Analysis of early synthetic dyes with HPLC-DAD-MS: an important database for analysis of colorants used in cultural heritage

Presented thesis at Faculdade de Ciências e Tecnologias, Universidade de Lisboa, to obtain the Master Degree in Conservation and Restoration of Textiles

Pharmacological evaluation of plant extracts from Chapada Diamantina (Bahia, Brazil): Focus on antioxidant and cytotoxic properties

Tese de Doutoramento em Biologia de Plantas MAP - Bioplant

Evolució i estabilitat d’àcids grassos i vitamines antioxidants a la llet materna

Recerca realitzada durant el periode Gener 2006-Abril 2007, a la Facultat de Farmàcia de la Universitat de Barcelona sota la direcció de la Dra. M. Carmen López Sabater. La nutrició durant els primers anys de vida té una enorme importància, amb repercussions en el creixement i desenvolupament del nen i en les possibles malalties futures. Tenint en compte que la llet materna és l’aliment ideal per al nadó durant els primers sis mesos de vida, és important conéixer la composició nutricional de la mateixa i la seva evolució al llarg de la lactància (calostre, llet de transició i madura). La llet conté nutrients majoritaris (proteïnes, carbohidrats i lípids) i nutrients minoritaris, com vitamines i minerals. S'ha desenvolupat i validat un mètode per a la determinació d'àcids grassos (AG) i Àcid Linoleic Conjugat (CLA) en llet materna per Cromatografia de Gasos "fast" (fast-GC), permetent reduir el temps d'anàlisi considerablement en comparació amb els mètodes utilitzats fins el moment. La repetibilitat i reproducibilitat trobades han estat bones, amb coeficients de variació inferiors al 10% en tots els casos. L'aplicació a 6 mostres de llet materna ha demostrat que es tracta d'un mètode senzill, ràpid, eficient i pràctic per a l'anàlisi rutinari d'un elevat nombre de mostres. També s'ha començat a desenvolupar un mètode per a l'anàlisi dels anàlegs de tocoferol de la llet materna, degut a l'interès de la vitamina E com a antioxidant natural. És un mètode per Cromatografia Líquida d'Ultra Resolució (UPLC) amb detecció per Fluorescència (FD) i per Photodiode Array (PDA) que permet treballar amb quantitats de mostra petites mantenint o inclús millorant la sensibilitat i reduint el temps d’anàlisi.

Avaluació de vitamines i antioxidants en aliments vegetals mitjançant tècniques HPLC-DAD i UPLC-MS/MS

El programa experimental s’ha portat a terme dins el marc de les activitats del projecte TRUEFOOD, finançat per la UE per als anys 2007-2010. L’objectiu principal d’aquesta activitat ha estat l’avaluació dels continguts en àcid ascòrbic (vitamina C), polifenols totals, àcids fenòlics i flavonoides en mostres de tomàquet i enciam, produïts sota diferents condicions de camp (producció ecològica i convencional). Per aconseguir els resultats s’han utilitzat mètodes analítics basats en tècniques de cromatografia líquida d’alta eficàcia (HPLC) i d’ultra-alta eficàcia (UHPLC) acoblades a sistemes de detecció de diode array (DAD) i espectrometria de masses (MSn). Per a l’àcid ascòrbic, s’ha desenvolupat un mètode ràpid que ha permès la determinació d’aquest compost en diferents matrius vegetals amb el mínim pretractament de les mostres, utilitzant una fase estacionària HILIC (Fluorinated Stationary Phase). Els mètodes desenvolupats d’anàlisi de compostos fenòlics han permès realitzar les anàlisi de forma ràpida, a fi de processar el màxim nombre de mostres per a obtenir resultats representatius. S’ha realitzat una completa caracterització dels extractes de tomàquet i enciam, ampliant el coneixement descrit en la bibliografia sobre la seva composició fenòlica. En el cas de l’enciam, s’ha identificat quatre compostos fenòlics que mai abans han estat descrits i quantificats en aquesta hortalissa. La definició, amb precisió, dels continguts en vitamina C i compostos fenòlics en les mostres analitzades ha permès comparar els efectes de diferents tècniques de cultiu sobre les característiques nutricionals dels vegetals objecte de l’estudi. Els mètodes d’anàlisi desenvolupats i els resultats derivats del projecte seran publicats properament en revistes científiques de reconegut prestigi.

