Nos últimos anos, a pesquisa ambiental tem se defrontado com a questão dos chamados disruptores endócrinos (EDCs). A estes compostos, tais como: produtos farmacêuticos, hormônios naturais e sintéticos, pesticidas, substâncias tensoativas, polímeros de baixa massa molecular e diversos outros contaminantes orgânicos presentes em efluentes municipais e industriais, atribui-se à capacidade de alterar o funcionamento do sistema endócrino. Estrogênios e progestogênios, naturais ou sintéticos, são excretados pela urina de mamíferos, e uma pequena porção nas fezes, e via efluentes de estações de tratamento de esgoto (ETE) entram em vias aquáticas, podendo causar alterações em organismos aquáticos, tais como feminização ou hermafroditismo. Neste contexto, no presente trabalho foi descrito uma metodologia analítica para a extração em fase sólida empregando cartucho C18, dos hormônios naturais, estrona (E1) e 17?-estradiol (E2), e dos hormônios sintéticos, levonorgestrel e 17?-etinilestradiol (EE2) (presentes em anticoncepcionais orais), a partir de uma matriz de esgoto sintético. Foram utilizados dois sistemas cromatográficos neste estudo, ambos de mesmo modelo (SLC-10A, Shimadzu), os quais consistiram em um injetor manual (seringa), com um volume de injeção ajustado para 20µL, e duas bombas modelo LC-10ADVP (Shimadzu). Foram utilizados dois tipos de detectores, um sistema DAD modelo SPD-M10AVP (Shimadzu) e um espectrofluorímetro modelo RF-551 versão 2.4 (Shimadzu). A separação foi feita em coluna C18 (250 X 4,6 mm, 5 µm) com um fluxo de 1 mL min-1. A condição ideal para separação foi o modo isocrático: 48/52 ACN:H2O. O comprimento de onda selecionado para quantificação no sistema de detecção DAD foi: 240nm para o levonorgestrel e 280nm para E1, E2 e EE2. No detector espectrofluorímetro, os comprimentos de onda selecionados para a excitação e emissão dos analitos E1, E2 e EE2 foram: 280nm e 306nm, respectivamente. Para se efetuar o estudo de recuperação, uma matriz simulando esgotos sanitários foi utilizada com o intuito de se ter uma amostra controle (testemunha) livre dos analitos de interesse, devido à dificuldade em se obter uma amostra de esgoto “real” livre destes hormônios. As amostras de esgoto sintético foram fortificadas em três níveis de concentração. Tomou-se 5 replicatas de 100,0mL de amostra testemunha (esgoto sintético) para cada nível de fortificação, estas amostras foram dopadas com os hormônios estudados. Após adaptações de metodologias descritas na literatura, a extração foi feita segundo o procedimento: condicionamento do cartucho (500mg/6mL) com 7mL de acetonitrila, 5mL de metanol e 5mL de água em uma razão de fluxo de 3mL min-1; percolagem de 250,0mL de amostras de esgoto bruto e efluentes tratados pela ETE-Araraquara com fluxo de 1mL min-1; secagem do cartucho por 1 hora a vácuo; eluição dos analitos com 6mL de acetonitrila com fluxo de 1mL min-1; secagem do extrato eluído em corrente de nitrogênio; reconstituição da amostra em 0,5mL de metanol. As amostras de esgoto sintético foram dopadas com os padrões estudados, para análise realizada no sistema DAD, a 0,250?g L-1 para levonorgestrel e 2,50?g L-1 para E1, E2 e EE2 (nível 1); 0,375 ?g L-1 para levonorgestrel e 3,75 ?g L-1 para E1, E2 e EE2 (nível 2); 0,500?g L-1 para levonorgestrel e 5,00?g L-1 para E1, E2 e EE2 (nível 3). Para análise realizada no sistema fluorescente, as amostras de esgoto sintético foram dopadas com os padrões estudados a 0,750?g L-1 para E1; 0,150?g L-1 para E2 e 0,250?g L-1 para EE2 (nível 1); 1,00 ?g L-1 para E1; 0,150?g L-1 para E2 e 0,200?g L-1 para EE2 (nível 2); 1,25?g L-1 para E1; 0,250?g L-1 para E2 e 0,300 ?g L-1 para EE2 (nível 3). Valores de recuperação entre 83-123% com coeficientes de variação menores do que 13,5% foram obtidos para todos os hormônios analisados pelos dois sistemas de detecção (DAD e Fluorescente). Esses dados demonstram a eficiência do método quanto à exatidão e precisão para os níveis de fortificação estudados. O método proposto foi utilizado para avaliar a presenças dos hormônios em afluentes (esgoto bruto) e efluentes da ETE-Araraquara. As amostras coletadas na ETE foram analisadas em triplicata. Foi identificado e quantificado o hormônio natural E2 (31ng L-1) em amostras obtidas antes do tratamento de esgoto. Não foram detectadas concentrações dos analitos em amostras obtidas após o tratamento. / In recent years environmental research has been faced with the issue of the endocrine disrupting chemicals (EDCs). Such compounds, such as: pharmaceutical products, natural and synthetic hormones, pesticides, tensive active substances, low mass molar polymers and many other organic contaminants that appear in municipal and industrial effluents, have the capacity of altering the manner in which the endocrine system works. Natural or synthetic estrogens and progestogens are excreted through the urine of mammals, and a small portion through faeces, and via effluents from sewage treatment plants (STP) flow into aquatic ducts, with the possibility of causing alterations in the aquatic organisms, such as feminization or hermaphroditism. Within this context, the present work describes an analytic methodology for solid phase extraction (SPE) using C18 cartridge of the natural hormones, estrone (E1) and 17?-estradiol (E2), and the synthetic hormones levonorgestrel and 17?-ethinylestradiol (EE2) (found in oral contraceptives) from a synthetic waste matrix. Two chromatographic systems were used in this study, both from the same model (SLC-10A, Shimadzu), a DAD system model SPD-M10AVP (Shimadzu) and a spectrofluorimeter model RF-551 type 2.4 (Shimadzu). Separation was performed in column C18 (250 X 4,6 mm, 5 ?m) with a flux of 1 mL min-1. The ideal separation condition was the isocratic mode: 48/52 ACN:H2O. To carry out the recuperation study, a matrix simulating sewers was used with the objective of having a control sample (witness) free of the samples under scrutiny of the difficulty in obtaining a “real” sewage sample free of these hormones. The samples of synthetic sewage were boosted in three levels of concentration. Recuperation values between 83-123% with variation coefficients lower than 13,5% were obtained for all studied hormones by both systems of detections (DAD and Fluorescent). These data demonstrate the efficiency of the method concerning the accuracy and precision for the fortification levels that were studied. The proposed method was used to assess the presence of hormones in inffluents (raw sewage) and effluents of Araraquara-STP. The natural hormone E2 (31ng L-1) was identified and quantified in samples obtained prior to sewage treatment. No concentrations of analytes in the samples were obtained after the treatment.

