Propolis is an approved food additive containing flavonoids as a major active constituent. Variability has been found in the composition of propolis in distinctive regions and it was noticed that there are limitations in the analysis of propolis. In this study, the identification of ten flavonoids and residual antibiotics in propolis was investigated by using several liquid chromatography techniques, including reversed-phase high-performance liquid chromatography (RP-HPLC), microemulsion LC (MELC) and ultra-performance LC (UPLC). The ten flavonoids that were selected for this research include rutin, myricetin, quercetin, apigenin, kaempferol, pinocembrin, CAPE, chrysin, galangin and acacetin while chlortetracycline, oxytetracycline and doxycycline were selected to examine the residual antibiotics in propolis. For the analysis of the selected flavonoids, routine RP-HPLC method was found to be the best method, while MELC technique was found more efficient for the analysis of the selected antibiotics. Solid phase extraction with HLB sorbent was utilised in the analysis of antibiotics for clean-up of propolis. In method development studies for flavonoids and antibiotics, one-factor-at-a-time (OFAT) approach was followed. The final optimised method for the analysis of flavonoids as well as the method. for the analysis of antibiotics was validated using the ICH guidelines, and various aspects, such as the linearity, selectivity, accuracy, recovery, robustness and stability parameters, were examined. Development of efficient conventional method for the extraction of flavonoids from propolis was studied extensively in the present research work using different extraction techniques such as maceration, hot extraction, ultrasound assisted extraction. Among all extraction experiments, ethanolic extraction using ultrasound extraction method was the best efficient approach. This thesis shows that, in general, the performance of O/W MELC is superior to that of conventional HPLC for the determination of residual antibiotics in propolis. UPLC was not suitable for the analysis of flavonoids and antibiotics. The conventional LC was the only technique to separate the ten flavonoids but MELC was able to separate nine of the flavonoids with faster analysis time. This work also showed that MELC uses cheaper solvents. This considerable saving in both cost and time will potentially improve efficiency within quality control. / Social Justice Department, Government of Maharashtra, India.

Oxytetracycline (OTC) is one of two antibiotics currently available and approved by the U.S. Food and Drug Administration for use as a chemotherapeutic agent in food fish and is widely used in the aquaculture industry. Previous pharmacokinetic studies of OTC have been conducted in cold water and warm water species of fish. However, no pharmacokinetic studies have been conducted on a cool water species such as yellow perch (Perca flavescens). The yellow perch is a cool water game and commercial species with high aquaculture potential. The pharmacokinetic profiles of oxytetracycline (OTC) was determined by measuring plasma concentrations in yellow perch following intraperitoneal (i.p.), intramuscular (i.m.), per os (p.o.), and intracardiac (i.c.) administration at a single dose of 50 mg/kg body weight. Using a modification of a high-performance-liquid-chromatographic (HPLC) technique, the plasma OTC concentrations were determined for each of the four routes of administration. Plasma concentrations were also evaluated in yellow perch exposed to a static 48-hour OTC water bath (100 mg/l). The terminal half-lives (t1/2) of OTC in yellow perch for i.p., i.m., p.o., and i.c. administrations were 112, 124, 50, and 28 h, respectively. The t1/2 for the i.m. route of administration was significantly longer than in any of the published i.m. OTC fish studies to date. However, the times of maximum OTC concentration (tmax) for the i.p., i.m. and p.o. administrations (2, 4, and 15 h, respectively) occurred relatively early in the plasma concentration-time curves. This suggests, that in yellow perch, OTC is initially absorbed very rapidly. The area under the plasma concentration-time curves (AUC) for the i.p., i.m., p.o., and i.c. routes of administration were 1718, 2659, 383, and 134 mcg·h/ml, respectively. No OTC was detected in the plasma of yellow perch following the water bath route of exposure. Finally, in yellow perch, effective therapy (plasma OTC concentrations above MIC values for most bacteria pathogenic to fish — 4 mcg/ml) would be achieved for up to 168 hours following a single i.p. or i.m. injection of 50 mg/kg and for up to 15 hours following a single p.o dose of 50 mg/kg. / Master of Science

Thesis (MScFoodSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The phenolic composition of Cyclopia species is believed to be partially responsible for the numerous health promoting properties associated with their extracts. Current quality control measures do not accommodate variation in phenolic profiles of Cyclopia species. In this study, comprehensive high performance liquid chromatography (HPLC) methods were developed for the improved characterisation of the phenolic composition of aqueous extracts of two Cyclopia species (C. subternata and C. maculata). The methods were developed to be suitable for both routine quantitative analysis on conventional HPLC instrumentation, and the construction of chromatographic fingerprints for further data analysis. The latter entailed similarity analysis and prediction of total antioxidant capacity (TAC). Using a methodical approach, two separate HPLC methods, using diode array detection (DAD), were developed and validated for the analysis of aqueous extracts prepared from unfermented (green) and fermented plant material of C. subternata and C. maculata. Separation was achieved using the same method parameters (column, temperature, mobile phases), except