In vivo antileishmanial activity and chemical profile of polar extract from Selaginella sellowii

The polar hydroethanolic extract from Selaginella sellowii(SSPHE) has been previously proven active on intracellular amastigotes (in vitro test) and now was tested on hamsters infected with Leishmania (Leishmania) amazonensis (in vivo test). SSPHE suppressed a 100% of the parasite load in the infection site and draining lymph nodes at an intralesional dose of 50 mg/kg/day × 5, which was similar to the results observed in hamsters treated with N-methylglucamine antimonate (Sb) (28 mg/Kg/day × 5). When orally administered, SSPHE (50 mg/kg/day × 20) suppressed 99.2% of the parasite load in infected footpads, while Sb suppressed 98.5%. SSPHE also enhanced the release of nitric oxide through the intralesional route in comparison to Sb. The chemical fingerprint of SSPHE by high-performance liquid chromatography with diode-array detection and tandem mass spectrometry showed the presence of biflavonoids and high molecular weight phenylpropanoid glycosides. These compounds may have a synergistic action in vivo. Histopathological study revealed that the intralesional treatment with SSPHE induced an intense inflammatory infiltrate, composed mainly of mononuclear cells. The present findings reinforce the potential of this natural product as a source of future drug candidates for American cutaneous leishmaniasis.

Natural aphrodisiacs: studies of commercially-available herbal recipes, and phytochemical investigation of "Erythroxylum vacciniifolium" Mart. (Erythroxylaceae) from Brazil

De tout temps, hommes et femmes ont cherché par tous les moyens à développer, préserver ou recouvrer leurs propres capacités sexuelles mais également à stimuler le désir du partenaire. L?utilisation d?aphrodisiaques naturels a été l?un des recours les plus répandus. De nos jours, la commercialisation de nouvelles "love drugs" de synthèse, e.g. Viagra®, Cialis®, Levitra®, a remis au goût du jour les aphrodisiaques classiques et à relancer la recherche sur des molécules nouvelles. La pratique croissante de l?automédication, le matraquage publicitaire sur les aphrodisiaques naturels, la prolifération sur le marché de compléments alimentaires non contrôlés et l?absence de véritable législation accroissent les risques qui pèsent sur la santé publique. Dans le but d?évaluer les risques potentiels sur le consommateur de produits aphrodisiaques commercialisés, le développement et la validation d?une méthode rapide d?analyse qualitative et quantitative de la yohimbine dans ces préparations du marché sont exposés dans la première partie de ce travail. La yohimbine est un antagoniste ?2-adrénocepteur du système nerveux central et périphérique, elle est employée depuis plus d?un siècle dans le traitement des dysfonctionnements érectiles. Cette méthode analytique utilise la chromatographie liquide couplée à l?ultraviolet et à la spectrométrie de masse (LC-UV-MS) et au total, vingt préparations aphrodisiaques ont été étudiées. La dose journalière de yohimbine mesurée s?est révélée très variable selon les produits puisqu?elle varie de 1.32 à 23.16 mg. La seconde partie de ce travail concerne l?étude phytochimique et pharmacologique d?Erythroxylum vacciniifolium Mart. (Erythroxylaceae), une plante, appelée localement catuaba, utilisée dans la médecine traditionnelle brésilienne comme tonique et aphrodisiaque. Dans un premier temps, l?extrait alcaloïdique a été analysé par chromatographie liquide haute performance (HPLC) couplée soit à un détecteur UV à barrette d?iode (LC-UV-DAD), soit à un spectromètre de masse (LC-MS), ou soit à un spectromètre de résonance magnétique nucléaire (LC-RMN). L?interprétation de ces données spectrales enregistrées en ligne a permis