Made available in DSpace on 2014-06-12T23:03:23Z (GMT). No. of bitstreams: 2 arquivo8761_1.pdf: 714218 bytes, checksum: d20a16bb335ecdf9b608fa2fd54b1c1f (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2005 / O leite humano muda a sua composição ao longo da lactação, enquanto aumenta o teor de lipídeos decresce o de vitaminas lipossolúveis, se diferenciando basicamente em três fases distintas: colostro, transição e maduro. O desenvolvimento fisiológico da mãe e sua alimentação comprometem a composição e a qualidade do leite, desta forma, estratificou-se as mães em primíparas e multíparas. Este trabalho teve como objetivo de mensurar as variações de vitaminas A e E no leite de diferentes fases, bem como avaliar a influência da dieta nos níveis destas vitaminas e sua adequação nutricional à dieta do lactente. Tomou-se amostras de leite humano de mães primíparas (n=9) e multíparas (n=9), doadoras dos Bancos de leite humano do Recife. Foi levantado inquérito alimentar das mães previamente selecionadas. A extração das vitaminas A (retinoltrans) e E ( -tocoferol) foram obtidas por extração líquido-líquido, após saponificação metanólica, conforme método proposto por Paixão & Stamford (2002); Paixão & Campos, (2003). A quantificação das vitaminas foi obtida por HPLC (High Performance Liquid Chromatography) em uma corrida cromatográfica de 8.5 minutos, eluidas em metanol, numa coluna ODS-II, 150 mm x 3,2 mm, 5 m, lidas em seus máximos de absorção de 325nm, até 5 min, 292nm, de 5.01 a 8.5 min, respectivamente. As multíparas apresentaram níveis de retinol (334±142μg/dL, 208±122μg/dL, 116±38μg/dL) superiores aos da primíparas (179±57μg/dL, 109±64μg/dL, 89±18μg/dL) para colostro, transição e maduro, respectivamente, diferindo significativamente (p 0,001) entre fases. As multíparas diferiram menos, nos níveis de tocoferol, nas fases colostro (1335±906μg/dL) e transição (367±98μg/dL), enquanto no maduro (412±82μg/dL) foi ligeiramente superior, inclusive de modo significante (p 0,001). Ao longo da lactação no período avaliado, o percentual de redução entre as fases, colostro e transição, e colostro e maduro foi mais acentuado para o tocoferol, superiores a 60% em ambos os grupos. A redução mais expressiva de retinol foi obtida entre o leite colostro e maduro (~50%). Na avaliação do inquérito alimentar, observouse o baixo consumo de alimentos ricos em vitamina A, principalmente nas primíparas, e elevado consumo de alimentos ricos em vitamina E em ambos grupos. Existem diferenças nos níveis de retinol e tocoferol entre leite materno de primíparas e multíparas, nas distintas fases. Do leite colostro a maduro, observou-se uma redução nos teores bem como em variabilidade, justificando diferenças no material biológico e tendendo a estabilização no leite maduro

