection methods of SN with numerous other peptide and steroid hormones in the same tissue sample in mice and zebrafish. The extraction efficiency for both peptide and steroid analytes is over 85%. The standard deviation for extraction and LC-MS/MS analysis for each compound varies between 5-10%. The steroid hormones can be quantified in the low to medium fmol/µL range. We quantified peptide hormones in the high fmol/µL to low pmol/µL range. Mouse SN levels were measured and compared against the levels of GnRH 1, oxytocin, vasopressin, E2, and P4 in multiple tissues at 3 important periods through the estrous cycle. In addition, SN levels were found to be moderately related to GnRH 1 levels in the hypothalamus in the estrous cycle. This is important because it is GnRH 1 that stimulates the luteinizing hormone surge in the pituitary that regulates ovulation in all vertebrate species. I also determined that SNa and SNb were both within the 2-8 pmol/µL range in the brain or pituitary harvested from a single female zebrafish. This makes it feasible for the first time to study the correlation between the SNs and other peptides and steroid hormones by quantifying them simultaneously in very small tissue samples. Untargeted peptidomics determined that the SN peptides in zebrafish can be further processed into smaller discrete fragments. This implies not only active synthesis and selective peptide processing but suggests that the are unknown functions of the SN peptide fragments that await discovery. This cost-effective package was used for the detailed assessment of hypothalamic-pituitary-gonadal function in mice and zebrafish and may be adaptable to many other hormones across species.

No description available.

No description available.

In the early 19th century a ground-breaking discovery was made that linked a dietary deficiency of a fat-soluble vitamin with the childhood disease known as rickets. The vitamin was named vitamin D and extensive research regarding the physiological importance of this vitamin followed ever since. It is currently known that vitamin D plays an important role in maintaining the calcium and phosphate homeostasis in the human body. Less clear evidence states the medical importance of vitamin D in the prevention and cancer, autoimmune disease and diabetes. Current literature shows that vitamin D has five distinct forms, vitamin D1 to D5, of which vitamin D2 and D3 are the most studied forms. The term “vitamin D” is often wrongfully used to include the vitamin D mother molecule, the vitamin D status indicator (25(OH)D), the biologically active form (1,25(OH)2D) and biologically inactive form (24,25(OH)2D). The interest for measurement of these vitamin D analogues is a continuously growing field both on individual and epidemiological level. For decades laboratories have struggled to produce a robust method capable of quantifying these different vitamin D analogues and uncovered a new form of complexity regarding the analysis of these analogues. The identification of the C3-epimeric forms of vitamin D metabolites has forced laboratories to rethink their analytical methods and several concerns were raised regarding the overestimation of the true vitamin D status by current analytical methods. The quantification of the biologically active and inactive forms of vitamin D is reported to be difficult and to date very few LC-MS/MS methods reported in the literature are able to quantify various vitamin D analogues. However, to our knowledge none of these methods are able to include the precursor vitamin D, the 25-hydroxylated metabolites, the biologically active and inactive metabolites, C3-epimers and isobaric compounds in a single run. Therefore the aim of this study was to develop, optimise and validate a LC-MS/MS method for the quantification of twelve vitamin D analogues in a single run. This was done by optimising the underlying LC-MS/MS parameters to ensure optimal analytical sensitivity in positive ESI mode and sufficient chromatographic separation between analytes with similar chemical properties. Furthermore, the optimised method was validated to ensure the accuracy and precision of the method before implementation into a clinical environment. The vitamin D analogues included in this study were vitamin D2, vitamin D3, 25(OH)D2, 25(OH)D3, 1,25(OH)2D2, 1,25(OH)2D3, 24,25(OH)2D2, 24,25(OH)2D3, 3-epi-25(OH)D2, 3-epi- 25(OH)D3, 7(OH)4C3 and 1α(OH)D3. A double liquid-liquid extraction with hexane and ethyl acetate were found to be the most efficient at extracting the vitamin D analogues from a serum matrix after matrix modification with sodium hydroxide. Recoveries of > 95 % (CV <10 %) were achieved for all the analytes. It was noted that a precursor adduct other than the molecular mass ion for a specific vitamin D analogue can produce a more abundant MS1 signal and that the ESI source parameters vary between analytes with different chemical properties and should therefore be optimised individually for each analyte. Various columns were assessed and sufficient chromatographic separation between the relevant analytes was achieved with an Agilent