d?obtenir des informations structurales et d?identifier partiellement près de 24 alcaloïdes appartenant à la classe des tropanes et potentiellement originaux. Par des méthodes classiques de chromatographie liquide sur l?extrait alcaloïdique de la plante, dix sept tropanes nouveaux ont ensuite été isolés dont les catuabines et leurs dérivés, et les vaccinines. Tous ces composés sont des tropane-diols ou triols estérifiés par au moins un groupe acide 1-méthyl-1H-pyrrole-2-carboxylique. Un de ces composés a été identifié comme un tropane N-oxyde. Toutes les structures ont été déterminées par spectrométrie de masse haute résolution et spectroscopie RMN multi-dimensionnelle. Parmi les nombreux tests biologiques réalisés sur ces tropanes, seuls les tests de cytotoxicité se sont révélés faiblement positifs pour certains de ces composés.<br/><br/>Throughout the ages, men and women have incessantly pursued every means to increase, preserve or recapture their sexual capacity, or to stimulate the sexual desire of selected individuals. One of the most recurrent methods has been the use of natural aphrodisiacs. Nowadays, the commercialization of new synthetic "love drugs", e.g. Viagra®, Cialis® and Levitra®, has fascinated the public interest and has led to a reassessment of classical aphrodisiacs and to the search for new ones. The practice of self-medication by an increasing number of patients, the incessant aggressive advertising of these herbal aphrodisiacs, the invasion of the medicinal market with uncontrolled dietary supplements and the absence of real directives amplifies the potential health hazards to the community. In order to evaluate the possible risks of commercialized aphrodisiac products on consumer health, the development and validation of a rapid qualitative and quantitative method for the analysis of yohimbine in these products, is reported in the first part of the present work. Yohimbine, a pharmacologically well-characterized ?2-adrenoceptor antagonist with activity in the central and peripheral nervous system, has been used for over a century in the treatment of erectile dysfunction. The analytical method is based on liquid chromatography coupled with ultraviolet and mass spectrometry (LC-UV-MS) and in total, 20 commercially-available aphrodisiac preparations were analyzed. The amount of yohimbine measured and expressed as the maximal dose per day suggested on product labels ranged from 1.32 to 23.16 mg. The second part of this work involved the phytochemical and pharmacological investigation of Erythroxylum vacciniifolium Mart. (Erythroxylaceae), a plant used in Brazilian traditional medicine as an aphrodisiac and tonic, and locally known as catuaba. With the aim of obtaining preliminary structure information on-line, the alkaloid extract was analyzed by high performance liquid chromatography (HPLC) coupled to diode array UV detection (LC-UVDAD), to mass spectrometry (LC-MS) and to nuclear magnetic resonance spectroscopy (LCNMR). Interpretation of on-line spectroscopic data led to structure elucidation and partial identification of 24 potentially original alkaloids bearing the same tropane skeleton. Seventeen new tropane alkaloids were then isolated from the alkaloid extract of the plant, including catuabines D to I, their derivatives and vaccinines A and B. All compounds were elucidated as tropane-diol or -triol alkaloids esterified by at least one 1-methyl-1H-pyrrole-2-carboxylic acid. One of the isolated compounds was identified as a tropane alkaloid N-oxide. Their structures were determined by high resolution mass spectrometry and multi-dimensional NMR spectroscopy. Among the numerous bioassays undertaken, only the cytotoxicity tests exhibited a weak positive activity of certain compounds.

