Consumers view natural antioxidants as a safe means to reduce spoilage in foods. In addition, these compounds have been reported to be responsible for human health benefits. Identification of these compounds in peanut skins may enhance consumer interest, improve sales, and increase the value of peanuts. This study evaluated analytical methods which have not been previously incorporated for the analysis of peanut skins. Toyopearl size-exclusion chromatography (SEC) was used for separating phenolic size-classes in raw methanolic extract from skins of Gregory peanuts. This allowed for an enhanced analysis of phenolic content and antioxidant activity based on compound classes, and provided a viable preparatory separation technique for further identification. Toyopearl SEC of raw methanolic peanut skin extract produced nine fractions based on molecular size. Analysis of total phenolics in these fractions indicated Gregory peanut skins contain high concentrations of phenolic compounds. Further studies revealed the fractions contained compounds which exhibited antioxidant activities that were significantly higher than that of butylated hydroxyanisole (BHA), a common synthetic antioxidant used in the food industry. This indicates peanut skin extracts are a viable antioxidant source, and that synthetic antioxidants can be replaced with those naturally-derived from peanut by-products. Structures contained in each fraction were identified using high performance liquid chromatography (HPLC) coupled with electrospray ionization (ESI) ion trap mass spectrometry (MSn). Prior to this study, approximately 20 compounds have been identified in peanut skins. The combination of Toyopearl SEC with ESI-HPLC-MSn allowed for the identification of 314 phenolic-based compounds, most of which are newly discovered compounds in peanut skins. Many compounds identified are known to have powerful antioxidant effects, and also have been reported to exhibit numerous beneficial chemical and biological activities, including the treatment of various human health-related conditions. It is evident that peanut skins may be a potential untapped source for the extraction of natural food antioxidants, nutracueticals, and even pharmaceuticals. Because peanut skins are largely a wasted resource to peanut processors, the novel polyphenols identified in this research could have a significant financial impact on the peanut industry. / Ph. D.

Ellagitannins are plant phenolics characterized by biaryl-coupled gallic acid moieties esterified to a D-glucose core. They are widely distributed through higher plants. In the case of oaks, ellagitannin concentrations in heartwood can reach up to 10% (dry wt. basis). These secondary metabolites are not only important physiologically but also influence the economic value and quality of wood products that contain them. Efforts were made to develop and validate the methods used to quantify both soluble and insoluble ellagitannins. First, the efficiencies of the two commonly used extraction solvents, aqueous acetone and aqueous methanol were evaluated. The results showed that aqueous acetone is superior to aqueous methanol in obtaining higher vescalagin and castalagin yields. In a separate study, the method used for determining insoluble ellagitannins was found to under-estimate the contents of insoluble ellagitannins in wood products. Anhydrous methanolic HCl was found to be an excellent reagent for releasing insoluble ellagic acid and gallic acid (as methyl gallate) from biomass substrates. Optimization of both the reaction conditions and the gradient HPLC analysis has led to the development of a robust and reliable protocol. The chemical stability of the two predominant ellagitannins in oaks (vescalagin and castalagin) were evaluated in aqueous methanol and water. It was found that oxygen, pH and higher temperature (60 °C) affect their stability with higher temperature being the most prominent factor. Both vescalagin and castalagin were found unstable in methanolic solutions. Vestalagin, however, is less stable than castalagin. In the course of finding alternative models for ellagitannin biosynthesis study, both callus tissues and suspension cell cultures of white oak (Quercus alba) and sweetgum (Liquidambar styraciflua) were investigated for their possible use as models for ellagitannin biosynthesis. It was found that oak callus tissue cultures (Quercus alba) are capable of producing ellagitannins, and the production and profile of ellagitannins can be modified by adjusting the media composition. Comparison of extracts from the heartwood of Quercus alba with those from callus tissue reveals that they have similar ellagitannin profiles. Through manipulation of the media nitrogen and copper concentrations the callus tissue produced almost 3 times as much castalagin and vescalagin. Suspension cells of Quercus alba and Liquidambar styraciflua were found to be unsuitable for the study of biosynthesis of ellagitannins. These cells either did not produce any detectable level of ellagitannins or the production was unstable. Although the suspension cells could be elicited to produce ellagic acid with glycanases (Driselase), the levels of ellagic acid were too low for quantitative metabolic studies. A method using high performance liquid chromatography – mass spectrometry was developed and optimized with purified ellagitannins. Ellagitannins analyzed under the optimal conditions all provide base peaks of (M-H)- from which the molecular weights of the ellagitannins can be determined. Mild fragmentation was also achieved to give fragments characteristic of ellagitannins (loss of ellagic acid and gallic acid if present). These characteristic peaks allow for rapid identification of ellagitannins from other secondary metabolites present in the samples. Application of the HPLC/ESI-MS in the identification of monomeric ellagitannins in white oak heartwood extracts revealed that it can unambiguously identify the two monomeric ellagitannins, castalagin and vescalagin, and their degradation product, ellagic acid. The key fragmentation pathways of the ellagitannins are also described. Finally, preliminary work using proteomics to study the heartwood formation was conducted. Proteins from transition zone and sapwood were determined and resolved with two-dimensional electrophoresis. It was found that both sapwood and transition woods contain active enzyme(s) capable of catalyzing formation of ellagic acid from pentagalloylglucose. Preliminary results from the 2-D gel separation of sapwood and transition wood proteins showed more protein spots in sapwood than in transition wood, suggesting that sapwood not only had higher protein levels but also a great total number of proteins. The lower complexity of the transition wood proteome suggests that this material may be a good substrate for studying the biaryl-coupling process. / Ph. D.