for differing mobile phase gradients. Hyphenation of the developed HPLC methods with mass spectrometry (MS) and tandem MS allowed the confirmation of phenolic compounds previously identified in Cyclopia, and the tentative identification of several additional compounds in Cyclopia species, which are reported here for the first time. These included apigenin-6,8-di- C-glucoside, 3-hydroxyphloretin-30,50-di-C-hexoside, eriodictyol-di-C-glucoside, iriflophenone-di-O,C-hexoside, hydroxymangiferin and hydroxyisomangiferin. Subsequently, a large number of aqueous extracts of randomly selected green C. subternata (n = 64) and C. maculata (n = 50) plant material samples were analysed. Large quantitative variations were observed on intra- and inter-species levels. Cyclopia maculata extracts contained almost six times more mangiferin than extracts from C. subternata. HPLC-DAD analysis produced duplicate fingerprints for each extract which were consequently used for further analysis. The chromatographic fingerprint of a bioactive extract of each species was included in the respective data sets. Similarity analysis was conducted between the fingerprints from the randomly selected extracts and the corresponding active extract. For each species several extracts were determined to have similar “activity” as that of the active extract (n = 15 for C. subternata and n = 45 for C. maculata). Compounds potentially responsible for the activity were tentatively identified with the aid of principal component analysis (PCA) in combination with similarity analysis. PCA was more effective in identifying small differences between fingerprints than similarity analysis based on the correlation coefficients (r) alone. Furthermore, multivariate data analysis was used to construct partial least squares (PLS) regression models for the prediction of TAC from fingerprint data of each species, and available data from two microplate TAC assays. The construction of the models was successful with reasonable errors (< 10%), and permitted the determination of compounds of interest for future research. These included compounds of known identity that had large positive contributions toward the predictions of TAC, or unknown compounds that had small UV signals, but relatively large positive contributions to the models. / AFRIKAANSE OPSOMMING: Die talle gesondheidbevorderingseienskappe van ekstrakte van Cyclopia spesies word gedeeltelik geassosieer met hul fenoliese samestelling. Huidige kwaliteitskontrolemaatreëls is nie in staat om die variasie wat in die fenoliese profiele van die spesies voorkom, te akkommodeer nie. Omvattende hoë druk vloeistof chromatografiese (HPLC) metodes is vir twee Cyclopia spesies, naamlik C. subternata en C. maculata, in hierdie studie ontwikkel vir beter karakterisering van die fenoliese samestelling van waterekstrakte van dié spesies. Die metodes moes ook geskik wees vir roetine analise van C. subternata en C. maculata ekstrakte op konvensionele HPLC instrumentasie, en vir die opstel van chromatografiese vingerafdrukke (fenoliese samestellingsprofiele) vir verdere data analise, soos gelykvormigheidsanalise en die voorspelling van die totale antioksidantkapasiteit (TAC). Twee HPLC metodes, wat van ’n ultraviolet-diode detektor (DAD) gebruik maak, is ontwikkel deur ’n sistematiese benadering te volg. Die onderskeie metodes is vir die ontleding van waterekstrakte van groen (ongefermenteerde) en gefermenteerde plantmateriaal van C. subternata en C. maculata gevalideer. Ongeag die spesie is optimale skeiding met dieselfde kolom, mobiele fase en kolom-temperatuur bereik, maar met verskillende mobiele fase gradiënte. Analise met massaspektrometrie (MS) en tandem MS het die teenwoordigheid van fenoliese verbindings, wat voorheen in Cyclopia spesies geidentifiseer is, bevestig. Verder is ook ’n aantal verbindings vir die eerste keer in Cyclopia tentatief geidentifiseer. Dit sluit apigenien-6,8-di-C-glukosied, 3- hidroksiefloretien-30,50-di-C-heksosied, eriodiktiol-di-C-glukosied, iriflofenoon-di-O,C-heksosied, hidroksiemangiferien en hidroksie-isomangiferien in. Vervolgens is ’n groot aantal ewekansig gekose waterekstrakte van beide groen C. subternata (n = 64) en C. maculata (n = 50) plantmateriaal geanaliseer, en groot kwantitatiewe variasie op intra- en inter-spesievlak waargeneem. Cyclopia maculata ekstrakte het byvoorbeeld byna ses maal die mangiferieninhoud van C. subternata ekstrakte gehad. HPLC-DAD analise van die ekstrakte het duplikaat vingerafdrukke van elke ekstrak geproduseer, wat vir verdere data analise gebruik is. Die chromatografiese vingerafdruk van ’n bioaktiewe ekstrak van elke spesie was by die onderskeie datastelle ingesluit. Gelykvormigheidsanalise is tussen vingerafdrukke van die ewekansig gekose ekstrakte en die ooreenstemmende aktiewe ekstrak uitgevoer. Vir elke spesie is ’n aantal “aktiewe” ekstrakte aangewys (n = 15 vir C. subternata en n = 45 vir C. maculata). Die verbindings wat potensieel verantwoordelik kan wees vir die aktiwiteite is met behulp van hoofkomponentontleding (PCA) in kombinasie met gelykvormigheidsanalise, tentatief aangewys. PCA was egter meer effektief om klein verskille tussen vingerafdrukke aan te dui, in vergelyking met gelykvormigheidsanalise wat slegs op die korrelasie koëffisiënt (r) gebaseer is. Meerveranderlike data analiese is gebruik om “gedeeltelike kleinste kwadrate” (PLS) regressiemodelle, vir die voorspelling van die TAC van beide spesies te bou. Die voorspelling is gebaseer op hul vingerafdruk data en TAC data van twee TAC mikroplaat metodes. Die model-konstruksie was suksesvol met aanvaarbare voorspellingsfoute (< 10%). Verbindings van belang kon ook bepaal word. Dit sluit bekende verbindings in wat groot positiewe bydraes ten opsigte van die voorspelling van TAC getoon het, asook ongeidentifiseerde verbindings wat klein UV-seine getoon het, maar relatiewe groot bydraes tot die modelle gehad het.

