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CALCIUM CARBONATE OR CALCIUM CHELATE? ELUCIDATING THIS QUESTION USING A CASE STUDY. Analytical Chemistry disciplines are present in the curriculum of several undergraduate courses. However, based on our experience, students not always are able to realize the association of the scientific concepts included in these disciplines with the social context and, specially, with their future field of work. In the present report, we used a case study about calcium supplementation by calcium carbonate and calcium chelate that included a science outreach activity, which was applied to students enrolled in three undergraduate courses: Pharmacy, Biomedicine, and Chemistry. We noticed that students not only got actively involved with the case, but also highlighted the importance of this activity, took the case beyond the classroom, and requested the inclusion of other similar activities in the discipline. Although different answers were presented for the case, the results obtained bring a positive impact on the instructors’ teaching practice and on the courses as a whole.

Objective We aimed to assess which metabolic pathways would be implicated in the phenotypic changes of the epithelial lining of odontogenic keratocyst after marsupialization, comparing pre- and post-marsupialized lesions with adjacent oral mucosa. Materials and methods Eighteen formalin-fixed and paraffin-embedded tissues from six subjects were divided into three paired groups: odontogenic keratocyst pre (n=6) and post marsupialization (n=6), and adjacent oral mucosa (n=6). The metabolic pathways found in these groups were obtained by high-performance liquid chromatography-mass spectrometry-based untargeted metabolomics performed. Results Through putative metabolite annotation followed by pathway enrichment and predictive analysis with automated algorithms (Mummichog and Gene Set Enrichment Analysis), we found differences in many cellular processes that may be involved in inflammation, oxidative stress response, keratinocyte-basal membrane attachment, differentiation, and proliferation functions, all relevant to odontogenic keratocyst pathobiology and the phenotype acquired after marsupialization. Conclusion Our study was able to identify several metabolic pathways potentially involved in the metaplastic changes induced by marsupialization of odontogenic keratocysts. An improved comprehension of this process could pave the way for the development of targeted therapies.

Iodine is a trace micronutrient that is critical for normal thyroid function and human health. Inadequate dietary intake is associated with cognitive impairment, infertility, growth retardation and iodine deficiency disorders in affected populations. Herein, we examined the prevalence of iodine deficiency in adults (median age of 61 years) based on the analysis of 24 h urine samples collected from 800 participants in four clinical sites across Canada in the Prospective Urban and Rural Epidemiological (PURE) study. Urinary iodide together with thiocyanate and nitrate were measured using a validated capillary electrophoresis assay. Protective/risk factors associated with iodine deficiency were identified using a binary logistic regression model, whereas daily urinary iodine concentration (24 h UIC, μg/L) and urinary iodine excretion (24 h UIE, μg/day) were compared using complementary statistical methods with covariate adjustments. Overall, our Canadian adult cohort had adequate iodine status as reflected by a median UIC of 111 μg/L with 11.9% of the population

Giant cell granuloma of the jaws are benign osteolytic lesions of the jaws. These lesions are genetically characterized by mutually exclusive somatic mutations at TRPV4, KRAS, and FGFR1, and a fourth molecular subgroup which is wild-type for the three mutations. Irrespective of the molecular background, giant cell granulomas show MAPK/ERK activation. However, it remains unclear if these mutations lead to differences in their molecular signaling in giant cell granulomas.Metabolomics, proteomics, and phosphoproteomics analyses were carried out in formalin-fixed paraffin-embedded samples of giant cell granuloma of the jaws. The study cohort consisted of five lesions harboring mutations in FGFR1, six in KRAS, five in TRPV4, and five that were wild-type for these mutations.Lesions harboring KRAS or FGFR1 mutations showed overall similar proteomics and metabolomics profiles. In all four groups, metabolic pathways showed similarity in apoptosis, cell signaling, gene expression, cell differentiation, and erythrocyte activity. Lesions harboring TRPV4 mutations showed a greater number of enriched pathways related to tissue architecture. On the other hand, the wild-type group presented increased number of enriched pathways related to protein metabolism compared to the other groups.Despite some minor differences, our results revealed an overall similar molecular profile among the groups with different mutational profile at the metabolic, proteic, and phosphopeptidic levels.

Objectives Cystic fibrosis (CF) is a debilitating genetic disorder that benefits from early detection. CF diagnosis relies on measuring elevated sweat chloride that is difficult in neonates with low sweat rates. We introduce a new method for sweat chloride determination from volume-limited specimens, and explore the potential utility of sweat bicarbonate in neonatal CF screening. Methods A rapid assay ( 5 min) was developed to analyze chloride and bicarbonate using capillary electrophoresis with indirect UV detection (CE-iUV). Pilocarpine-stimulated sweat samples from screen-positive CF infants were collected at two hospital sites, including confirmed CF (n = 12), CF screen-positive inconclusive diagnosis (n = 4), and unaffected non-CF cases (n = 37). All sweat chloride samples were analyzed by a coulometric titrator and CE-iUV, and the viability to measure acid-labile bicarbonate was also evaluated. Results Stability studies revealed that bicarbonate can be reliably assessed in sweat if acidification and heating were avoided. Method validation demonstrated that sweat chloride and bicarbonate were quantified with acceptable accuracy (recovery of 102%), precision (CV = 3.7%) and detection limits (∼ 0.1 mM). An inter-laboratory comparison confirmed a mean bias of 6.5% (n = 53) for sweat chloride determination by CE-iUV relative to a commercial chloridometer. However, sweat bicarbonate did not discriminate between CF and non-CF infants (AUC = 0.623, p = 0.215) unlike chloride (AUC = 1.00, p = 3.00 × 10