No / A sensitive and rapid oil-in-water (O/W) microemulsion high performance liquid chromatography (MELC) method has been developed. The water-in-oil (w/o) microemulsion was used as a mobile phase in the determination of salbutamol in aqueous solutions. In addition, the influence of operating parameters on the separation performance was examined. The samples were injected into C18, (250mmx4.6mm) analytical columns maintained at 25(o)C with a flow rate 1 ml/min. The mobile phase was 95.5% v/v aqueous orthophosphate buffer 20 mM (adjusted to pH 3 with orthophosphoric acid), 0.5% ethyl acetate, 1.5% Brij35, and 2.5% 1-butanol, all w/w. The salbutamol and internal standard peaks were detected by fluorescence detection at the excitation and emission wavelengths of 267 and 313 nm respectively. The method had an accuracy of > 97.78% and the calibration curve was linear (r2 = 0.99) over salbutamol concentrations ranging from 25 to 500 ng/mL. The intra-day and inter-day precisions (CV %) were <1.6 and <1.8, respectively. The limit of detection (LOD) and limit of quantitation (LOQ) were 9.61ng/ml and 29.13ng/ml, respectively. The method reported is simple, precise and accurate, and has the capacity to be used for determination of salbutamol in the pharmaceutical preparation.

No / An isocratic oil-in-water microemulsion High Performance Liquid Chromatography (MELC) was developed and validated for the determination of terbutaline in urine samples. A solid phase extraction (SPE) method which used Oasis HLB cartridges was optimised to isolate terbutaline from a urine matrix followed by HPLC with fluorescence detection. The urinary assay was performed in accordance with FDA and ICH regulations for the validation of bioanalytical samples. The method uses the isocratic oil-in-water micro emulsion to separate the terbutaline from the endogenous urine components. The chromatographic separation was carried out on C18-Spherisorb (250mm×4.6mm) analytical column maintained at 30 °C. The mobile phase was 94.4% aqueous orthophosphate buffer (adjusted to pH 3 with orthophosphoric acid), 0.5% ethyl acetate, 1.5% Brij35, 2.5% 1-butanol and 1.1% Octanesulfonic acid (OSA), all w/w. The terbutaline peak was detected by fluorescence detection, using excitation and emission wavelengths of 267 and 313 nm, respectively. The linearity of response was demonstrated at six different concentrations of terbutaline which were extracted from spiked urine, ranging from 60 to 1000ng/ml. The terbutaline was extracted from urine by a solid phase extraction clean-up procedure on Oasis HLB cartridges, and the relative recovery was >87.64% (n = 5). The limit of detection (LOD) and limit of quantitation (LOQ) in urine were 20.21 and 61.24ng/ml, respectively. The intra-day and inter-day precisions (in term of % coefficient of variation) were <3.56% and <2.87%, respectively. In the method development the influence of the composition of the microemulsion system was also studied and the method was found to be robust with respect to changes of the microemulsion components.