Orientador: Felix Guillermo Reyes Reyes / Dissertação (mestrado) - Universidade Estadual de Campiknas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-19T16:49:25Z (GMT). No. of bitstreams: 1 DelBianchi_Michelle_M.pdf: 2100554 bytes, checksum: 574b7310661133a3b473c024dee257a0 (MD5) Previous issue date: 2012 / Resumo: Edulcorantes sao compostos quimicos de origem sintetica ou natural que tem a propriedade de adocar alimentos, em substituicao total ou parcial a sacarose ou outros acucares e reduzem o teor calorico do produto resultante. A ingestao de adocantes artificiais no mundo tem crescido nos ultimos anos, com o objetivo principal de reduzir a ingestao calorica. O aumento global de doencas cronicas relacionadas com a ingestao de acucar, particularmente a obesidade e a diabetes, e tambem uma razao para o aumento do consumo de adocantes artificiais. O objetivo deste estudo foi avaliar a ingestao de adocantes artificiais atraves do consumo de adocantes de mesa liquidos e avaliar a possibilidade de consumo superior a sua ingestao diaria aceitavel (IDA) estabelecida por FAO/WHO comite de peritos em aditivos alimentares (JECFA). Inicialmente, uma pesquisa de campo foi realizada para avaliar qual marca de adocante de mesa liquido mais consumido no Brasil e as caracteristicas de uso do mesmo. Para tanto, um questionario foi enviado por correio eletronico, para individuos, com questoes relativas ao consumo de adocantes e, caso consumidos, aspectos de uso incluindo marca do adocante, ingestao e motivo de consumo. O questionario tambem continha perguntas sobre questoes socioeconomicas, demograficas e de saude. Em base a esta pesquisa, verificou-se quais sao as marcas de adocantes liquidos de mesa mais consumidas. Os edulcorantes presentes nesses adocantes mais consumidos sao: sacarina, ciclamato, aspartame e acessulfame-K. Foi realizada a quantificacao desses edulcorantes nos adocantes de maior consumo. Para a quantificacao de sacarina, aspartame e acessulfame-K foi utilizada a cromatografia liquida de alta eficiencia acoplada ao detector de arranjo de diodos (CLAE-DAD). A quantificacao de ciclamato foi realizada por espectrofotometria UV/Vis, em 314 nm, apos derivatizacao para N, Ndiclorociclohexilamina. Os metodos analiticos foram validados de acordo com os seguintes parametros de validacao: linearidade, faixa linear, especificidade, precisao (repetibilidade intra e inter dias), limite de deteccao (LOD) e limite de quantificacao (LOQ). Todos os metodos mostraram parametros de validacao de acordo com o guia para validacao de metodos analiticos estabelecidos pela Agencia Nacional de Vigilancia Sanitaria (ANVISA). A maioria das respostas ao questionario foram do genero feminio (70,2%) e provenientes da regiao Sudeste do Brasil (70,8%). Entre os entrevistados, 47% usavam adocantes liquidos de mesa. A maioria que relatou o uso de adocantes artificiais tinham entre 21 e 32 anos ou acima de 45 anos de idade. Destes, 83% afirmaram nao ter qualquer tipo de patologia e que a principal razao para o consumo de adocantes liquidos de mesa foi a preferencia em relacao ao acucar. Cerca da metade (54%) dos consumidores relataram sentir sabor desagradavel ou amargo quando consomem adocante e 32% nao sabiam qual era o edulcorante artificial contido no adocante. Os resultados da analise dos adocantes liquidos de mesa provenientes do mercado varejista indicou variacao significativa (p<0,05) na concentracao de edulcorante entre marcas e lotes da mesma marca. Com base nos resultados do questionario verificou-se qual e a marca mais consumida. Essa marca contem sacarina e ciclamato. Uma amostra dessa marca foi entregue a individuos adultos consumidores deste produto, para calcular o nivel de consumo dos edulcorantes artificiais. Foi verificado que a ingestao de sacarina e ciclamato atraves do consumo de adocantes liquidos de mesa representa, em media, 38,7% e 19,8% do valor de IDA do edulcorante, respectivamente. Considerando a variacao na concentracao de adocantes entre as marcas e lotes da mesma marca e que cada edulcorante artificial tem um valor de IDA estabelecido, recomenda-se que as concentracoes dos edulcorante em adocantes liquidos de mesa sejam apresentados na etiqueta da embalagem, para fornecer ao consumidor informacoes sobre seu consumo, e permitir controlar a sua exposicao total a estas substancias. Em geral, os resultados sugerem uma substituicao cada vez maior de acucar pelos adocantes artificiais, que indicam a necessidade de se obter dados sobre a ingestao total de adocantes artificiais, a fim de avaliar o risco de que estas substancias apresentam para a saude dos consumidores / Abstract: Artificial sweeteners are chemical compounds of synthetic or natural origin that have sweetening properties, as full or partial replacement of sucrose or other sugars, and reduce the caloric content of the resulting product. The intake of artificial sweeteners in the world has grown in recent years, with the primary goal of reducing caloric intake. The overall increase of chronic diseases relating to sugar intake, particularly obesity and diabetes, is also a reason for the increased consumption of artificial sweeteners. The aim of this study was to evaluate the intake of artificial sweeteners through the consumption of