In this work multivariate strategies were employed in order to highlight new potential biomarkers of interest to detect the exogenous treatment of steers intramuscularly treated with boldenone undecylenate. Serum samples collected from treated (n = 4) and control (n = 8) crossbred animals of varying ages and weights were extracted using a simple sample preparation procedure based on salt assisted liquid-liquid extraction. Data acquisition was performed using liquid chromatography and Q-Exactive™ Orbitrap mass spectrometry. Data processing and treatment were performed using two non-targeted workflows: (1) Compound Discoverer software and (2) XCMS package on the open-source R software combined with MetaboAnalyst. Three potential biomarkers were highlighted taking into account the chromatographic shapes, the feature location on the generated s-plots, the fold change, the adjusted p values, the coefficient of variation in the QC samples and the area under the ROC curves. Predicted formulas based on mass accuracy, structural composition and spectra similarity were proposed. A robust statistical model to predict the boldenone treatment was further developed based on the weighted abundances of the selected biomarkers. The requirements for screening methods were successfully fulfilled, together with a wider detection window in comparison with the monitoring of the deconjugated metabolite boldenone, although biomarker identification studies are still ongoing.

Metabolomics studies rely on the availability of suitable analytical platforms to determine a vast collection of chemically diverse metabolites in complex biospecimens. Liquid chromatography-mass spectrometry operated under reversed-phase conditions is the most commonly used platform in metabolomics, which offers extensive coverage for nonpolar and moderately polar compounds. However, complementary techniques are required to obtain adequate separation of polar and ionic metabolites, which are involved in several fundamental metabolic pathways. This chapter focuses on the main mass-spectrometry-based analytical platforms used to determine polar and/or ionizable compounds in metabolomics (GC-MS, HILIC-MS, CE-MS, IPC-MS, and IC-MS). Rather than comprehensively describing recent applications related to GC-MS, HILIC-MS, and CE-MS, which have been covered in a regular basis in the literature, a brief discussion focused on basic principles, main strengths, limitations, as well as future trends is presented in this chapter, and only key applications with the purpose of illustrating important analytical aspects of each platform are highlighted. On the other hand, due to the relative novelty of IPC-MS and IC-MS in the metabolomics field, a thorough compilation of applications for these two techniques is presented here.

Metabolomics is a discipline that offers a comprehensive analysis of metabolites in biological samples. In the last decades, the notable evolution in liquid chromatography and mass spectrometry technologies has driven an exponential progress in LC-MS-based metabolomics. Targeted and untargeted metabolomics strategies are important tools in health and medical science, especially in the study of disease-related biomarkers, drug discovery and development, toxicology, diet, physical exercise, and precision medicine. Clinical and biological problems can now be understood in terms of metabolic phenotyping. This overview highlights the current approaches to LC-MS-based metabolomics analysis and its applications in the clinical research.

New experiments for undergraduate students are needed to stimulate experiential learning in the laboratory while providing valuable training for future career development. Iodine deficiency remains a major public health concern that is monitored by measuring the median urinary iodide concentration of a population. In this context, we have introduced a kinetic spectrophotometric experiment based on the classic Sandell–Kolthoff reaction for second-year undergraduate students in analytical chemistry. This two-day laboratory experiment incorporates principles of quantitative chemical analysis, redox chemistry, reaction kinetics, optical spectroscopy, sample pretreatment, external calibration, method validation, statistical analysis, and quality assurance. Additionally, students gain real-world experience of implementation of a reliable analytical method used in global health initiatives to combat iodine deficiency, including participation in a round-robin study with the CDC. This colorimetric assay is widely ...

Signal variation is a common drawback in untargeted metabolomics using liquid chromatography-mass spectrometry (LC-MS), mainly due to the complexity of biological matrices and reduced sample preparation, which results in the accumulation of sample components in the column and the ion source. Here we propose a simple, easy to implement approach to improve data quality in untargeted metabolomics by LC-MS. This approach involves the use of a divert valve to direct the column effluent to waste at the beginning of the chromatographic run and during column cleanup and equilibration, in combination with longer column cleanups in between injections. Our approach was tested using urine samples collected from patients after renal transplantation. Analytical responses were contrasted before and after introducing these modifications by analyzing a batch of untargeted metabolomics data. A significant improvement in peak area repeatability was observed for the quality controls, with relative standard deviations (RSDs) for several metabolites decreasing from ∼60% to ∼10% when our approach was introduced. Similarly, RSDs of peak areas for internal standards improved from ∼40% to ∼10%. Furthermore, calibrant solutions were more consistent after introducing these modifications when comparing peak areas of solutions injected at the beginning and the end of each analytical sequence. Therefore, we recommend the use of a divert valve and extended column cleanup as a powerful strategy to improve data quality in untargeted metabolomics, especially for very complex types of samples where minimum sample preparation is required, such as in this untargeted metabolomics study with urine from renal transplanted patients.

The sweat chloride test remains the gold standard for confirmatory diagnosis of cystic fibrosis (CF) in support of universal newborn screening programs. However, it provides ambiguous results for intermediate sweat chloride cases while not reflecting disease progression when classifying the complex CF disease spectrum given the pleiotropic effects of gene modifiers and environment. Herein we report the first characterization of the sweat metabolome from screen-positive CF infants and identify metabolites associated with disease status that complement sweat chloride testing. Pilocarpine-stimulated sweat specimens were collected independently from two CF clinics, including 50 unaffected infants (e.g., carriers) and 18 confirmed CF cases. Nontargeted metabolite profiling was performed using multisegment injection–capillary electrophoresis–mass spectrometry as a high throughput platform for analysis of polar/ionic metabolites in volume-restricted sweat samples. Amino acids, organic acids, amino acid derivatives, dipeptides, purine derivatives, and unknown exogenous compounds were identified in sweat when using high resolution tandem mass spectrometry, including metabolites associated with affected yet asymptomatic CF infants, such as asparagine and glutamine. Unexpectedly, a metabolite of pilocarpine, used to stimulate sweat secretion, pilocarpic acid, and a plasticizer metabolite from environmental exposure, mono(2-ethylhexyl)phthalic acid, were secreted in the sweat of CF infants at significantly lower concentrations relative to unaffected CF screen-positive controls. These results indicated a deficiency in human paraoxonase, an enzyme unrelated to mutations to the cystic fibrosis transmembrane conductance regulator (CFTR) and impaired chloride transport, which is a nonspecific arylesterase/lactonase known to mediate inflammation, bacterial biofilm formation, and recurrent lung infections in affected CF children later in life. This work sheds new light into the underlying mechanisms of CF pathophysiology as required for new advances in precision medicine of orphan diseases that benefit from early detection and intervention, including new molecular targets for therapeutic intervention., We report the first characterization of the sweat metabolome from screen-positive cystic fibrosis infants and identify novel metabolites associated with disease status relative to unaffected carriers that complement sweat chloride testing.