Introduction : La présence des moisissures et de leurs mycotoxines dans les matrices complexes de papiers est peu étudiée. Notre travail a porté sur la recherche de mycotoxines sur les papiers patrimoniaux altérés par le foxing et les papiers peints moisis issus de logements dont les habitants ont été diagnostiqués comme porteurs de symptômes allergiques et du syndrome des bâtiments malsains. Objectifs : Identifier les espèces fongiques de ces deux types de supports papiers et y déterminer la production de métabolites fongiques. Matériels et Méthodes : Le foxing a été caractérisé par des techniques pluridisciplinaires (physiques, biologiques, bioanalytiques, tests de cytotoxicité). Les métabolites fongiques dans les extraits hydro-organiques de ces papiers ont été recherchés et identifiés par spectrométrie de masse afin d'évaluer s'ils pouvaient être reliés aux espèces fongiques détectées par microbiologie. Puis le risque toxique de ces extraits de papiers a été évalué sur un modèle cellulaire in vitro. Résultats : Pour le foxing, nous avons pu en exclure une origine métallique et montrer que les micro-organismes présents dans celui-ci sont essentiellement des espèces fongiques. Pour les papiers peints, des pics relatifs à des métabolites fongiques ont été retrouvés. Grâce à l'ensemencement individuel d'espèces fongiques sur papier peint et à l'aide de la MS2, nous avons pu relier les composés de m/z 401 et 487 à S. chartarum. Les tests de cytoxicité ont montré une augmentation significative de la cytotoxicité des cellules A549 avec certains papiers peints moisis par rapport au papier peint témoin. Les cellules A549 ont montré une surexpression du TNF-α, de l'IL-8 et du CYP 1A1, après contact avec ces mêmes papiers peints. Discussion-Conclusion : Nous n'avons détecté aucune mycotoxine dans le foxing excluant ainsi d'éventuels liens entre inhalation de mycotoxines émanant de vieux manuscrits et symptomatologie respiratoire. Pour les habitants des logements et leurs symptômes respiratoires, il est difficile de les relier à une espèce fongique donnée ou à un métabolite donné, bien que S. chartarum ait pu être mis en cause. Ces premiers résultats doivent être confirmés par des études ultérieures / Introduction: Little study has been carried out on the presence of fungi and their mycotoxins in complex paper matrices. Our work has focused on finding mycotoxins on heritage paper altered by the foxing and wallpapers from moldy homes whose residents have been diagnosed with symptoms of allergies and sick building syndrome. Objectives: To identify the fungal species of these two types of paper and to determine the production of fungal metabolites. Materials and methods: The foxing has been characterized using multi-disciplinary technics (physical, biological, bioanalytical, cytotoxicity assays). The fungal metabolites in the hydro-organic extracts of these papers were sought and identified by mass spectrometry to assess whether they could be related to fungal species detected by microbiology. The toxicological risk of wallpaper extracts was then evaluated on model in vitro cells. Results: For the foxing, we could exclude a metal origine and we showed that the microorganisms present are mainly fungal species. For wallpapers, fungal metabolites were found. By seeding individual fungal species on wallpaper and using MS2, we were able to link the compounds of m/z 401 and 487 to S. chartarum. Cytotoxicity tests showed a significant increase in cytotoxicity of A549 cells with moldy wallpaper in comparison with the control wallpaper. A549 cells showed an overexpression of TNF-α after contact with these wallpapers as well as a significant overexpression of IL-8 and CYP 1A1.Discussion-Conclusion: We detected no mycotoxin in foxing, thus excluding possible links between inhalation of mycotoxins from old manuscripts and respiratory symptoms. For people in their homes and respiratory symptoms, it is difficult to draw any relationships to a given fungal species or a metabolite although S. chartarum has been implicated. These initial results should be confirmed by further study

Charles University in Prague Pharmaceutical Faculty in Hradec Králové Department of Pharmaceutical Botany and Ecology Candidate: Miloslava Ďuráčová Consultant: RNDr. Jitka Vytlačilová, Ph.D. Title of thesis: HPLC-MS determination of active biological compounds in Elbe river Biologically active compounds have a various ways to enter environment. They can occur as pesticides, cosmetics and pharmaceutics and their metabolites. Such compounds are classified as environmental contaminants. There is a increased environmental concentration connected with increasing consumption of biologically active compounds. There is a urgent need to follow the effect on the different parts of ecosystem and levels of biologically active compounds. This work was prepared during the cooperation with Povodí Labe state enterprise. A novel analytical method was described in the experimental part of this thesis. I was employed to evaluate biologically compounds levels in the surface water samples. This method is now routinely used. 10 out of 19 evaluated compounds reached concentrations higher than LOD. Acetaminophene (559 ng/l, Králický stream), gabapentine (457 ng/l Elbe, LB Schmilka), cotinine (139 ng/l, Králický potok) were the biologically active compounds with the highest found concentrations. Keywords: HPLC-MS analysis,...