liquid tabletop sweeteners and evaluate the possibility of consumption exceeding their Acceptable Daily Intake (ADI) established by the FAO/WHO Joint Expert Committee on Food Additives (JECFA). Initially, a field survey was undertaken to assess which liquid tabletop sweetener brand had the largest market share of the retail market in Brazil and the characteristics of usage. For this purpose, a questionnaire was submitted by electronic mail to individuals with questions relating to their consumption of artificial sweeteners, and if consumed, aspects of usage including brand, frequency and motivation of consumption. In addition, the questionnaire contained questions regarding socio-economic, demographic and health status. Based on the results of the survey the specific artificial sweeteners present in the most widely used liquid tabletop sweetener brands were identified and quantified. The artificial sweeteners present in the most widely consumed brands are: saccharin, cyclamate, aspartame and acesulfame-K. Quantification of saccharin, aspartame and acesulfame-K in the commercial products was determined by high performance liquid chromatography coupled to a diode array detector (HPLC-DAD). Quantification of cyclamate was carried out by spectrophotometry UV/Vis, at 314 nm, after derivatization to N, N diclorocyclohexilamine. The analytical methods employed were validated according to the following validation parameters: linearity, linear range, specificity, accuracy, precision (repeatability intra-and inter-day), limit of detection (LOD) and limit of quantification (LOQ). All the methods showed validation parameters in accordance to the Guide for the Validation of Analytical Methods established by the Brazilian Agency of Sanitary Surveillance (ANVISA). Most of the responses to the questionnaire came from females (70.2%), from the southeast region of Brazil (70.8%). Among the respondents, 47% used artificial tabletop sweeteners. The majority of the respondents who reported using artificial sweeteners were between 21 and 32 years old or above 45 years old. Among these 83% reported not having any type of pathology and that the primary reason for artificial sweetener consumption was the preference in relation to sugar. Approximately half (54%) of consumers reported an unpleasant or bitter aftertaste when consuming the sweetener and 32% did not know which artificial sweetener was in the sweetener they used. The results of the analysis of the liquid tabletop sweeteners samples from the retail market indicated there is a statistical variation (p<0.05) in the sweeteners concentration between brands and batches from the same brand. Based on the questionnaire results, it was verified the most consumed brand. The brand contains saccharin and cyclamate. A sample of this brand was provided to adult volunteers to estimate the level of consumption of those artificial sweeteners. It was found that the intake of saccharin and cyclamate through the consumption of the liquid tabletop sweetener represent, on average, 38.7% and 19.8% of their ADI values, respectively. Considering the variation found in the sweeteners concentration between brands and batches from the same brand and that each artificial sweetener has an ADI value established, it is recommended that the concentrations of the artificial sweeteners in the liquid tabletop sweeteners be shown on the packaging label, to allow the consumer to make informed decisions regarding their intake and to allow them to better control their total exposure to these substances. In general, the results suggest an increasing replacement of sugar by artificial sweeteners, which indicate the need to obtain up to date data on the total intake of artificial sweeteners in order to assess the risk that these substances present to the consumers health / Mestrado / Ciência de Alimentos / Mestre em Ciência de Alimentos

M. Tech (Department of Chemistry, Faculty of Applied and Computer Sciences) Vaal University of Technology. / A simple and rapid method for determination of estrone (E1) and β-estradiol (E2) was developed and validated using high performance liquid chromatography (HPLC). The solutions of standards and sample were prepared with distilled water. HPLC separation was performed in isocratic method 50/50 (water/methanol) using 4.6 mm x 250 mm id film thickness 5 µm) XDB-C18 capillary column, detector DAD, UV on 254 nm, temperature 20 ºC with flow rate of 2 mL/min, sample volume 20 µL and run time of 10 min. Calibration curves were linear between concentration range 1.0 - 15.0 ppm. The method was validated for limit of detection and quantification, linearity, precision, trueness and specificity. Also the method was applied to directly and easily to the analysis of the E1 and E2. Adsorption experiments were carried out in batch mode using multistirrer in a series of Erlenmeyer flasks of 50 ml capacity covered to prevent contamination having concentration ranges of E1 and E2 from 1 to 10 mg/L with adsorbent dose range 0.01 to 1 g at pH range 1 to 10 and temperature range 15°C to 35°C, placed on multistirrer. The results of the batch studies showed that simultaneous adsorption shows the maximum percent (91%) removal of E1 and (86 %) E2 at optimum temperature 25 °C of adsorbent dose 