Iodine deficiency is the most common preventable cause of intellectual disabilities in children. Global health initiatives to ensure optimum nutrition thus require continuous monitoring of population-wide iodine intake as determined by urinary excretion of iodide. Current methods to analyze urinary iodide are limited by complicated sample pretreatment, costly infrastructure, and/or poor selectivity, posing restrictions to large-scale epidemiological studies. We describe a simple yet selective method to analyze iodide in volume-restricted human urine specimens stored in biorepositories by capillary electrophoresis (CE) with UV detection. Excellent selectivity is achieved when using an acidic background electrolyte in conjunction with dynamic complexation via α-cyclodextrin in an unmodified fused-silica capillary under reversed polarity. Sample self-stacking is developed as a novel online sample preconcentration method to boost sensitivity with submicromolar detection limits for iodide (S/N ≈ 3, 0.06 μM) directly in urine. This assay also allows for simultaneous analysis of environmental iodide uptake inhibitors, including thiocyanate and nitrate. Rigorous method validation confirmed good linearity (R(2) = 0.9998), dynamic range (0.20 to 4.0 μM), accuracy (average recovery of 93% at three concentration levels) and precision for reliable iodide determination in pooled urine specimens over 29 days of analysis (RSD = 11%, n = 87).

Background A capillary electrophoresis (CE) assay was recently introduced as a new method for monitoring iodine nutrition in large-scale epidemiological studies. However, further tests revealed unanticipated matrix-dependent interferences when analyzing submicromolar levels of iodide in human urine as the predominate ionic form of dietary iodine. Herein, we describe a rigorous validation study that was used to identify sources of bias and establish modifications to the original CE method to improve method accuracy. Methods An interlaboratory method comparison using CE with UV detection and inductively coupled plasma–mass spectrometry (ICP-MS) was performed to quantify urinary iodide concentrations (n = 71) independently at McMaster University and Hamilton General Hospital, as well as the CDC as part of their quality assurance program. A positive bias in the original CE method was indicated, and buffer conditions were subsequently optimized to overcome matrix interferences for reliable iodine status determination. Results Positive bias in CE was attributed to variable concentrations of sulfate, a major urinary anion interference with similar mobility to iodide under the conditions originally reported. By increasing the concentration of α-cyclodextrin in the background electrolyte, the CE method was able to tolerate urinary sulfate over its normal physiological range without loss in signal response for iodide. The optimized CE assay generated results that were consistent with ICP-MS using 2 different internal standards (187Re and 130Te) with a median bias under 10%. Conclusions CE offers a simple, selective, and cost-effective separation platform for surveillance of the iodine status of a population requiring only small volumes (

New methods for quantitative analysis of strong anions are required for diagnostic testing of human diseases. Current techniques suffer from poor selectivity and/or long analysis times that are not amenable for labile anions in high-saline or volume-restricted samples. We introduce a rapid assay (5 min) based on capillary electrophoresis (CE) with indirect UV detection for simultaneous analysis of sulfate, sulfite, and chloride in human urine, plasma, and sweat specimens. Remarkable selectivity for strong anions is achieved by using an acidic background electrolyte under reversed polarity that results in electrokinetic rejection of matrix interferences at the capillary inlet. A dual co-ion probe system consisting of 5 mM naphthalene disulfonate (NDS) and 5 mM naphthalene trisulfonate (NTS) in 0.4 M formic acid, pH 2.0 is developed for detection of UV transparent anions (S/N ≈ 3, 60 μM with a 25 μm inner diameter fused-silica capillary) with good peak symmetry and baseline stability. Due to the chemical reactivity of sulfite, dilute formaldehyde is used as a reagent to form an acid-stable hydroxymethylsulfonate adduct. Method validation confirmed excellent linearity (R(2)0.999), good accuracy (mean bias ≈7%), and acceptable long-term reproducibility (CV10%) over 20 days. The assay allows for artifact-free determination of sulfate and sulfite with consistent results for chloride when compared to standard electrochemical methods (R(2)0.975). Preliminary data suggest that kidney-stone formers have lower urinary sulfate excretion relative to non-kidney-stone patient controls (p = 0.0261). CE offers a selective yet robust platform for routine analysis of strong anions that is needed for confirmatory testing of cystic fibrosis, sulfite oxidase deficiency, urolithiasis, and other disorders of sulfur metabolism and/or anion transport.