Technologies Pentafluorophenyl column. Baseline separation was achieved between 25(OH)D3 and 3-epi-25(OH)D3 as well as 25(OH)D2 and 3-epi-25(OH)D2, which is a requirement for this method to be viable. The method was subjected to a series of validation steps to ensure the accuracy and precision of the method. These included the assessment of the analytical range, LOD, LOQ, inaccuracy, imprecision, stability, interference and recovery. It was found that the optimised method had good linearity (r > 0.995), acceptable repeatability (CV < 10 %) and within-lab precision (CV < 15%) and excellent method accuracy (systematic error < 6.60 %). Furthermore, all the analytes proved to be stable for 48 hours after sample preparation with no interferences found for co-eluting analytes. Finally, based on the sigma metric scale specifications, it was calculated that this method proved to be “world class” and very little QC is needed to ensure the quality of the data derived from this method. Based on the findings in this study, it was concluded that a novel LC-MS/MS method for the quantification of twelve vitamin D analogues in a single run was successfully developed. All the LC-MS/MS parameters were optimised to ensure optimal analytical sensitivity for each analyte and the method was validated based on a series of method validation steps required for implementation into a clinical laboratory. This validation proved this method to be ready for implementation into a clinical environment. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015

In the early 19th century a ground-breaking discovery was made that linked a dietary deficiency of a fat-soluble vitamin with the childhood disease known as rickets. The vitamin was named vitamin D and extensive research regarding the physiological importance of this vitamin followed ever since. It is currently known that vitamin D plays an important role in maintaining the calcium and phosphate homeostasis in the human body. Less clear evidence states the medical importance of vitamin D in the prevention and cancer, autoimmune disease and diabetes. Current literature shows that vitamin D has five distinct forms, vitamin D1 to D5, of which vitamin D2 and D3 are the most studied forms. The term “vitamin D” is often wrongfully used to include the vitamin D mother molecule, the vitamin D status indicator (25(OH)D), the biologically active form (1,25(OH)2D) and biologically inactive form (24,25(OH)2D). The interest for measurement of these vitamin D analogues is a continuously growing field both on individual and epidemiological level. For decades laboratories have struggled to produce a robust method capable of quantifying these different vitamin D analogues and uncovered a new form of complexity regarding the analysis of these analogues. The identification of the C3-epimeric forms of vitamin D metabolites has forced laboratories to rethink their analytical methods and several concerns were raised regarding the overestimation of the true vitamin D status by current analytical methods. The quantification of the biologically active and inactive forms of vitamin D is reported to be difficult and to date very few LC-MS/MS methods reported in the literature are able to quantify various vitamin D analogues. However, to our knowledge none of these methods are able to include the precursor vitamin D, the 25-hydroxylated metabolites, the biologically active and inactive metabolites, C3-epimers and isobaric compounds in a single run. Therefore the aim of this study was to develop, optimise and validate a LC-MS/MS method for the quantification of twelve vitamin D analogues in a single run. This was done by optimising the underlying LC-MS/MS parameters to ensure optimal analytical sensitivity in positive ESI mode and sufficient chromatographic separation between analytes with similar chemical properties. Furthermore, the optimised method was validated to ensure the accuracy and precision of the method before implementation into a clinical environment. The vitamin D analogues included in this study were vitamin D2, vitamin D3, 25(OH)D2, 25(OH)D3, 1,25(OH)2D2, 1,25(OH)2D3, 24,25(OH)2D2, 24,25(OH)2D3, 3-epi-25(OH)D2, 3-epi- 25(OH)D3, 7(OH)4C3 and 1α(OH)D3. A double liquid-liquid extraction with hexane and ethyl acetate were found to be the most efficient at extracting the vitamin D analogues from a serum matrix after matrix modification with sodium hydroxide. Recoveries of > 95 % (CV <10 %) were achieved for all the analytes. It was noted that a precursor adduct other than the molecular mass ion for a specific vitamin D analogue can produce a more abundant MS1 signal and that the ESI source parameters vary between analytes with different chemical properties and should therefore be optimised individually for each analyte. Various columns were assessed and sufficient chromatographic separation between the relevant analytes was achieved with an Agilent Technologies Pentafluorophenyl column. Baseline separation was achieved between 25(OH)D3 and 3-epi-25(OH)D3 as well as 25(OH)D2 and 3-epi-25(OH)D2, which is a requirement for this method to be viable. The method was