In der Promotion wird die Entwicklung, Optimierung und Validierung einer Reversed-phase-Chromatography Methode zur Messung des Ribavirinplasmaspiegels beschrieben. Diese wurde mit einer Solid Phase Extraction zur Probenvorbereitung kombiniert. Zudem finden sich zahlreiche Auswertungen von gemessenen Patienenchromatogrammen zu ausgewählten, klinisch relevanten Fragestellungen, wie beispielsweise die Darstellung des Ribavirinplasmaspiegels im Tagesverlauf, im Verlauf der ersten sechs Therapiewochen, im Vergleich von Männern und Frauen, sowie bei einem niereninsuffizienten Patienten. Zu den erhobenen Ergebnissen wird Stellung genommen, und daraus resultierende Schlussfolgerungen bezüglich einer zukünftigen Optimierung der Hepatitis-C-Therapie kommentiert. / The doctorate program will describe the development, optimization and validation of a reversed phase chromatography method for measuring ribavirinplasma levels. This has been combined with solid phase extraction for preparing samples. In addition, numerous analyses of patients’ chromatograms are included relative to clinically relevant questions such as the depiction of ribavirinplasma levels during the course of a day, during the course of the first six weeks of therapy, in comparison to other men and women as well as patients suffering from kidney insufficiencies. The collected data will be commented on, as well as the resulting implications relative to a future optimization of hepatitis-C therapy.

Residuos de fármacos estão presentes em diversas matrizes ambientais e estudos focados na determinação destes tem ganhado grande importância nos últimos anos, devido ao aumento do consumo de medicamentos pela população. A questão do controle de resíduos de compostos farmacologicamente ativos no meio ambiente aquático foi reconhecida como uma das questões emergentes na Química Ambiental, e tem-se dado maior importância visto que os fármacos são encontrados em matrizes em estudos em concentrações de &mu;gL-1 e ngL-1. Nesta pesquisa estudou-se três fármacos antiflamatórios que são amplamente consumidos pela população: diclofenaco, nimesulida e paracetamol. O método analítico foi desenvolvido e validado para a determinação destes fármacos em amostras de águas superficiais da cidade de São Carlos (SP). Inicialmente foi feita a validação do método proposto segundo a Resolução DOQ-CGCRE-008 do INMETRO. Os limites de detecção, e de quantificação e inferior de quantificação do método para a determinação do diclofenaco, nimesulida e paracetamol, foram, respectivamente, 0,5; 1,1 e 1,1 &mu;gL-1. A linearidade, desvio-padrão relativo, exatidão e recuperação média para o diclofenaco foram, respectivamente, R de 0,99, 3,03%, 100,55% e 97,94%. Para a nimesulida, os valores de linearidade, desvio-padrão relativo, exatidão e recuperação, foram, R de 0,98, 2,43%, 101,46% e 100,67%. Já para o paracetamol obteve-se os seguintes valores para linearidade, desvio-padrão relativo, exatidão e recuperação, R de 0,99, 3,50%, 97,94% e 93,17%, respectivamente. Na segunda etapa deste estudo aplicou-se o método validado na análise de amostras de águas coletadas na cidade de São Carlos (SP). Para o método de extração utilizou-se a extração em fase sólida (SPE) e como técnica analítica utilizou-se o HPLC/DAD. Os resultados não indicaram a presença dos fármacos diclofenaco, nimesulida e paracetamol até o limite de detecção do método empregado. / Residues of drugs are present in various environmental matrices and studies focused on the determination of these have gained in importance in recent years, due to increased drug consumption by the population. The issue of control of residues of pharmacologically active compounds in the aquatic environment was recognized as one of the emerging issues in environmental chemistry, and has given greater importance since the drugs are found in studies in matrices at concentrations &mu;gL-1 and ngL-1. In this study was studied three drugs that are widely consumed by the population: diclofenac, nimesulide and acetaminophen. The analytical method was developed for the determination of these drugs in surface water samples from São Carlos (SP). Initially, made a validation of the method proposed second resolution DOQ-008-CGCRE INMETRO. The detection, quantification and lower quantification limits of method for determining of diclofenac, nimesulide and paracetamol were 0.5, 1.1 and 1.1 &mu;gL-1, respectively. The linearity, relative standard , accuracy and average recovery of the method for diclofenac were, respectively, R equal to 0.99, 3.03%, 100.55% and 97.94%. For nimesulide, the values of linearity, relative standard, accuracy and recovery were R equal to 0.98, and 2.43%, 101.46% and 100.67%. For acetaminophen obtained the following values for linearity, relative standard, accuracy and recovery, R equal to 0.99, 3.50%, 97.94% and 93.17% respectively. In the second stage of the study applied the validated method in the analysis of water samples collected in the São Carlos (SP). For extracting the drugs, SPE cartridges were used followed by HPLC / DAD. The results indicate the absense of the studied drugs diclofenac, nimesulide and acetaminophen down to the detection limits of the method employed.