A utilização de medicamentos veterinários é de grande importância na produção animal, porém também pode trazer conseqüências negativas, como a presença de resíduos em alimentos, como o leite bovino. As sulfonamidas são uma classe de antibióticos utilizados no tratamento veterinário e humano, sendo de grande importância sua análise em alimentos de origem animal, como o leite, uma vez que podem provocar resistência bacteriana, reações alérgicas e intoxicações, comprometendo a segurança alimentar da população e prejudicando a fabricação de derivados lácteos. A maioria dos estudos vem utilizando a extração em fase sólida (SPE), na extração das sulfonamidas e no clean up da amostra. Entretanto, o método QuEChERS, introduzido em 2003, vem sendo considerado promissor na extração de diversos compostos em matrizes alimentícias. Assim, a presente pesquisa teve o objetivo de desenvolver e validar métodos analíticos, comparando os métodos de extração SPE e QuEChERS, com posterior análise por cromatografia líquida de alta eficiência com detecção de arranjo de diodos, para a determinação de sulfonamidas (sulfatiazol, sulfametazina e sulfadimetoxina) em leite bovino. Na otimização das condições cromatográficas e do método de extração QuEChERS, utilizou-se planejamentos experimentais. Primeiramente, foram otimizadas as condições cromatográficas, selecionando-se coluna C18 Luna (100 x 4,6 mm, 5 µm), 17 µL de volume de injeção, comprimento de onda de 290 nm, vazão de 0,50 mL min-1, temperatura de 20 °C e eluição gradiente com os solventes água e etanol. No método de extração QuEChERS, o procedimento otimizado utilizou 6 g de leite, 3 mL de água, 6 mL de acetonitrila contendo 1% de ácido acético, 2,4 g de MgSO4 e 0,6 g de acetato de sódio, sendo que na etapa de clean up da amostra utilizou-se 37,5 mg de amina primária e secundária, 25 mg de octadecilsilano e 150 mg de MgSO4. O planejamento experimental foi considerado eficiente na otimização das condições cromatográficas e no procedimento de extração QuEChERS, obtendo-se boa separação entre os picos e valores de recuperação entre 95 e 98%, com desvio padrão relativo inferior a 8,25% para os analitos na concentração de 0,05 mg kg-1. No desenvolvimento do método de extração SPE não foi possível obter boa recuperação dos analitos e clean up da amostra. Acredita-se que as sulfonamidas estudadas sejam perdidas na etapa de pré-tratamento do leite, a qual é importante para a realização da SPE. Desse modo, a extração usando o QuEChERS foi selecionada para a análises das sulfonamidas em leite bovino. Os ensaios de validação demonstraram que o método é adequado para a análise das sulfonamidas, dentro do limite máximo de resíduos (0,1 mg kg-1), estabelecido pela Agencia Nacional de Vigilância Sanitária, com exatidão e precisão dentro da faixa aceitável pelos guias de validação seguidos. Além disso, foi possível utilizar apenas etanol e água como fase móvel cromatográfica, o que é uma vantagem em relação a métodos tradicionais, pois utiliza um solvente orgânico menos tóxico e renovável. A vazão de fase móvel é relativamente baixa, o que contribui para a diminuição do volume de solvente orgânico consumido e de resíduos gerados. The use of veterinary medicines is of great importance in animal production, but also can have negative consequences, such as the occurrence of residues in food of animal origin, among which bovine milk stands out. Sulfonamides are an antibiotic class used in animal and human treatments, so it is important to analyze this kind of residue in food of animal origin, such as milk, because it can cause allergic reactions and poisoning, affecting food safety and damaging the manufactory of dairy products. Most of the studies have used solid-phase extraction (SPE) in the extraction of sulfonamides and clean up of the sample. However, the QuEChERS method, introduced in 2003, has been considered promising in the extraction of various compounds in food matrices. Thereby, this research aimed to develop and validate analytical methods comparing the extraction methods SPE and QuEChERS, followed by high performance liquid chromatography with diode array detection for the analysis of sulfonamides (sulfathiazole, sulfamethazine and sulfadimethoxine) in bovine milk. In the optimization of the chromatographic conditions and QuEChERS method, we used experimental design. Chromatographic conditions were optimized, selecting column C18 Luna (100 x 4.6 mm, 5 µm), injection volume of 17 µL, wavelength of 290 nm, flow rate of 0.5 mL min-1, temperature of 20 °C and gradient mode, using water and ethanol as mobile phase. In the QuEChERS method, the optimized procedure used 6 g of milk, 3 mL of water, 6 mL of acetonitrile with 1% of acetic acid, 2.4 g of MgSO4 and 0.6 g of sodium acetate, and in the clean up step it was used 37.5 mg of primary and secondary amine, 25 mg of octadecylsilane and 150 mg of MgSO4. Experimental design was considered efficient in the optimization, since it was obtained good separation of the chromatographic peaks and recovery between 95 and 98% with relative standard deviation below 8.25% for the analytes in the concentration of 0.050 mg kg-1. In the development of the extraction method SPE, it was not possible to obtain good recovery for the analytes and clean up of the sample. It is believed sulfonamides are lost in the sample pre-treatment step, which is important in performing SPE. Therefore, the QuEChERS method was selected for this analysis. The validation tests demonstrate the method is appropriate, within the maximum limit residue (0.1 mg kg-1), established by the National Health Surveillance Agency, with accuracy and precision within the accepted limit, according to the followed validation guides. Moreover, it was possible to use just water and ethanol as mobile phase, what is an advantage in relation to traditional methods, because it uses an organic solvent less toxicant and renewable. Rate flow is also relatively low, which contributes to decrease the required volume of organic solvent and the amount of waste produced.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior The objective of this thesis was the application of multivariate statistical methods for honey and coffee, essentially parallel factor analysis (PARAFAC), a second-order chemometric method. PARAFAC was utilized in the applications as a curve resolution method for the identification of fluorescent compounds present in the samples and their quantification exploring the second-order advantage, i.e., the ability to quantify an analyte in the presence of uncalibrated interferences. Three applications were developed. The first application of this thesis presented a tutorial for the construction and use of a PARAFAC model exemplified by a case study of sensory analysis of coffee capsules. The application aimed to demonstrate the use of PARAFAC for professionals unacquainted with this chemometric method. The methods of data preprocessing, the imposition of mathematical constraints and the criteria for guiding the choice of the number of factors in the model are addressed, such as the core consistency diagnostic (CORCONDIA) and the property of uniqueness by the split-half analysis. In the second application, PARAFAC and spectrofluorimetry were used to develop a method for the direct determination of phenylalanine in honey. PARAFAC models were constructed using molecular excitation-emission matrices (EEM) obtained by a standard addition analytical curve. The concentration of phenylalanine in the samples varied from 5.7 mg kg-1 to 32.5 mg kg-1 and the results were validated by a chromatographic method. In the last application, a method was developed to identify samples of Aroeira honey adulterated with corn syrup, sugar cane molasses, and polyfloral honey. Aroeira honey is a promising commodity from the northern region of Minas Gerais State, Brazil, which has gained prominence for its antimicrobial potential. In this application, PARAFAC was used as an exploratory method to identify the fluorescent compounds present in pure Aroeira honey. Supervised classification models (PLS-DA, U-PLSDA and N-PLSDA) were built with the EEMs of the pure and adulterated samples. The UPLS-DA models presented the best classification results with misclassification rates of 4% and 8% for the training and test sets, respectively. O objetivo desta tese foi a aplicação de métodos de estatística multivariada para análise de mel e café, essencialmente análise de fatores paralelos (PARAFAC), método quimiométrico de segunda ordem. O PARAFAC foi utilizado nas aplicações como método de resolução de curvas para a identificação de compostos fluorescentes presentes nas amostras e quantificação destes explorando a vantagem de segundaordem, ou seja, a capacidade de quantificar um analito na presença de interferências não calibradas. Três aplicações foram desenvolvidas. A primeira aplicação desta tese apresentou um tutorial para a construção e utilização de um modelo PARAFAC exemplificado por um estudo de caso de análise sensorial de cápsulas de café. A aplicação visou demonstrar o emprego de PARAFAC para profissionais não familiarizados com este método quimiométrico. São abordados os métodos de préprocessamento de dados, a imposição de restrições matemáticas e os critérios que norteiam a escolha do número de fatores do modelo, como o teste de consistência trilinear (CORCONDIA) e a propriedade de unicidade pela análise de split-half. Na segunda aplicação, PARAFAC e espectrofluorimetria foram empregados no desenvolvimento de um método para a determinação direta de fenilalanina em mel. Modelos PARAFAC foram construídos utilizando matrizes de excitação-emissão molecular (EEM) obtidas por uma curva analítica de adição-padrão. A concentração de fenilalanina nas amostras variou de 5,7 mg kg-1 a 32,5 mg kg-1 e os resultados foram validados por um método cromatográfico. Na última aplicação, foi desenvolvido um método para a identificação de amostras de mel de Aroeira adulteradas com xarope de milho, melaço e mel polifloral. O mel de Aroeira é um promissor commodity oriundo da região norte de Minas Gerais que tem ganhado destaque por seu potencial antimicrobiano. Nesta aplicação, o PARAFAC foi utilizado como um método exploratório visando identificar os compostos fluorescentes presentes no mel de Aroeira puro. Modelos de classificação supervisionada (PLS-DA, U-PLSDA e NPLSDA) foram construídos com as EEMs das amostras puras e adulteradas. Os modelos UPLS-DA apresentaram os melhores resultados de classificação com taxas de classificação incorreta de 4% e 8% para os conjuntos de treinamento e de teste, respectivamente.