0.1 g, and pH 7. The mechanism, isotherms and kinetics of removal of two endocrine disrupting chemicals, estrone (E1) and β-estradiol (E2) by activated carbon adsorption were investigated in an agitated non-flow batch adsorption studies. Mathematical models were used to describe the adsorption phenomenon with the kinetic and thermodynamic parameters evaluated using the adsorption equilibrium data at varying temperatures. Higher adsorption rates were achieved at acidic to neutral pH ranges, with the sorption kinetic data showing a good fit to the pseudo second order rate equation and the Langmuir adsorption isotherm model for both E1 and E2. The Gibbs free energy were –16.68 kJ/mol and –17.34 kJ/mol for E1 and E2 respectively. The values of enthalpy for both E1 (84.50 kJ/mol) and E2 (90 kJ/mol) indicated a chemical nature of the sorption process. Both the isotherm and thermodynamic data obtained all supported the mechanism of adsorption of E1 and E2 to be mainly chemisorption’s supported by some physical attractions.

M. Tech (Department of Chemistry, Faculty of Applied and Computer Sciences) Vaal University of Technology. / In this study, analytical method for determining the chlorinated phenols in water was developed using High Performance Liquid Chromatography. The following four compounds which are 2, 4, 6- Trichlorophenol (2, 4, 6 TCP), 3-chlorophenol (3CP), 2, 4- Dichlorophenol (2, 4 DCP) and 4-chloro-3-methylphenol (4C3MP) were identified and quantified with a High Performance Liquid Chromatography (HPLC). The validation parameters tested were,: linearity, trueness, precision, detection limit of quantitation, sensitivity, specificity, selectivity. The linear calibration ranges of five standard solution from 1-10 ppm. The linearity ranges between 0.9298-0.9813. The activated carbon based on the waste macadamia nutshell activated carbon (MAC) was investigated for its potential uses as an adsorbent for chlorinated phenols removal and compared with grafted macadamia nutshell activated carbon (GMAC). The adsorbent was characterized with Fourier transform infrared spectrophotometer (FTIR), scanning electron microscope (SEM) and thermo gravimetric analysis (TGA). The parameters such as pH, temperature, contact time, concentration and adsorbent were investigated by adsorption technique. The strata C18E has been used before for the same reason and therefore the research was based on mimic the functional group of solid phase extraction (SPE) into macadamia activated carbon (MAC). The functional groups in SPE C18E are benzene and octadecyl. MAC was grafted with strata C18E functional groups to compare its potential with the SPE. The pseudo-first-order and pseudo-second-order kinetic models were applied to verify the experimental data. The pseudo-second order exhibited the best fit for the kinetic studies for MAC adsorption. Chemical removal of chlorinated phenols from wastewater is necessary to reduce harmful products on the environment and human health. Chlorinated phenols have been previously listed as some of the highest priority contaminants and as well as mainly important capability carcinogenic toxins released from chemical plants. Their availability in water supplies was perceived by their bad taste and smell. The acceptable chlorinated phenols concentration in portable water is 1 (mg/l) base on the approval of world health organization. The permanent checking of chlorinated phenols in environmental samples has a greater significance and stresses highly effectiveness, common selectively and great sensitively methods. The maximum uptake of Phenol using weighed mass of MAC was found to be 78 % and for GMAC was 84% for both 2,4,6TCP. t=250 min, pH=5, Co=1mg/l, T = 25 oC and m = 0.3 g/l were the optimum condition for Phenol-MAC system and GMAC system. Over all analysis of equilibrium model analysis indicates the fitness of Langmuir isotherm model to Phenol MAC adsorption system, suggesting a monolayer adsorption of phenol on the surface of MAC. Phenol adsorption capacity of MAC was found to be decreasing with increase in temperature suggesting that the adsorption process was exothermic in nature, which was further supported by the negative values of change in enthalpy. Characterization of MAC and GMAC confirmed the mesoporous texture, highly carbonaceous nature and a higher effective surface area of 912 m2/g. The highest phenol uptake capacity of GMAC was found to be 8.0049 mg/g. The optimal conditions for various process parameters are t = 250 min, pH=5, Co=1mg/l, T = 25 oC and m = 0.3 g/l were the optimum condition for Phenol-GMAC system. Like Phenol-MAC system, the kinetics studies confirmed that Phenol-GMAC adsorption system can be described by pseudo- second-order kinetics model. Equilibrium model analysis indicates the fitness of Langmuir isotherm model to Phenol-MAC adsorption system, suggesting a monolayer adsorption of phenol on the surface of GMAC. Phenol adsorption capacity of GMAC was found to be decreasing with increase in temperature suggesting that the adsorption process was exothermic in nature, which was further supported by the negative values of change in enthalpy. The negative values of Gibb’s free energy suggested that adsorption of phenol onto GMAC was a spontaneous process.