This work is focused on determination of the concentration of rutin in the fruits of elderberry (Sambucus nigra L.). The theoretical part provides an overview of the properties, occurrence and use of elderberry in natural healing, medicine and food industry. Furthermore, important substances contained in elderberry, especially flavonoids, including rutin, are mentioned. Properties, biological effects and the possibility of determination of rutin are briefly described. In the experimental part the optimization and validation of the method of high performance liquid chromatography (HPLC), which is used for identification and quantification of rutin in fruits of 18 elderberry species, was performed. The pressurized hot water extraction was used for the preparation and solid phase extraction (SPE) for purification of the sample. The largest amount of rutin was found in a variety of elderberry Albida, 6,70 mg per 1 g of dry matter. High concentrations of rutin included varieties Haschberg, Sambu, Pregarten, Sambo and Sampo. On the other hand, the lowest concentration was found in variety Aurea, 1,24 mg.g-1. Finally, rutin content in elderberries was compared with the content of rutin in elder leaves and branches andwith significant sources of rutin – buckwheat and rue.

This work focuses on the determination of inositol in drugs and food supplements using high-performance liquid chromatography in HILIC mode. The work is divided into several sections, it discusses the characteristics of analyzed myo-inositol including methods for saccharides determination. A chapter is detached for high-performance liquid chromatography. One of the work goals was also chromatography columns comparison. The experimental part includes measurement conditions, methods of sample preparation evaluated data and results including the discussion. Two real samples were analyzed, in both of them the content of myo-inositol was declared by the producer.

This thesis focuses on analysis of reaction products of phthalaldehyde with selected amino acids using the combination of high-performance liquid chromatography with mass spectrometry. Only one product with dihydroisoindole structure is formed in the case of simple amino acids (glycine, glycine ethyl ester, alanine, α-aminobutyric acid, valine, leucine and isoleucine). Reactions of phthalaldehyde with amino acids with two amino groups (lysine, asparagine, glutamine and arginine) yield different types of compounds. Main products are formed by the interaction of both of the amino groups with one molecule of phthalaldehyde. Apart from this type of product, these reactions also result in formation of the expected analogous structures of dihydroisoindoles. Moreover, the formation of products containing one molecule of amino acid and two molecules of phthalaldehyde is not excluded. The products were structurally analysed by the use of high-performance liquid chromatography and tandem mass spectrometry. Reaction products of phthalaldehyde with α-aminobutyric acid, valine, leucine, isoleucine and lysine were isolated and further analyses such as electron ionisation with double focusing sector analyser and nuclear magnetic resonance were performed. Pieces of information resulting from the analysis of...

Charles University, Faculty of Pharmacy in Hradec Králové Department of: Pharmaceutical Chemistry and Pharmaceutical Analysis Student: Bc. Martin Novák Supervisor: doc. PharmDr. Radim Kučera, Ph.D. Consultant: Mgr. et Mgr. Rafael Doležal, Ph.D. Title of diploma thesis: Optimization of chromatographic parameters for chiral separation of biologically active compounds. The diploma thesis was focused on the development of a HPLC-UV method for the determination of K 1277 enantiomers of systematic name N-(2-((6-chloro-1,2,3,4-tetra- hydroacridin-9-yl)amino)hexyl-2-amino-3-(1H-indole-3-yl) propylamide dihydrochloride, which is one of the compounds from the tacrine-tryptophan hybrids group. These tacrine-tryptophan hybrids could be considered as promising candidates of potential drugs against Alzheimer's disease. The thesis brings an explanation of basic characteristic of chiral molecules, principles of chiral separation, pathophysiology, clinical manifestation and treatment of Alzheimer's disease and short characteristic of tacrine-tryptophan hybrids in the theoretical section. The aim of my diploma thesis was to find the optimal chromatographic conditions for separation of K 1277 enantiomers synthesized from tacrine and tryptophan fragments. The experimental part deals with the development of the chiral...