As abelhas têm um papel muito importante na sustentação das florestas e da produção agrícola, onde através da polinização mantêm a biodiversidade e aumentam o rendimento de sementes e frutas. No entanto, nos últimos anos a população das abelhas está em declínio, em parte, devido aos efeitos de agrotóxicos, principalmente os neonicotinóides. No Brasil tem se tornado cada vez mais comuns episódios de mortalidade de abelhas e abandono de colmeias como consequência do uso extensivo de agrotóxicos. Apesar disso, existem poucos estudos sobre os efeitos dessas substâncias nas espécies nativas e híbridas. Assim, o presente estudo teve como finalidade desenvolver um método de extração QuEChERS miniaturizado seguido da cromatografia líquida de ultra eficiência acoplada à espectrometria de massas sequencial (UPLC-MS/MS), para a determinação dos neonicotinóides imidacloprida e tiametoxam no pólen forrageado e nos tecidos das abelhas africanizadas (Apis mellifera L.) e Tetragonisca angustula Latreille (1811). Este método permitiu estimar resíduos dos agrotóxicos em níveis de nanograma/grama (ng g-1), usando menores quantidades de amostra e reagentes em comparação com os métodos tradicionais. Dessa forma, foram empregadas 50 vezes menos abelhas africanizadas, 500 vezes menos T. angustula e 150 vezes menos pólen como amostras. Em relação aos reagentes, foram usados 15 vezes menos sais e solvente. Obtiveram-se recuperações médias entre 64,5 e 105,5 %, com boa repetibilidade (RSD