Etude de la toxicité hépatique de la cocaïne associée à de l'alcool: Effets de l'éthanol sur le métabolisme de la cocaïne dan les hépatocytes de rat en suspension et dans un modèle enzymatique humain

Le but essentiel de notre travail a été d?étudier la capacité du foie, premier organe de métabolisation des xénobiotiques, à dégrader la cocaïne en présence d?éthanol, à l?aide de deux modèles expérimentaux, à savoir un modèle cellulaire (les hépatocytes de rat en suspension) et un modèle acellulaire (modèle reconstitué in vitro à partir d?enzymes purifiées de foie humain). La première partie a pour objectifs de rechercher les voies de métabolisation de la cocaïne qui sont inhibées et / ou stimulées en présence d?éthanol, sur hépatocytes isolés de rat. Dans ce but, une méthode originale permettant de séparer et de quantifier simultanément la cocaïne, le cocaéthylène et huit de leurs métabolites respectifs a été développée par Chromatographie Phase Gazeuse couplée à la Spectrométrie de Masse (CPG / SM). Nos résultats préliminaires indiquent que l?éthanol aux trois concentrations testées (20, 40 et 80 mM) n?a aucun effet sur la cinétique de métabolisation de la cocaïne. Notre étude confirme que l?addition d?éthanol à des cellules hépatiques de rat en suspension supplémentées en cocaïne résulte en la formation précoce de benzoylecgonine et de cocaéthylène. L?apparition retardée d?ecgonine méthyl ester démontre l?activation d?une deuxième voie de détoxification. La production tardive d?ecgonine indique une dégradation de la benzoylecgonine et de l?ecgonine méthyl ester. De plus, la voie d?oxydation intervenant dans l?induction du stress oxydant en produisant de la norcocaïne est tardivement stimulée. Enfin, notre étude montre une métabolisation complète de la concentration initiale en éthanol par les hépatocytes de rat en suspension. La deuxième partie a pour but de déterminer s?il existe d?autres enzymes que les carboxylesterases formes 1 et 2 humaines ayant une capacité à métaboliser la cocaïne seule ou associée à de l?éthanol. Pour ce faire, une méthode de micropurification par chromatographie liquide (Smart System®) a été mise au point. Dans le cadre de nos dosages in situ de la cocaïne, du cocaéthylène, de la benzoylecgonine, de l?acide benzoïque et de la lidocaïne, une technique par Chromatographie Liquide Haute Performance couplée à une Détection par Barrette de Diode (CLHP / DBD) et une méthode de dosage de l?éthanol par Chromatographie Phase Gazeuse couplée à une Détection par Ionisation de Flamme équipée d?un injecteur à espace de tête (espace de tête CPG / DIF) ont été développées. La procédure de purification nous a permis de suspecter la présence d?autres enzymes que les carboxylesterases formes 1 et 2 de foie humain impliquées dans le métabolisme de la cocaïne et déjà isolées. A partir d?un modèle enzymatique reconstitué in vitro, nos résultats préliminaires indiquent que d?autres esterases que les formes 1 et 2 de foie humain sont impliquées dans l?élimination de la cocaïne, produisant benzoylecgonine et ecgonine méthyl ester. De plus, nous avons montré que les sensibilités de ces enzymes à l?éthanol sont variables.<br/><br/>The main purpose of our work was to study the ability of the liver, as the first organ to metabolise xenobiotic substances, to degrade cocaine in the presence of ethanol. In order to do this, we used two experimental models, namely a cellular model (rat liver cells in suspension) and an a-cellular model (model reconstructed in vitro from purified human liver enzymes). The purpose of the first part of our study was to look for cocaine metabolising processes which were inhibited and / or stimulated by the presence of ethanol, in isolated rat liver cells. With this aim in mind, an original method for simultaneously separating and quantifying cocaine, cocaethylene and eight of their respective metabolites was developed by Vapour Phase Chromatography coupled with Mass Spectrometry (VPC / MS). Our preliminary results point out that ethanol at three tested concentrations (20, 40 et 80 mM) have no effect on the kinetic of metabolisation of cocaine. Our study confirms that the addition of alcohol to rat liver cells in suspension, supplemented with cocaine, results in the premature formation of ecgonine benzoyl ester and cocaethylene. The delayed appearance of ecgonine methyl ester shows that a second detoxification process is activated. The delayed production of ecgonine indicates a degradation of the ecgonine benzoyl ester and the ecgonine methyl ester. Moreover, the oxidising process which occurs during the induction of the oxidising stress, producing norcocaine, is stimulated at a late stage. Finally, our study shows the complete metabolisation of the initial alcohol concentration by the rat liver cells in suspension. The second part consisted in determining if enzymes other than human carboxylesterases 1 and 2, able to metabolise cocaine on its own or with alcohol, existed. To do this, a micropurification method us ing liquid phase chromatography (Smart System®) was developed. A technique based on High Performance Liquid Chromatography coupled with a Diode Array Detection (HPLC / DAD) in the in situ proportioning of cocaine, cocaethylene, ecgonine benzoyl ester, benzoic acid and lidocaine, and a method for proportioning alcohol by quantifying the head space using Vapour Phase Chromatography coupled with a Flame Ionisation Detection (head space VPC / FID) were used. The purification procedure pointed to the presence of enzymes other than the human liver carboxylesterases, forms 1 and 2, involved in the metabolism of cocaine and already isolated. The preliminary results drawn from an enzymatic model reconstructed in vitro indicate that human liver carboxylesterases, other than forms 1 and 2, are involved in the elimination of cocaine, producing ecgonine benzoyl ester and ecgonine methyl ester. Moreover, we have shown that the sensitivity of these enzymes to alcohol is variable.

Carotenoids profile and total polyphenols in fruits of Pereskia aculeata Miller

Pereskia aculeata Mill. (Ora-pro-nóbis) is a native cactaceae from tropical America, whose leaves have high protein content. In Brazil it is found in all territorial extension between the states of Bahia and Rio Grande do Sul. Most studies have focused on chemical characterization of the leaves of this specie. The objective was to assess the carotenoids profile and the total polyphenols present in the fruits of P. aculeate. Carotenoids were determined by HPLC-PAD (high performance liquid chromatography - photodiode array detector), total polyphenols were determined by Folin-Ciocalteu and vanillin methods. Trans-β-carotene was the main carotenoid, followed by α-carotene, lutein and other minor carotenoids. It was found 64.9 ± 1.1 mg.100g-1 of gallic acid equivalent, 14.8 ± 0.2 mg.100g-1 of catechin equivalent. Carotenoid identification of P. aculeate fruits are presented here by the first time and indicate that these fruits can be researched as source of bioactive substances, especially antioxidant and provitamin A carotenoids.