subjected to a series of validation steps to ensure the accuracy and precision of the method. These included the assessment of the analytical range, LOD, LOQ, inaccuracy, imprecision, stability, interference and recovery. It was found that the optimised method had good linearity (r > 0.995), acceptable repeatability (CV < 10 %) and within-lab precision (CV < 15%) and excellent method accuracy (systematic error < 6.60 %). Furthermore, all the analytes proved to be stable for 48 hours after sample preparation with no interferences found for co-eluting analytes. Finally, based on the sigma metric scale specifications, it was calculated that this method proved to be “world class” and very little QC is needed to ensure the quality of the data derived from this method. Based on the findings in this study, it was concluded that a novel LC-MS/MS method for the quantification of twelve vitamin D analogues in a single run was successfully developed. All the LC-MS/MS parameters were optimised to ensure optimal analytical sensitivity for each analyte and the method was validated based on a series of method validation steps required for implementation into a clinical laboratory. This validation proved this method to be ready for implementation into a clinical environment. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015

As elevadas taxas de colesterol plasmático representam um grande risco à saúde, uma vez que podem causar doenças cardiovasculares. Para o tratamento e prevenção da dislipidemia são utilizados medicamentos reguladores do colesterol, como as estatinas. Embora eficazes e extensamente utilizados, esses fármacos apresentam efeitos adversos se administrados na dosagem errada. Assim, faz-se necessário o desenvolvimento de um método de monitorização terapêutica a fim de se ajustar a concentração desses compostos no sangue. Este trabalho visa o desenvolvimento de um método para análise de pravastatina (PRA), atorvastatina (AT), fluvastatina (FLV) e sinvastatina (SV) em plasma humano usando cromatografia líquida acoplada à espectrometria de massas (LC-MS). Na etapa de preparo de amostras, de forma inédita, utilizou-se a técnica microextração por sorvente empacotado (MEPS) para a análise de plasma humano contendo quatro estatinas. Para a otimização das condições de extração avaliaram-se, por experimentos univariados, parâmetros como fase extratora, composição do solvente de eluição e de lavagem. Outros fatores como volume de amostra, ciclos de amostragem, ciclos de eluição e etapas de eluição foram avaliados empregando-se planejamento experimental multivariado. A extração foi realizada utilizando-se uma fase estacionária C18 Chromabond como sorvente. O método MEPS-LC-MS/MS desenvolvido foi validado baseando-se nas recomendações da agência nacional de vigilância sanitária (ANVISA) e apresentou linearidade, seletividade, precisão, exatidão e recuperação adequadas para as estatinas, excetuando-se para a sinvastatina. A faixa de linearidade obtida foi de 10-200 ng mL-1 (FLV e AT) e 20-200 ng mL-1 (PRA). Os limites de quantificação obtidos foram da ordem de 10 ng mL-1 (AT e FLV) e 20 ng mL-1 (PRA). Desta forma o método desenvolvido poderá ser utilizado para a determinação dos níveis de pravastatina, fluvastatina e atorvastatina em amostras de plasma humano. / Elevated plasma cholesterol level is a risk factor for coronary diseases, which are the most deadly sickness according to the World Health Organization (WHO). In order to fight the hypercholesterolemia in patients, statins are a well-established class of drugs to be prescribed. Even though they are efficient, some side effects can be associated with statin therapy, especially when interactions with other drugs occur. In these cases, monitoring the concentration can optimize the drug dosage to therapeutic effectiveness whilst minimizing the adverse effects. The aim of this work was to develop a method for analysis of pravastatin (PRA), atorvastatin (AT), fluvastatin (FLV) and simvastatin (SV) in human plasma. The experimental means chosen to attain the goal was liquid chromatography-tandem mass spectrometry (LC-MS/MS) and for the sample preparation, microextraction by packed sorbent (MEPS). To optimize the extraction conditions, parameters such as sorbent, elution and washing solution were evaluated. Other parameters such as sampling, elution cycles, sample volume and elution steps were evaluated using multivariate experimental design. The extraction was performed using C18 Chromabond as sorbent. The method was validated based on ANVISA recommendations and featured appropriated linearity, selectivity, accuracy, precision, and recovery, except for simvastatin. The calibration curve in plasma was obtained in the concentration range 10-200 ng mL-1 (FLV and AT) and 20-200 ng mL-1 (PRA) and the limit of quantification (LOQ) was 10 ng mL-1 (FLV and AT) and 20 ng mL-1 (PRA). The method developed proved to be suitable for the analysis of pravastatin, fluvastatin and atorvastatin in human plasma sample, but not simvastatin, and it can contribute to a more efficient usage of the statins in the treatment of hypercholesterolemia.