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior A metabolômica investiga modificações de metabólitos em uma célula, tecido ou organismo. Os aminoácidos representam uma classe de metabólitos importantes em estudos voltados para o diagnóstico precoce de doenças, incluindo o câncer, cujo principal modelo experimental celular é a levedura Saccharomyces cerevisiae. O benzo[a]pireno (BaP) é considerado um contaminante prioritário para diferentes agências de controle ambiental por ser um composto comprovadamente carcinogênico para humanos. Portanto, é importante avaliar a interferência de BaP no metabolismo de aminoácidos com a finalidade de encontrar biomarcadores de câncer. Neste trabalho, foi desenvolvido e otimizado um novo procedimento de extração de aminoácidos (derivatizados com propil cloroformato/propanol) usando nanotubos de carbono magnéticos funcionalizados. Foi possível concluir que o nanotubo híbrido A15E15 e a dessorção sem aquecimento promoveram a melhor extração dos analitos. Nos estudos de exposição de Saccharomyces cerevisiae, verificou-se, através de curvas de crescimento celular, que a concentração de BaP de 25,0 µmol/L provocou alterações na viabilidade celular, indicando alterações metabólicas. Um método de cromatografia a gás com detector de espectrometria de massas utilizando coluna Phenomenex ZB-AAA foi otimizado possibilitando a determinação de 16 aminoácidos (AA) em leveduras expostas e não expostas ao BaP. O estudo de alguns parâmetros de mérito mostrou que o método apresentou boa seletividade e linearidade na faixa de 0,004 a 50,00 mg/L (R2 > 0,9800). Os limites de detecção (LOD) ficaram na faixa de 0,002 a 0,31 mg/L e os limites de quantificação (LOQ), na faixa de 0,004 a 0,41 mg/L. A determinação de AA em células expostas e não expostas ao BaP mostrou, por meio de ANOVA com teste post-hoc de Bonferroni, com intervalo de confiança de 95%, que os aminoácidos glicina (Gly), serina (Ser), fenilalanina (Phe), prolina (Pro), asparagina (Asn), ácido aspártico (Asp), ácido glutâmico (Glu), tirosina (Tyr) e leucina (Leu) diferenciaram estatisticamente os dois grupos de amostras. Esse resultado confirmou estudos anteriores que revelaram o potencial desses AA como candidatos a biomarcadores de câncer. Metabolomics investigates changes in metabolites in a cell, tissue or organism. Amino acids represent a class of important metabolites in studies aimed at the early diagnosis of diseases, including cancer, whose main experimental cellular model is the yeast Saccharomyces cerevisiae. Benzo[a]pyrene (BaP) is considered a priority contaminant for different environmental control agencies because it is a proven carcinogenic compound for humans. Therefore, it is important to evaluate the BaP interference in the metabolism of amino acids in order to find cancer biomarkers. In this work, a new amino acid extraction procedure (derivatized with propyl chloroformate/propanol) using functionalized magnetic carbon nanotubes was developed and optimized. It was possible to conclude that the hybrid nanotube A15E15 and desorption without heating promoted the best analytes extraction. In studies of exposure of Saccharomyces cerevisiae, it was found, through cell growth curves, that the BaP concentration of 25.0 µmol/L caused changes in cell viability, indicating metabolic changes. A gas chromatography method with mass spectrometry detector using a Phenomenex ZB-AAA column was optimized, enabling the determination of 16 amino acids (AA) in yeasts exposed and not exposed to BaP. The study of some parameters of merit showed that the method showed good selectivity and linearity in the range of 0.004 to 50.00 mg/L (R2 > 0.9800). The limits of detection (LOD) were in the range of 0.002 to 0.31 mg/L and the limits of quantification (LOQ) in the range of 0.004 to 0.41 mg/L. The AA determination in cells exposed and not exposed to BaP showed, through ANOVA with Bonferroni post-hoc test, with a confidence interval of 95%, that the amino acids glycine (Gly), serine (Ser), phenylalanine (Phe), proline (Pro), asparagine (Asn), aspartic acid (Asp), glutamic acid (Glu), tyrosine (Tyr) and leucine (Leu) statistically differentiated the two groups of samples. This result confirmed previous studies that revealed the potential of these AAs as cancer biomarkers candidates.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico Esse trabalho teve como objetivo investigar as interações antagônicas e sinérgicas entre As e Se em plantas de linhaça dourada (Linum usitatissimum L.), buscando entender como o Se altera os mecanismos de tolerância e o metabolismo da planta na presença do As. A absorção, toxicidade, translocação e a distribuição de nutrientes essenciais (Fe, Mn e Zn) nas plantas foram avaliados em experimentos de exposição ao AsV, SeIV e SeVI e co-exposição ao AsV + SeIV. Para isso, as plantas foram cultivadas em solução de Hoagland 10% v v-1 suplementada com AsV, SeIV, SeVI ou AsV + SeIV em diferentes concentrações. As quantificações de As e Se foram realizadas por espectrometria de massas com plasma acoplado indutivamente (ICP-MS). As alterações provocas pelo Se nos mecanismos de tolerância e no metabolismo da planta quando co-exposto ao As também foram investigadas utilizando duas técnicas de espectrometria de massas com ionização ambiente PS-MS (paper spray mass spectrometry) e DESI-MSI (desorption electrospray ionization mass spectrometry imaging). A identificação de compostos tiólicos (fitoquelatinas e seus metabólitos) e de compostos inorgânicos e orgânicos de As e Se em extratos de raízes de plantas co-expostas foram feitas por PS-MS. Alterações metabólicas provocadas em folhas de plantas expostas a AsV, SeIV e co-expostas a AsV + SeIV foram verificadas por imagens químicas obtidas por DESI-MSI. Os experimentos de exposição e co-exposição demonstraram que o SeVI é a espécie mais tóxica e com maior translocação. O SeIV na concentração de 5 µmol L-1 foi capaz de reduzir o acúmulo de As e a co-exposição alterou a absorção de Fe, Mn e Zn. A identificação de fitoquelatinas (PCs), que são compostos tiólicos responsáveis por complexar metais e metaloides em plantas e diminuir sua toxicidade, foi realizada por PS-MS. Nos extratos de raízes de plantas co-expostas foram identificados glutationa (GSH), reduzido-PC2, reduzido-PC3, SeII-PC2 e AsIII-PC3. Compostos inorgânicos (AsV e AsIII) e orgânicos (ácido dimetilarsínico – DMA e ácido monometilarsônico - MMA) de As também foram identificados por PS-MS. Nas folhas foi possível verificar quatro perfis de alterações metabólicas que ocorreram em função das diferentes concentrações de exposição e co-exposição. O SeIV diminui a toxicidade do AsV apesar da absorção e translocação de As terem aumentado. Não foi possível associar a diminuição da toxicidade do AsV na presença de SeIV com a formação de PCs. As alterações metabólicas mais evidentes observadas nas folhas das plantas indicam a formação de metabólitos relacionados ao estresse abiótico. The goal of this work was to investigate the antagonistic and synergistic interactions between As and Se in golden flaxseed plants (Linum usitatissimum L.) in order to understand how Se changes the tolerance mechanisms and metabolism in the presence of As. Absorption, toxicity, translocation and the distribution of essential nutrients (Fe, Mn and Zn) in plants were evaluated in the experiments exposure to AsV, SeIV and SeVI and co-exposure to AsV + SeIV . Seedlings