Os ovinos domésticos (Ovis aries) estão entre os primeiros animais domesticados para lã, leite, carne e pele. Novas biotecnologias de avaliação e manipulação do sêmen estão sendo estudadas para a elucidação de causas de infertilidade em machos. Sabe-se que danos causados na membrana espermática diminuem a qualidade seminal. Considerando que as membranas são compostas por uma bicamada de fosfolipídios e que a peroxidação lipídica é uma das principais causadoras de lesão celular, explica-se a importância de estudos sobre os lipídios constituintes do sêmen. A peroxidação lipídica é conseqüente da reação entre os lipídios e as espécies reativas de oxigênio que são fisiologicamente produzidas pelo metabolismo celular. Esse quadro pode ser diminuído pela presença de antioxidantes no sêmen, como a vitamina E. O sêmen foi coletado através da técnica da vagina artificial e após análise imediata e mediata, foi centrifugado para a obtenção de duas frações: plasma seminal e pellet de espermatozóide. Ambas tiveram seus lipídios extraídos por dois métodos diferentes e foram qualificados e quantificados pela especificidade e sensibilidade da Cromatografia Liquida de Alta Eficiência e assim pudemos escolher qual o melhor processo de extração. A análise estatística dos dados foi realizada através do programa SAS for Windows. O AG saturado predominante no espermatozóide é o mirístico e o AG insaturado predominante é o DHA, em ambas as extrações. No plasma seminal, nos dois métodos, o AG saturado que prevalece é o palmítico e o insaturado é o oléico. Dentre os métodos Folch (1957) modificados estudados, o que obtivemos melhores resultados na identificação e quantificação dos AG foi a extração 1. / The domestic sheep (Ovis Aries) is one of the first domesticated animals for wool, milk and meat production. The study of technologies for assessment and handling of semen are in course for the elucidation of male infertility causes. It is well known that damage to the sperm membrane decreases semen quality. Considering that the membranes are composed by a phospholipid bilayer, lipid peroxidation is a major cause of cell damage and explains the importance of studies on lipid components of semen. Lipid peroxidation is a consequence of the reaction between lipids and reactive oxygen species that are physiologically produced by cellular metabolism. This event may be reduced in the presence of antioxidants in semen, such as tocopherol. Semen was collected by artificial vagina and, after sperm evaluation, samples were centrifuged resulting in two fractions: seminal plasma and spermatozoa pellet. Both had their lipids extracted by two different methods and were qualified and quantified for the sensitivity and specificity to the high performance liquid chromatography in order to determine the most efficient extraction technique. Statistical analysis was performed using SAS software for Windows. The predominant saturated fatty acid in sperm is the myristic and the most abundant insaturated fatty acid in both extractions was DHA. In seminal plasma, in both methods, the prevailing fatty acid is the saturated palmitic and the unsaturated oleic. Among the methods evaluated, we obtained the best results of identification and quantification of fatty acids in extraction 1.