O quadro evolutivo da agricultura brasileira resulta em benefícios à população exigindo crescentes avanços tecnológicos no setor. Constantemente, novos agrotóxicos são introduzidos estimulando estudos científicos com a finalidade de determinar e avaliar os impactos na população e no meio ambiente. No presente trabalho, a matriz avaliada foi o lodo gerado no processo de tratamento de água para consumo humano, coletado na região do Vale do Ribeira, SP. A técnica empregada foi a cromatografia líquida de fase reversa acoplada à espectrometria de massas triploquadrupolar em tandem com ionização por electrospray. Os compostos foram extraídos previamente da matriz. O desenvolvimento da metodologia exigiu tratamento dos dados para que esses pudessem ser utilizados e transformados em informações confiáveis. Os processos envolvidos foram avaliados usando o conceito da validação de ensaios químicos. Os indicadores avaliados foram seletividade, linearidade, intervalo de trabalho, sensibilidade, exatidão, precisão, limite de detecção, limite de quantificação e robustez. Esses indicadores produziram valores quantitativos e qualitativos que foram estatisticamente evidenciados de forma objetiva. A metodologia desenvolvida e validade é simples. Como resultado, mesmo explorando a sensibilidade da técnica, os compostos estudados não foram encontrados no lodo da ETA de Registro. Isso leva a crer que esses compostos podem estar presentes em concentrações muito baixas, podem sofrer degradação durante o tratamento da água ou não são retidos completamente pela ETA. 7 / The evolving scenario of Brazilian agriculture brings benefits to the population and demands technological advances to this field. Constantly, new pesticides are introduced encouraging scientific studies with the aim of determine and evaluate impacts on the population and on environment. In this work, the evaluated sample was the sludge resulted from water treatment plant located in the Vale do Ribeira, São Paulo, Brazil. The technique used was the reversed phase liquid chromatography coupled to electrospray ionization tandem mass spectrometry. Compounds were previously liquid extracted from the matrix. The development of the methodology demanded data processing in order to be transformed into reliable information. The processes involved concepts of validation of chemical analysis. The evaluated parameters were selectivity, linearity, range, sensitivity, accuracy, precision, limit of detection, limit of quantification and robustness. The obtained qualitative and quantitative results were statistically treated and presented. The developed and validated methodology is simple. As results, even exploring the sensitivity of the analytical technique, the work compounds were not detected in the sludge of the WTP. One can explain that these compounds can be present in a very low concentration, can be degraded under the conditions of the water treatment process or are not completely retained by the WTP.