were cultivated in a 10 % v v-1 Hoagland solution supplemented with AsV, SeIV, SeVI or AsV + SeIV at different concentrations. Elemental quantification was performed by inductively coupled plasma mass spectrometry (ICP-MS). The changes caused by Se in the tolerance mechanisms and metabolism when co-exposed to As were also investigated using two mass spectrometry with ambient ionization techniques PS-MS (paper spray mass spectrometry) and DESI-MSI (desorption electrospray ionization mass spectrometry imaging). The detection and identification of thiolic compounds (phytochelatins and their metabolites) and inorganic and organic compounds of As and Se in extracts from co-exposed root plant were made by PS-MS. Metabolic changes caused in plant leaves exposed to AsV, SeIV and co-exposed to AsV + SeIV were verified by chemical images obtained by DESI-MSI. Exposure and co-exposure experiments demonstrated that SeVI was the most toxic species with the highest translocation by the plant. SeIV at a concentration of 5 µmol L-1 was able to reduce the accumulation of As and the co-exposure altered the absorption of Fe, Mn and Zn. The detection and identification of phytochelatins (PCs), that are thiolic compounds responsible for complexing metals and metalloids in plants and reducing their toxicity was possible using PS-MS. Glutathione (GSH), reduced-PC2, reduced-PC3, SeII-PC2 and AsIII-PC3 were identified in the extracts of co-exposed plants. Inorganic compounds (AsV and AsIII) and organic compounds (dimethylarsinic acid - DMA and monomethyarsonic acid - MMA) from As were also identified by PS-MS. In the leaves it was possible to verify four profiles of metabolic changes that occurred after exposure and co-exposure at different concentrations of As and Se. SeIV decreases AsV toxicity although As absorption and translocation have increased. It was not possible to associate the decrease in AsV toxicity in the presence of SeIV with the PCs formation. The most evident metabolic changes observed in the leaves indicated the formation of metabolites related to abiotic stress.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Os antidepressivos tricíclicos (TCAs) são substâncias usadas para o tratamento de distúrbios de ansiedade e depressão. E apesar de sua eficácia, apresentam muitos efeitos colaterais. Devido a isso e também à janela terapêutica estreita desses fármacos faz-se necessário a sua determinação em amostras biológicas. Para isso, um sistema multipoços de eletroextração (EE) multifases foi empregado para extração dos TCAs: doxepina, imipramina, amitriptilina, nortriptilina e clomipramina em fluido oral. Em seguida, o extrato obtido foi analisado por cromatografia líquida de ultra eficiência acoplada à espectrometria de massas sequencial (UHPLC-MS/MS) além da espectrometria de massas com ionização por paper spray (PS-MS). A influência do campo elétrico na extração foi avaliada, sendo esse um fator crucial para uma boa recuperação dos TCAs. Os analitos foram extraídos de uma solução doadora composta pela amostra e acetonitrila 1:1 (v/v), migrando através de uma membrana livre de 1-octanol, chegando à fase aceptora. Essa última foi constituída de papel cromatográfico embebido em ácido acético 400 mmol L-1 possuindo uma ponta triangular propícia para técnica de PS-MS. Estudos univariados foram realizados para otimização dos parâmetros de preparo de amostra: tipo de solvente de dessorção dos analitos, tipos de papel e de eletrólito usados na fase aceptora, solvente orgânico usado como filtro, presença de carreador no filtro orgânico e tipo de solvente orgânico na fase doadora. Uma otimização multivariada também foi realizada para avaliação do tempo de extração, diferença de potencial elétrico e percentual de solvente orgânico na fase doadora. Após otimização, o percentual de extração alcançado para cada um dos analitos foi cerca de 55% com um tempo de extração de 10 minutos a 300 V e percentual de acetonitrila de 50% (v/v). O método EE-UHPLC-MS/MS otimizado foi validado na faixa de concentração de TCAs de 2 a 12 ng mL-1, com LD de 0,13 a 0,25 ng mL-1 e LQ de 0,44 a 0,84 ng mL-1. A precisão e exatidão foram avaliadas em três níveis de concentração (2, 6 e 10 ng mL-1) alcançando coeficientes de variação menores que 26% e recuperações maiores que 75%. O método validado foi aplicado a amostras reais de fluido oral fortificadas, e os resultados tiveram baixa variação entre as amostras de diferentes voluntários. O método mostrou-se seletivo e ausente de efeito matriz. Uma nova configuração para a eletroextração foi realizada a fim de reduzir fontes de variação no posicionamento das fases aceptoras e ainda ser facilmente acoplada à espectrometria de massas com ionização por paper spray. Nessa configuração o percentual de extração de TCAs foi de 35% e os coeficientes de variação para a análise por PS-MS foram menores que 20%. Tricyclic antidepressants (TCAs) are substances used for the treatment of anxiety disorders and depression. And despite its effectiveness, have many side effects. Due to this and also to narrow therapeutic window of these drugs it is necessary to its determination in biological samples. For this reason, a multiphase multiwell plate for electroextraction was employed to extract the tricyclic antidepressants: doxepin, imipramine, amitriptyline, nortriptyline, and clomipramine from oral fluid. Afterwards the extract was analyzed by ultra-high liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and also by paper spray mass spectrometry analysis. The effect of electric field in the extraction was evaluated since it is a crucial factor to achieve good recovery of TCAs. The analytes were extracted from donor solution consisted of a mixture of a sample and acetonitrile 1:1 (v/v), across the free liquid membrane made of 1-octanol, arriving in the acceptor phase. This last one was constituted of chromatographic paper embedded in acetic acid 400 mmol L-1 with a triangular tip suitable to PS-MS technique. Univariate studies were carried out to optimize the parameters: desorption of analytes from acceptor phase, chromatography paper and electrolyte types used in the acceptor phase, the organic solvent used as organic filter, presence of carrier on the organic filter and organic solvent type used in the donor phase. A multivariate optimization was performed to evaluate the extraction time, applied electric potential difference and the solvent percentage in the donor phase. After optimization, extraction efficiency was about 55% with 10 minutes of extraction at 300 V and 50% (v/v) of acetonitrile in the donor phase. The optimized EE-UHPLC-MS/MS method was validated in the range 2 – 12 ng mL-1 to TCAs, with LD was 0,13 to 0,25 and LQ was 0,44 to 0,84. The precision and accuracy were assessed at three concentration levels (2, 6 and 10 ng mL) with relative standard deviations under 26% and recovery higher than 75%. The method validated was applied to real fortified samples with low variation among different sample sources. The method was selective and unaffected by matrix effect. A new electroextraction configuration has been tested in an attempt to reduce variation sources on the paper position of the acceptor phase and to easily and directly couple electroextraction to paper spray mass spectrometry. Using this system, the extraction efficiency was about 35%. And the relative standard deviations were lower than 20% in the analysis by PS-MS.