This work is concerned with lipidic substances, in particular vegetable oils, used as pigment binders or as a protective varnishes for finishing artwork. The introduction reviews the recent knowledge of this subject, in particular with respect to the identification of the most used drying oils, and of the methods to study their changes in the course of time. Both can be achieved using determination of the relative representation of fatty acids, most characteristic being the ratio of stearic/palmitic a oleic/palmitic acids. This parameter is changing as the artwork is getting older and the oils are drying. Because of the availability of the instrumentation needed, we have chosen the high- performance liquid chromatography method for further experimental work. The first step was optimalization of the analytical method by standards of fatty acids. For derivatization of fatty acids, reagents 2,4-dinitrophenylhydrazine and 2-nitrophenylhydrazine were examined. Whereas we were unable to get any results with the first one (the method based on the article: Bravo, B. et al., Talanta 64, 1329-1334, 2004) for unknown reasons, good results were obtained with the second one. Derivates of fatty acids have been analyzed by high performance liquid chromatography (HPLC) on column with revesed phase (C18). The...

Um método para determinação de praguicidas das classes organofosforados, carbamatos, triazinas e ureias por HPLC-MS/MS em urina de pessoas expostas em decorrência de atividades agrícolas é proposto neste trabalho. O modo de eluição isocrática e gradiente em cromatografia líquida de fase reversa com a coluna Synergi Max RP (150 x 4,6 mm, 4 &#181;m) foram experimentados para a separação dos componentes da amostra. O modo isocrático de eluição nas condições 45:55, 60:40 e 70:30 de ACN/água foram insuficientes para eluição dos componentes da amostra com fatores de retenção aceitáveis entre 0,5 e 20. Em modo gradiente, os efeitos do tempo de gradiente (30 a 100 min), a concentração inicial de ACN (8 a 14%), vazão da fase móvel (0,5 a 1,0 mL min-1) e temperatura (30 a 40 &#176;C) foram verificados para aumento de resolução e diminuição do tempo de análise. Em composições distintas de fase móvel (ACN/água), o logaritmo dos fatores de retenção foi colocado em gráfico em função da acidez (&#945;) e a basicidade (&#946;) da ligação de H, constante dielétrica (&#949;) e parâmetro de solubilidade de Hildebrand (&#948;H). Dimetoato e metomil tiveram comportamentos diferenciados, ambos explicados pelas ligações de H intramoleculares. Através de um planejamento fatorial completo de dois níveis, usando-se as funções COF e CRS como resposta, variáveis como vazão da fase móvel, tempo de gradiente, concentração inicial de ACN e temperatura foram investigados. A melhor resposta experimental forneceu como condições de análise: 0,5 mL min-1, 40 &#176;C, 75 min e 5% de ACN, para vazão, temperatura, tempo de gradiente e concentração inicial de ACN, respectivamente. Em um sistema LC-MS/MS usando o ion trap como analizador de massas, a infusão direta dos padrões de praguicidas, para conhecimento do pico do íon molecular e das transições MS/MS, foi estudada. Experimentos de quantificação em UPLC-MS/MS foram baseados na separação em fase reversa com a coluna ACQUITY UPLC HSS T3 (50 x 2,1 mm, 1,8 &#181;m), espectrômetro de massas equipado com analizador de massas triplo quadrupolo e modo positivo de ionização. Transições MRM de quantificação e de confirmação foram monitoradas para cada praguicida. O método apresentou respostas lineares no intervalo de 0,25 a 50 &#181;g L-1 para todos os praguicidas com coeficiente de determinação maiores que 0,999. O LD do método variou de 0,192 a 3,19 &#181;g L-1. O LQ do método variou entre 0,582 e 13,6 &#181;g L-1, com coeficiente de variação inferior a 20% CV. As recuperações em amostras fortificadas em 0,75 &#181;g L-1 variaram entre 55,7 e 72,6% e a precisão em termos de %CV entre 1,94 e 19,1%. As recuperações em amostras fortificadas em 10 &#181;g L-1 variaram entre 99,1 a 105% e a precisão entre 0,751 e 8,98% CV. As recuperações em amostras fortificadas em 50 &#181;g L-1 variaram entre 99,5 a 101% e a precisão entre 0,877 e 5,94% CV. Das 25 amostras quantificadas, 13 apresentaram concentrações dos praguicidas na faixa linear da curva de calibração. Estes valores de concentração variaram entre 10 e 50 &#181;g L-1. / In this work a method for determination of different chemical groupes of pesticides (organophosphorus, carbamates, triazines, organochlorine and ureas) is proposed using HPLC-MS/MS in samples from occupational exposed agriculture workers. Isocratic and gradien elution in reversed-phase chromatography using a Synergi Max RP (150 x 4,6 mm, 4 &#181;m) column were investigated for separation proposal. Isocratic elution conditions 45:55, 60:40, 70:30 ACN/water provided unsatisfactory results with retention factor from 0.5 to 20. In gradient