<p>U okviru ove doktorske disertacije pažnja je sa jedne strane posvećena razradi NMR&nbsp;spektrometrijskih i veoma osetljivih hromatografskih (HPLC/DADi HPLC/TLS) metoda, a sa&nbsp;druge strane razradi jednostavnijih spektrofotometrijskih i voltametrijskih metoda za određivanje&nbsp;odabranih neonikotinoida. Kao posebno vredan doprinos, razrađena je metoda pripreme ekolo&scaron;ki&nbsp;pogodne bizmut-film elektrode na staklastom ugljeniku i na planarnoj ugljeničnoj elektrodi.&nbsp;Efikasnost elektrode je upoređena sa elektrodom modifikovanom filmom od žive. Za ispitivanje&nbsp;povr&scaron;ine radnih elektroda primenjena je i (SEM/EDS) metoda. Razrađene metode su testirane za&nbsp;određivanje neonikotinoida iz komercijalnih formulacija, kao i iz uzoraka krompira, kukuruza,&nbsp;paprike, meda i povr&scaron;inske vode.</p> / <p>The Ph. D. thesis is concerned with the development of NMR spectrometric and very&nbsp;sensitive chromatographic (HPLC/DAD and HPLC/TLS) methods, on the one&nbsp; hand, and simpler&nbsp;spectrophotometric and voltammetric methods on the other, for the &nbsp;determination of selected&nbsp;neonicotinoids. As an especially valuable contribution, a procedure has&nbsp; been developed for the&nbsp;preparation of an environment-friendly bismuth film electrode on glassycarbon and planar&nbsp;carbon electrode. The achieved efficiency of such electrode was compared with that of mercury&nbsp;film electrode. Surface morphology of the working electrodes was studied by the SEM/EDS&nbsp;method. The developed methods have been tested for the determination of the neonicotinoids from&nbsp;their commercial formulations, as well as from samples of potato,&nbsp; maize, pepper, honey, and&nbsp;surface water.</p>

The Musculus complexus (pipping muscle) plays a primary role in the hatching of the chick from the eggshell at the end of its embryonic life. Various metabolic and cellular changes have been associated with the pipping muscle during development. These studies profiled the pipping muscle at the molecular level by identifying proteins which are associated with the developmental changes. In the first phase of the study, protein expression profile of the Day 13 chicken embryo pipping muscle was obtained using DDF with nano HPLC mass spectrometric analyzer. Identified proteins were categorized based on Gene Ontology. In the second phase, pipping muscle lymph was profiled from Day 20 chicken embryo using PCT with LTQ-Orbitrap mass spectrometric analyzer. The identification of constituent proteins of the piping muscle provides a better understanding of the complex cellular processes and functionality of the pipping muscle with potential benefits for improving hatchability in the poultry industry.

A high-performance liquid chromatography (HPLC) method with ultraviolet (UV) detection was developed and validated for the quantification of lorazepam in oral solution. The chromatographic conditions include an isocratic separation (25% water / 75% methanol at a flow rate of 0.500 ml/min) on a Waters X=Bridge C18 column (150 x 4.6 mm; 3.5-micron particle size). The method was validated using guidance from the United States Pharmacopeia (USP) chapter, including investigation of system suitability, precision, accuracy, linearity, and specificity. The calibration curves on three non-consecutive days met the linearity criteria R2 >0.99. Each chromatogram for 200 mcg/mL calibration samples, designated as 100% assay level, met system suitability criteria of resolution ≥2.0, tailing factor ≤2.0, column efficiency (theoretical plates) ≥2000, and precision of prior metrics % RSD ≤1.0. Three replicates of each concentration, 150 mcg/mL at 75% assay, 200 mcg/mL at 100% assay, and 250 mcg/mL at 125% assay were assessed for precision and accuracy over 3 days. Precision and accuracy were evaluated and met the inter-day (repeatability) criteria % RSD and % Error ≤ 2% and intra-day (intermediate) criteria % RSD and % Error ≤ 5% at the 75%, 100%, and 125% assay levels. To assess for specificity, 200 mcg/mL samples were assessed for degradation after being subjected to heat (>60°C), oxidation (3% H2O2), acidic (0.1M HCl), and basic (0.1M NaOH) environments. Samples from each condition were evaluated for lorazepam recovery at 0, 24, and 48 hours. Most drug loss was observed with the samples subjected to acidic and oxidative environments, with 14.71% and 13.16% drug loss, respectively after 48 hours. This method was developed to support the 30-day stability investigation of lorazepam oral solution when stored in oral syringes at room and refrigerated temperatures.

This diploma thesis is focused on theoretical and practical aspects of High performance liquid chromatography (HPLC). This method is introduced as one of the most frequently used current analytical methods. The theoretical part of thesis is focused on instrumentation of HPLC and particular components of HPLC analytical system. The most often used mobile phases and static phases are described as well. Based on these theoretical aspects, laboratory exercise using HPLC for future teachers is designed. Caffeine is used as ideal model material for this exercise. Caffeine is well-known substance, because of its traditional usage for example in food-processing industry. Final part of this thesis is brief view on framework educational programmes for primary and secondary education. As appendix of this thesis, manual for referenced laboratory exercise is provided. KEYWORDS chromatography, caffeine, HPLC, instrumentation of HPLC, teacher training, RVP