O número crescente de descobertas de substâncias bioativas produzidas pelo metabolismo secundário de cianobactérias tem despertado o interesse de grupos de pesquisa no mundo todo com o objetivo comum de descrever e explorar estas moléculas e entender a sua biossíntese. No Brasil, as pesquisas sobre moléculas bioativas produzidas por linhagens de cianobactérias nativas são escassas. Neste trabalho, utilizando iniciadores específicos da PCR e sequenciamento, a presença de genes envolvidos na biossíntese da neurotoxina saxitoxina (STX) foi confirmada em representantes dos gêneros Dolichospermum, Sphaerospermopsis, Cuspidothrix e Cylindrospermopsis, enquanto que genes da citotoxina cilindrospermopsina (CYN) foram detectados somente em representantes de Cylindrospermopsis. Genes envolvidos com a produção dos inibidores enzimáticos, microviridina (MDN) e a cianobactina microciclamida (MCA) foram sequenciados em isolados do gênero Microcystis. Os genomas das linhagens de Cylindrospermopsis raciborskii CENA302 e CENA303 foram sequenciados usando a plataforma HiScan SQ (Ilumina) com biblioteca pareada 2 x 100 pb. O genoma da Sphaerospermopsis torques-reginae ITEP-024 foi sequenciado utilizando a plataforma Ion Torrent (Life Technologies) com tamanhos de fragmentos de até 200 pb. As tentativas de montagem ab initio dos genomas foram realizadas e o agrupamento gênico da saxitoxina (28 kb) da linhagem C. raciborskii CENA302 foi identificado e caracterizado. As análises filogenéticas das sequências de aminoácidos envolvidos com a biossíntese das moléculas bioativas avaliadas demonstraram que os isolados brasileiros de cianobactérias formam clados com elevado valor de reamostragem com sequências homólogas de cianobactérias conhecidas como produtoras dessas moléculas. Neste estudo é relatada pela primeira vez a presença de genes cyr em linhagens da América do Sul de C. raciborskii e a presença simultânea de genes cyr e sxt em uma única linhagem de C. raciborskii. Além disso, este é o primeiro estudo que relata a presença de genes envolvidos na biossíntese de MDN e MCA nas espécies de cianobactérias M. protocystis, M. panniformis e M. wesenbergii. Análises por espectrometria de massas acoplada a cromatografia líquida (LC-MS) e imunoensaio enzimático (ELISA) foram utilizadas a fim de detectar e identificar variantes estruturais das moléculas bioativas das cianobactérias que tiveram os genes biossintéticos sequenciados. A análise de LC-MS mostrou a produção das variantes GTX2, GTX3, STX e dc-STX pela linhagem C. raciborskii CENA302, enquanto que a linhagem C. raciborskii CENA305 apresentou as variantes NEO, C1 e dcGTX3. As quatro novas variantes de MCY, [D-Val1]MC-RR, [D-Leu/Ile1]MC-RR, [D-Leu/Ile1]MC-YR e [D-Phe1]MC-LR, foram encontradas nas espécies M. panniformis SPC702 e M. protocystis SPC697. Este é o primeiro relato da produção de MCY por essas duas espécies de Microcystis. Dezesseis linhagens que ainda não possuíam as sequências do gene de RNAr 16S foram sequenciadas. O resultado da análise filogenética das sequências do gene de RNAr 16S foi coerente com as descrições morfológicas, sendo que todas as linhagens foram caracterizadas em nível de espécie. As informações geradas neste estudo contribuem para o aumento do conhecimento da diversidade metabólica dos isolados brasileiros de cianobactérias e trazem nova visão sobre a evolução dessas moléculas produzidas pelo metabolismo secundário / The growing numbers of discoveries of bioactive substances produced by cyanobacterial secondary metabolism has attracted the interest of research groups around the world with the common goal of describing and exploring these molecules and understanding their biosynthesis. In Brazil, researches on bioactive molecules produced by native cyanobacterial strains are scarce. In this work, using specific PCR primers and sequencing, the presence of genes involved in the biosynthesis of the neurotoxin saxitoxin (STX) was confirmed in representatives