elution, effects of gradient time (30 to 100 min), initial concentration of ACN (8 to 14%), flow rate (0.5 to 1 mL min-1) and temperature (30 to 40 &#176;C) were studied for increasing resolution and decreasing analysis time. For different proportion of ACN in the mobile phase, the logarithm of retention factor has been ploted against solvent hydrogen-bond donor (&#945;), solvent hydrogen-bond acceptor (&#946;), dieletric constant (&#949;) and Hildebrand solubility (&#948;H). Dimethoate and methomyl presented different behaviour in relation to others, explained by H intramolecular bond. Applying a two level experimental design having as response functions COF and CRS, combined factors such as flow rate, gradient time, ACN initial concentration and temperature were investigated. Optimal conditions were obtained in 0.5 mL min-1, 40 &#176;C, 75 min e 5% CAN, for flow rate, temperature, gradient time and ACN initial concentration, respectively. Applying a LC-MS/MS system equipped with an ion trap mass analyzer, direct infusion of standard solutions of pesticides for knowledge of ion molecular peak and their MRM transitions was obtained. Quantitative experiments using UPLC-MS/MS were based in reversed-phase separation with a Acquity UPLC HSS T3 (50 x 2,1 mm, 1,8 &#181;m) column, mass spectrometry instrument equipped with triple quadrupole mass analyzer at positive ion polarity. Quantifying and confirming ion transitions were monitored for each pesticide compound. Linearity in range of 0.25 to 50 &#181;g L-1 was established for all pesticides with high coefficients determination R2 0.999 were obtained. The method LOD varied between 0.192 to 3.19 &#181;g L-1. LOQ varied from 0.582 to 13.6 &#181;g L-1, with coefficient variation below 20% CV. Recoveries for samples spiked at 0.75 &#181;g L-1 concentration level varied from 55.7 to 72.6% and precision in terms of CV between 1.94 and 19.1% CV. Spiked samples at 10 &#181;g L-1 concentration level varied from 99.1 to 105% and precision between 0.751 and 8.98% CV. Recoveries for samples spiked at 50 &#181;g L-1 concentration level varied from 99.5 to 101% and precision from 0.877 to 5.94% CV. From 25 samples quantified, 13 presented concentration values in linear range of the calibration curve. These values varied from 10 to 50 &#181;g L-1.

A review of the main natural dyes (particularly yellow flavonoids and red anthraquinones) and proteinaceous substrates used in Historical Tapestries and North American porcupine quill work was undertaken, and is summarised in Chapter 1. The analysis of natural dyes which have been used on museum artefacts other than textiles has received little systematic study, particularly those of non-European origin. In this research, the use of Ultra Performance Liquid Chromatography (UPLC) for study of natural dyes found on historical textiles and ethnographical objects decorated with porcupine quill work is explored; this required a transfer of existing analytical protocols and methodology. The advantages of using Ultra Performance Liquid Chromatography (UPLC) was evaluated through a method development based on the separation and quantification of ten flavonoid and anthraquinone dyes as described in Chapter 2. These methods were then applied to the characterisation of the dye sources found on a group of sixteenth century historical tapestries which form an important part of the Burrell Collection in Glasgow and are believed to have been manufactured in an English workshop (Chapter 3) and also to the analysis of some late nineteenth century North American porcupine quill work from a collection owned by National Museums Scotland (Chapter 5); allowing exciting conclusions to be drawn in each case about the range of dyestuffs used in their manufacture. The second aim of this research was the development of methodology for the non-invasive quantification of metal ion residues on porcupine quill substrates. This was achieved through a comparative study of reference porcupine quills prepared in-house with dyebaths containing a range of metal ion concentrations (copper and tin). The concentration of metal ions sorbed by the porcupine quills was then quantified with Inductively Coupled Plasma (ICP) coupled to Optical Emission Spectrometry (OES) and non-invasive Particle Induced X-Ray Emission analysis (PIXE) coupled with Rutherford Backscattering Spectrometry (RBS) as described in Chapter 4. The responses provided by the different methods were compared and they were then applied to the study of micro-samples collected from mid-nineteenth century Northern Athapaskan porcupine quill work. Unexpectedly, the use of UPLC analysis and RBS-PIXE analysis allowed the characterisation of traded European natural dyes used with metallic mordants (copper and tin) on these samples, highlighting how European contact impacted on traditional Athapaskan porcupine quill work in the late nineteenth century (Chapter 5).