O objetivo desse trabalho foi demonstrar a viabilidade do acoplamento entre as técnicas de cromatografia líquida de alta eficiência com a espectrometria de emissão óptica com plasma indutivamente acoplado (HPLC - ICP OES) em um equipamento da Thermo Fisher Scientific, visando análise de especiação elementar de espécies com maiores concentrações na amostra. A identificação de espécies moleculares associadas aos elementos Cu, Fe, Mg, e Zn em amostras de castanha de caju foi adotada como modelo para comprovar tal acoplamento. Para que o ICP OES pudesse adquirir sinais de emissão ao longo de um tempo determinado, ou seja, para que o equipamento pudesse coletar um sinal transiente, fundamental para o acoplamento HPLC - ICP OES, foi necessário executar algumas modificações. Para isso, foram feitas alterações na placa eletrônica principal do equipamento, e trocada sua caixa controladora de gases. Os parâmetros instrumentais do ICP OES foram otimizados em uma série de ensaios com o intuito de estabelecer e avaliar desempenho do instrumento e a robustez do método empregado nas análises de castanha de caju. Determinações das concentrações totais de Ca, Cu, Fe, Mg, Mn, Ni, e Zn foram feitas em amostras de castanha de caju e nos materiais de referência NIST 1515 e NIST 1547, apresentando uma exatidão do método aceitável, levando-se em consideração o preparo de amostra e a medida no ICP OES. As determinações totais elementares nas amostras de castanha de caju indicaram que os teores de Ca foram de (0,04 ± 0,01) % ; Cu (26,3 ± 5,0) mg kg-1; Fe (47,9 ± 8,8) mg kg-1; Mg (0,31 ± 0,04) %; Mn (25,6 ± 3,9) mg kg-1; Ni (24,8 ± 1,9) mg kg-1; Zn (66,3 ± 2,4) mg kg-1. Para identificação de espécies moleculares associadas aos elementos Cu, Fe, Mg, e Zn, foi feita nas amostras de castanha de caju uma extração de metais e proteínas utilizando-se de uma solução extrato de NaOH 1 mol L-1. O extrato ainda foi submetido à uma etapa de clean-up, com precipitação das proteínas com acetona e ressupenção das mesmas com tampão Tris-HCl, pH 7,4. As proteínas e metais ressupensos em tampão foram analisados no sistema HPLC ICP OES, em que a separação cromatográfica ocorreu através de uma coluna de exclusão por tamanho (SEC), a detecção molecular através do detector UV-Vis do HPLC, e a detecção elementar através do ICP OES. As espécies moleculares identificadas estão no intervalo de massa molecular compreendido entre 2 e 177 kDa, sendo que Cu, Fe e Zn apresentam associação com essas espécies moleculares. Foram monitorados ainda, no ICP OES, os sinais de intensidade de emissão de P e S. Esses elementos também apresentaram associação com espécies moleculares com peso molecular entre 2 e 177 kDa, sugerindo que essas espécies se tratam de metaloproteínas. / The aim of this study was to demonstrate the capability of coupling between liquid chromatography techniques of high efficiency with the optical emission spectrometry with inductively coupled plasma (HPLC - ICP OES) in an equipment of Thermo Fisher Scientific, aiming elemental speciation analysis of species with higher concentrations in the sample. The identification of molecular species associated with elements Cu, Fe, Mg and Zn in cashew nuts samples was adopted as a model to prove such coupling. For ICP OES could get emission signal through a certain time, so that the equipment could collect a transient signal, critical to the HPLC coupling - ICP OES, it was necessary to perform some modifications in the instrument. For this, changes were made to the main electronic board of the equipment and also changed their gas controller box. The instrumental parameters of the ICP OES were optimized in a series of tests in order to establish and evaluate performance of the instrument and the robustness of the method employed in cashew nut analysis. Determination of the total concentration of Ca, Cu, Fe, Mg, Mn, Ni and Zn were made in cashew sample and reference materials NIST 1515 and NIST in 1547, with an accuracy acceptable method, taking into account the sample preparation and measurement by ICP OES. The elemental total determinations in cashew nut samples indicated that Ca were (0.04 ± 0.01)%; Cu (26.3 ± 5.0) mg kg-1; Fe (47.9 ± 8.8) mg kg-1; Mg (0.31 ± 0.04)%; Mn (25.6 ± 3.9) mg kg-1; Ni (24.8 ± 1.9) mg kg-1; Zn (66.3 ± 2.4) mg kg-1. For the identification of molecular species associated with the elements Cu, Fe, Mg, and Zn was performed on samples of cashew nuts an extract of metals and proteins using an extract solution of NaOH 1 mol L-1. The extract was further subjected to one-step of clean-up with protein precipitation with acetone following by a resuspension with Tris-HCl buffer, pH 7.4. Proteins and metals resuspended in buffer, were analyzed in the HPLC - ICP OES system, wherein the chromatographic separation took place by a size exclusion column (SEC), the molecular detection by UV-Vis detector of HPLC, and elemental detection by ICP OES. The molecular species were identified in the molecular weight range between 2 and 177 kDa, and Cu, Fe and Zn are associated with these molecular species. Were also monitored in the ICP OES, P and S emission intensity signals. These elements also associated with molecular species having a molecular weight between 2 and 177 kDa, suggesting that this species are metalloproteins.