of the genera Dolichospermum, Sphaerospermopsis, Cuspidothrix and Cylindrospermopsis, while genes of the cytotoxin cylindrospermopsin (CYN) were detected only in representatives of Cylindrospermopsis. Genes involved in the production of protease inhibitors, microviridin (MDN) and the cianobactin microciclamide (MCA), were sequenced in isolates of the genus Microcystis. The genomes of Cylindrospermopsis raciborskii strains CENA302 and CENA303 were sequenced using the high-throughput platform HiScan SQ (Illumina) as paired-ends 2 x 100 bp. The Sphaerospermopsis torques-reginae ITEP-024 genome was sequenced using the high-throughput platform Ion Torrent (Life Technologies) with fragment sizes up to 200 bp. Attempts of ab initio genomes assembly were performed and the 28 kb saxitoxin gene cluster of C. raciborskii strains CENA302 was identified and characterized. Phylogenetic analyses of amino acid sequences involved in the biosynthesis of the bioactive molecules evaluated showed that the Brazilian cyanobacterial isolates formed clades with high bootstrap values with homologous sequences of known cyanobacterial producers of these molecules. In this study is reported for the first time the presence of cyr genes in South America strains of C. raciborskii and the simultaneous presence of cyr and sxt genes in a single C. raciborskii strain. Furthermore, this is the first study reporting the presence of genes involved in the biosynthesis of MDN and MCA in the cyanobacterial species M. protocystis, M. panniformis e M. wesenbergii. Analyses by mass spectrometry coupled to liquid chromatography (LC-MS) and enzyme immunoassay (ELISA) were used to detect and identify structural variants of bioactive molecules of the cyanobacteria that had the biosynthetic genes sequenced. Analysis of LC-MS showed the production of the variants GTX2, GTX3, STX and dc-STX by the C. raciborskii strain CENA302, whereas the strain C. raciborskii CENA305 presented the variants NEO, C1 and dcGTX3. The new four MCY variants [D-Val1]MC-RR, [D-Leu/Ile1]MC-RR, [D-Leu/Ile1]MC-YR and [D-Phe1]MC-LR were found in the species M. panniformis SPC702 and M. protocystis SPC697. This is the first report of the MCY production by these two species of Microcystis. Sixteen strains that still lacked the 16S rRNA gene sequences were sequenced. The result of the phylogenetic analysis of 16S rRNA gene sequences was consistent with the morphological descriptions, and all strains were characterized to species level. The informations generated in this study contribute to the increase of knowledge on metabolic diversity of Brazilian cyanobacterial strains and bring new insight into the evolution of these molecules produced by secondary metabolism

A presen&ccedil;a de antibi&oacute;ticos em &aacute;guas superficiais e subterr&acirc;neas &eacute; motivo de preocupa&ccedil;&atilde;o, devido ao surgimento de bact&eacute;rias resistentes. Contudo, a maioria dos trabalhos publicados na literatura cient&iacute;fica mant&eacute;m o foco nos antibi&oacute;ticos inalterados, sendo que as mol&eacute;culas degradadas tamb&eacute;m est&atilde;o no ambiente. Esses produtos de degrada&ccedil;&atilde;o podem ser t&atilde;o ou mais prejudiciais do que as mol&eacute;culas que lhes deram origem. Esse trabalho teve por objetivo desenvolver m&eacute;todos de extra&ccedil;&atilde;o utilizando a microextra&ccedil;&atilde;o por sorvente empacotado (MEPS) para pr&eacute;-concentrar e recuperar produtos de fotodegrada&ccedil;&atilde;o de representantes das sulfonamidas (sulfametazina) e fluoroquinlonas (ciprofloxacino), duas das mais importantes classes de antibi&oacute;ticos. MEPS &eacute; considerada uma t&eacute;cnica promissora utilizando pequenos volumes de amostra e de solventes, al&eacute;m de empregar pequenas quantidades de fase extratora, que ainda pode ser reutilizada. Foram comparadas as fases Oasis&reg; HLB e nanotubos de carbono, sendo que a primeira apresentou melhores resultados. Seis