Within the field of depression the “monoamine hypothesis” has been the leading theory to explain the biological basis of depression. This theory proposes that the biological basis of depression is due to a deficiency in one or more of three key neurotransmitter systems, namely noradrenaline, dopamine and serotonin which are thought to mediate the therapeutic actions of virtually every known antidepressant agent. Citalopram is a selective serotonin-reuptake inhibitor (SSRI) used for the treatment of depression and anxiety disorders. Citalopram is a racemic compound, in other words composed of a 50:50 mixture of two enantiomers (S-(+)-citalopram and R-(-)-citalopram) and with one of the enantiomers (S-(+)-citalopram) accounting for the inhibitory effect. At the time of introduction of citalopram the physician needed a therapeutic drug monitoring service to identify patients with interactions, compliance problems and for handling questions concerning polymorphic enzymes and drug metabolism. An achiral analytical separation method based on solid-phase extraction followed by high-performance liquid chromatography (HPLC) was developed for routine therapeutic drug monitoring (TDM) of citalopram and its two main demethylated metabolites. As the data available on citalopram were from achiral concentration determinations and to be able to further investigate citalopram enantiomers effects and distribution, a chiral method for separation of the enantiomers of citalopram and its demethylated metabolites was established. The advances within chiral separation techniques have made measurement of the concentrations of the individual enantiomers in biological fluids possible. The process behind enantioselective separation is however not fully understood and the mechanism behind the separation can be further scrutinized by the use of multivariate methods. A study of the optimization and characterization of the separation of the enantiomers of citalopram, desmethylcitalopram and didesmethylcitalopram on an acetylated ß-cyclodextrin column, by use of two different chemometric programs - response surface modelling and sequential optimization was performed. Sequential optimization can be a quicker mean of optimizing a chromatographic separation; response surface modelling, in addition to enabling optimization of the chromatographic process, also serves as a tool for learning more about the separation mechanism. Studies of the antidepressant effect and pharmacokinetics of citalopram have been performed in adults, but the effects on children and adolescents have only been studied to a minor extent, despite the increasing use of citalopram in these age groups. A study was initiated to investigate adolescents treated for depression, with respect to the steady-state plasma concentrations of the enantiomers of citalopram and its demethylated metabolites. The ratios between the S- and R-enantiomers of citalopram and didesmethylcitalopram were in agreement with studies involving older patients. The concentrations of the S-(+)- and R-(-) enantiomers of citalopram and desmethylcitalopram were also in agreement with values from earlier studies. The results indicate that the use of oral contraceptives may have some influence on the metabolism of citalopram. This might be because of an interaction of the contraceptive hormones with the polymorphic CYP2C19 enzyme. Even though the SSRIs are considered less toxic compared with older monoamine-active drugs like the tricyclic/tetracyclic antidepressants, the risk of developing serious side effects such as ECG abnormalities and convulsions has been seen for citalopram, when larger doses have been ingested. Furthermore, fatal overdoses have been reported where citalopram alone was the cause of death. Data on the toxicity of each of the enantiomers in humans have not been reported and no data on blood levels of the enantiomers in cases of intoxication have been presented. An investigation was initiated on forensic autopsy cases where citalopram had been found at the routine screening and these cases were further analysed with enantioselective analysis to determine the blood concentrations of the enantiomers of citalopram and metabolites. Furthermore the genotyping regarding the polymorphic enzymes CYP2D6 and CYP2C19 were performed. In 53 autopsy cases, we found increasing S/R ratios with increasing concentrations of citalopram. We found also that high citalopram S/R ratio were associated with high parent drug to metabolite ratio and may be an indicator of recent intake. Only 3.8 % were found to be poor metabolizers regarding CYP2D6 and for CYP2C19 no poor metabolizer was found. Enantioselective analysis of citalopram and its metabolites can provide valuable information about the time that has elapsed between intake and death. Genotyping can be of help in specific cases but the possibility of pharmacokinetic interactions is apparently a far greater problem than genetic enzyme deficiency. / On the day of the public defence the status of article IV was: Submitted.