A poluição do ar por material particulado (MP) é uma preocupação mundial devido aos seus grandes malefícios à saúde humana, principalmente devido à capacidade do MP de adsorver substâncias tóxicas. Entres elas, estão os hidrocarbonetos policíclicos aromáticos (HPAs), que são reconhecidos como compostos carcinogênicos e mutagênicos. Material particulado com diâmetro aerodinâmico menor que 2,5 µm (PM2.5), presente no ar da cidade brasileira de Belo Horizonte - MG, foi coletado utilizando-se um amostrador de grande volume, por um período de um ano. A amostragem foi feita próximo a uma avenida de grande tráfego de veículos. A concentração média de PM2.5 neste período foi calculada e o valor encontrado foi de 36,39 µg m-3 . Esta concentração está muito acima do valor de 10 µg m-3 estabelecido pela Organização Mundial da Saúde (OMS) como limite máximo anual. O material particulado foi analisado e foi determinada a concentração dos 16 HPAs prioritários, de acordo com a Agência de Proteção Ambiental Americana. Para a análise, foi empregada a técnica de microextração em fase sólida por imersão direta com fibra resfriada, associada à cromatografia gasosa acoplada à espectrometria de massas. O valor da concentração anual de HPAs presentes no PM2.5 foi de 3,89 ng m-3 . Simultaneamente à coleta de PM2.5, foi realizada a coleta da fase gasosa, com auxílio de uma espuma de poliuretano. Utilizando-se um método semelhante ao que foi empregado para o PM2.5, foi determinada a concentração dos 8 HPAs de menor massa molecular, que normalmente estão presentes na fase gasosa na atmosfera. O valor encontrado foi de 12,55 ng m-3 . A partir das concentrações de HPAs, calculou-se a concentração de Benzo(a)pireno equivalente, cujo valor foi 0,94 ng m-3 . Este marcador é utilizado para o cálculo do risco estimado de câncer a que uma população está exposta, num período de 70 anos. Para este estudo, o valor encontrado foi 82/1.000.000. Foram também determinadas as possíveis fontes de emissão desses compostos, através das razões de isômeros. As principais fontes são a queima de combustíveis fósseis e de biomassa. Tais resultados confirmam a grande importância de se controlar este tipo de poluição. Simultaneous Determination of Polycyclic Aromatic Hydrocarbons on Atmospheric Particulate Matter and Gas Phase in Belo Horizonte, Brazil, Using CF-SPME-GC/MS. The atmospheric pollution by particulate matter (PM) is a worldwide concern due to its great impacts on human health, especially because of MPs ability to adsorb toxic substances. Among them there are the polycyclic aromatic hydrocarbons (PAHs), which are known carcinogenic and mutagenic compounds. Particulate matter with aerodynamic diameter smaller than 2.5 m (PM2.5), found in theatmosphere of the Brazilian city of Belo Horizonte MG, was collected using a high volume sampler, for a one-year period. The sampling was performed next to a high traffic avenue. The average concentration of PM2.5 in this period was calculated and found to be 36.39 g.m-3. Thisconcentration is way above the 10 g.m-3 value stablished by the World Health Organization (WHO) as the threshold for a one-year period.The particulate matter was analyzed and the concentration of 16 PAHs was determined. These PAHs are defined by the American Environmental Protection Agency as priority pollutants. For the analysis, the cold fiber direct immersion solid phase microextraction technique was employed, followed by gas chromatography coupled with mass spectrometry. The annual mean concentration of the PAHs on the PM2.5 was 3.89 ng m-3. Simultaneously to the sampling of PM2.5, the gas phase was also collected, using a polyurethane foam. A method, similar to the one utilizedwith the PM2.5, was employed for determining the concentration of the 8 PAHs of smaller molecular mass, normally found at the gas phase in the atmosphere. The concentration was 12.55 ng.m-3. With those PAHs concentrations, the Benzo(a)pyrene equivalent was calculated and foundto be 0,94 ng.m-3. This marker is employed to determine the incremental cancer risk to which a population is exposed in a lifetime period of approximately 70 years. For this study, the value found was 82/1,000,000. The possible emission sources of the PAHs were also determined, bytheir isomeric ratios. The main sources are fossil fuel and biomass burning. These results confirm the importance of controlling this kind of pollution.