produtos de degrada&ccedil;&atilde;o da sulfametazina (SMZ) e oito produtos do ciprofloxacino (CIP), al&eacute;m das mol&eacute;culas inalteradas, foram pr&eacute;-concentrados e analisados por cromatografia l&iacute;quida acoplada &agrave; espectrometria de massas de alta resolu&ccedil;&atilde;o (LC-ESI-ToF). Inicialmente, as concentra&ccedil;&otilde;es de f&aacute;rmaco inalterado utilizadas na fotodegrada&ccedil;&atilde;o foram de 25 mg L-1 (SMZ) e 10mg L-1 (CIP), para que os produtos fossem identificados. As taxas de recupera&ccedil;&atilde;o por MEPS ficaram acima de 50%, o que &eacute; um resultado promissor, considerando-se as diferen&ccedil;as estruturais dos produtos de degrada&ccedil;&atilde;o. Em concentra&ccedil;&otilde;es 100 vezes menores, MEPS conseguiu pr&eacute;-concentrar todos os produtos da SMZ e do CIP, facilitando sua detec&ccedil;&atilde;o. Ap&oacute;s desenvolver a pr&eacute;-concentra&ccedil;&atilde;o por MEPS em &aacute;gua purificada, foram realizados estudos de efeito de matriz em esgoto sint&eacute;tico. Enquanto somente um produto de degrada&ccedil;&atilde;o da SMZ sofreu supress&atilde;o de ioniza&ccedil;&atilde;o, todos os outros (inclusive os de CIP) experimentaram um aumento de sinal devido &agrave; presen&ccedil;a dos interferentes de matriz. O m&eacute;todo desenvolvido para MEPS tamb&eacute;m foi testado para pr&eacute;-concentrar produtos de degrada&ccedil;&atilde;o anaer&oacute;bica da SMZ em reator biol&oacute;gico, obtendo-se &ecirc;xito. Dessa forma, MEPS desponta como uma t&eacute;cnica promissora para pr&eacute;-concentrar produtos de degrada&ccedil;&atilde;o e para que pesquisadores possam acompanhar a degrada&ccedil;&atilde;o de reatores biol&oacute;gicos, visto que requer pequenas quantidades de amostra, n&atilde;o alterando significativamente o volume do meio reacional. / Antibiotics are present both surface water and groundwater and this is motive of concern, due to their ability to cause bacterial resistance. Nevertheless, most of publications in scientific area focuses in unchanged compounds, even knowing the presence of degraded molecules in the environment. These degradation products might be so or more dangerous than unchanged compounds. In this work, extraction methods for degradation products were developed using microextraction by packed sorbent (MEPS). MEPS is an eco-friendly technique due to little consumption of sample, solvents and sorbent. The degradation products of sulfamethazine (SMZ) and ciprofloxacin (CIP) were generated by photodegradation at 25 mg L-1 and <br clear=\"all\" /> 10 mg L-1 of unchanged drug, respectively. The number of degradation products was six for SMZ and eight for CIP. Oasis&reg; HLB and carbon nanotubes were tested as sorbents and the first got better results in preconcentration. The chromatographic analysis was performed by liquid chromatography coupled to high resolution mass spectrometry (LC-ESI-ToF). Recovery rates obtained by MEPS were greater than 50%, which is a significant result, considering structural differences among degradation products. After setting extraction conditions, MEPS was able to recover degradation products at concentrations 100 times lower and more akin to those find in the environment. Using synthetic sewage as medium, matrix effect studies were performed. The prevalent effect was an increase of ionization in degradation products (both SMZ and CIP), just one SMZ product experienced suppression of ionization. At last, SMZ was degraded in an anaerobic reactor and the degradation products were preconcentrated by MEPS. Although the biological degradation products were not the same of photodegradation (except by one), MEPS was capable to preconcentrate them. Thereby, MEPS starts to dawn as a promising technique to preconcentrate degradation products, specially for researchers using biological reactors, since MEPS requires low volumes of sample and almost do not change the final bulk of reactor.

Bisfenóis, filtros UV de benzofenona, parabenos e antimicrobianos como triclocarban são amplamente utilizados em produtos alimentares, farmacêuticos, cosméticos e de cuidados pessoais e também são encontrados como contaminantes. A maioria destes compostos apresentam possíveis efeitos adversos à saúde, principalmente devido ao fato de sua potencial atividade desreguladora endócrina. Neste sentido, é crescente o interesse pelo desenvolvimento de métodos analíticos que sejam mais simples e que possam ter a capacidade de detecção do maior número de analitos possíveis simultaneamente para uso em estudos de biomonitoramento humano. O objetivo deste projeto foi desenvolver um novo método de preparo de amostras em microextração em sorvente empacotado (MEPS, do inglês \"microextraction by packed sorbent\") para a extração simultânea de substâncias potencialmente desreguladoras endócrinas (parabenos, bisfenóis, benzofenonas e triclocarban). Em 250 ?L de urina sintética (pH 5,3) foram adicionados os analitos a uma concentração de 20 ng/mL e diluídos com 250 ?L de água. Após este procedimento, os compostos foram extraídos manualmente da amostra de urina usando MEPS C18 com 5 ciclos de aspirar-dispensar de 100 ?L. A eluição dos analitos foi realizada utilizando 100 ?L (metanol/água 80:20), seguido de análise em LC-MS/MS. O cartucho MEPS, após a limpeza, foi reutilizado para extrações múltiplas sem a presença de efeito de memória. As separações cromatográficas foram conduzidas em uma coluna C18 a 40 °C. A fase móvel foi composta por um gradiente de metanol e água a uma vazão de 500 ?L/min. Os dados foram adquiridos no modo MRM com íons negativos como precursores. O método MEPS-LC-MS/MS proposto apresentou curva de calibração linear para todos os analitos com coeficiente de correlação (r) maior que 0,99 no intervalo do limite inferior de quantificação (LIQ) a 20,0 ng/mL. Os LODs variaram de 0,005-0,1 ng/mL e os LIQ variaram de 0,5-2,5 ng/mL. Além disso, a precisão foi expressa como o coeficiente de variação, onde os valores obtidos foram menores que 20% (n=3) nos LIQ e menores que 15% (n=3) nas concentrações de 10,0 e 20,0 ng/mL. A exatidão foi expressa na forma de erro padrão relativo, onde os valores obtidos não foram superiores a ±20% (n=3) para os LIQ e ±15% (n=3) nas concentrações 10,0 e 20,0 ng/mL. O procedimento MEPS proposto apresentou vantagens que incluem a possibilidade de extração e determinação simultânea de 16 analitos em urina, com redução dos volumes de amostra e solvente utilizados, além de vantagens como tempo reduzido de preparo de amostra, clean up da matriz e dispensa etapa prévia ao preparo de amostra / Bisphenol, benzophenone UV filters, parabens and antimicrobials as triclocarban are widely used in food, pharmaceuticals products, cosmetic and personal care products or they are fond as contaminants. Most of these compounds show possible health adverse effects, mainly due to the fact of its potential endocrine disrupting activity. Thus, there is an increasing interesting in new analytical method development that will be simple and able to detect largest number of analytes possible simultaneously to be used in human biomonitoring studies. The project goal was developed a new sample preparation methody in Microextraction in packed sorbent (MEPS) to simultaneous extraction followed by liquid chromatography coupled to mass spectrometry in tandem analysis (LC-MS/MS) of 16 substances potentially endocrine disrupting (parabens, bisphenols, benzophenones and triclocarban). In 250 ?L of synthetic urine (pH 5.3) were spiked with compounds at a concentration of 20 ng/mL and diluted with 250 ?L of water. After this procedure, the compounds were extracted manually from urine sample using MEPS C18 with 5 draw-eject 100 ?L cycles. The elution was performed using 100 ?L (methanol/water 80:20) followed by LC-MS/MS analysis. The MEPS cartridge, after cleaned, was used for multiple extractions without any carry-over effect. Chromatographic separations were carried out on a C18 column at 40 oC. The Mobile phase was composed by a gradient methanol and water at a flow rate of 500 ?L/min. Data were acquired the MRM mode with negative ions as precursors. The proposed MEPS-LC-MS/MS method showed calibration curve linear to all analytes with correlation coefficient higher than 0,99 in the range from low limit of quantification (LIQ) to 20,0 ng/mL. The LODs range from 0,005-0,1 ng/mL and LIQ range from 0,5 to 2,5 ng/mL. Moreover, the precision was reported as variation coefficient, where the values were less than 20% (n=3) to the LIQ and less than 15% (n=3) to 10,0 and 20,0 ng/mL concentrations. The accuracy was reported as relative standard error, where the values were less than ±20% to the LIQ and less than ±15% (n=3) to 10,0 and 20,0 ng/mL concentrations. The MEPS procedure proposed showed advantages including possibility to extraction and simultaneous determination of 16 analytes in urine, with sample and solvents volume reduction besides it shows advantages as time reducing in sample preparation, matrix clean up and dispense previous sample preparation step.