Perfil cromatográfico de duas espécies de Plumbaginaceae: Plumbago scandens L. E Plumbago auriculata Lam

The genus Plumbago belongs to the family Plumbaginaceae, order Plumbaginales. Comparative chemical profile of P. scandens (native) and P. auriculata (cultivated) was obtained by normal and reversed-phase high performance liquid chromatography with photodiode array detector. Comparison of the ultraviolet espectra and the retention times for the compounds allowed to find similar metabolic patterns in roots, stems and leaves. Four flavonoids, one phenolic acid or derivative and the naphtoquinone plumbagin were comparatively identified to standards.

Investigation of the occurrence of pesticide residues in rural wells and surface water following application to tobacco

In this work the exposure of wells and surface water to pesticides, commonly used for tobacco cropping, was assessed. Water consumption wells and surface water flows were sampled at different times. After a preconcentration step with solid phase extraction (SPE), the selected pesticides were determined by gas chromatography with electron capture detection (GC-ECD) or high performance liquid chromatography with diode array detection (HPLC-DAD). No pesticides were detected in the well water samples and surface water flow in the winter season. However, in the spring and summer higher concentrations of chlorpyrifos and imidacloprid were found in the water source samples. Atrazine, simazine and clomazone were also found. The occurrence of pesticides in collected water samples was related with the application to tobacco.

High performance liquid chromatography method for quantification of the N-phenylpiperazine derivative LASSBio-579 in rat plasma

A rapid HPLC analytical method was developed and validated for the determination of the N-phenylpiperazine derivative LASSBio-579in plasma rat. Analyses were performed using a C18 column and elution with 20 mM sodium dihydrogen phosphate monohydrate - methanol. The analyte was monitored using a photodiode array detector (257 nm). Calibration curves in spiked plasma were linear over the concentration range of 0.3-8 mg/mL with determination coefficient > 0.99. The lower limit of quantification was 0.3 mg/mL. The applicability of the HPLC method for pharmacokinetic studies was tested using plasma samples obtained after administration of LASSBio-579 to Wistar rats, showing the specificity of the method.

Otimização de método para determinação de flavonóis e flavonas em frutas por cromatografia líquida de alta eficiência utilizando delineamento estatístico e análise de superfície de resposta

This work optimized the HPLC conditions for the simultaneous determination of luteolin, apigenin, myricetin, quercetin and kaempferol in aglycone form, as well defined the best conditions for hydrolysis/extraction of these flavonoids in fruits, using the statistical central composite design and response surface analysis. A reverse phase method was developed using a gradient of methanol/water acidified with 0.3% formic acid as mobile phase and a photodiode array detector. The samples were extracted with methanol/water (50:50 v/v) at 90 ºC. The optimum time and HCl concentration varied for the different fruits investigated, demonstrating the necessity of optimizing these conditions for each fruit analyzed. Good recovery (87.1 to 96.3%), repeatability and linearity were obtained.

Rapid and accurate hplc-dad method for the determination of the herbicide bispyribac-sodium in surface water, and its validation

A new method is described for the determination of the herbicide bispyribac-sodium in surface water, especially from river and irrigated rice water samples. The method involves extraction in solid phase and quantification by high performance liquid chromatography with diode array detection (HPLC-DAD). After optimization of the extraction and separation parameters, the method was validated. The method presented average recoveries of 101.3 and 97.7%, under repeatability and intermediate precision conditions, respectively, with adequate precision (RSD from 0.9 to 7.5%). The method was applied for the determination of bispyribac-sodium in surface water samples with a limit of detection of 0.1 μg L-1.

Determinação de compostos fenólicos por cromatografia líquida de alta eficiência isocrática durante estacionamento da erva-mate

Different phenolic compound, 5- caffeoylquinic acid (5-CQA), caffeic acid (AC) and rutin (Ru) contents of yerba-mate (Ilex paraguariensis) Brazilian samples of 06 different regions of São Mateus - Paraná, during natural and accelerated industrial storage, were evaluated. For quantification, a reverse phase HPLC isocratic method was developed and validated using methanol:water (35:65 v/v) acidified with 0.5% acetic acid as mobile phase and a photodiode array detector. The six sample global average contents were (34.90 and 36.10 mg g-1) for 5-CQA, (0.18 mg g-1 and 0.23 mg g-1) for AC and (7.12 and 7.18 mg g-1) for Ru, respectively, for the natural and accelerated storage systems. The results showed that the 5-CQA and Ru content are kept constant during the storage while AC content increase only during accelerated storage.

Principais técnicas de preparo de amostra para a determinação de resíduos de agrotóxicos em água por cromatografia líquida com detecção por arranjo de diodos e por espectrometria de massas

The determination of pesticide residues in water samples by Liquid Chromatography require sample preparation for extraction and enrichment of the analytes with the minimization of interferences to achieve adequate detection limits. The Solid Phase Extraction (SPE), Solid Phase Microextraction (SPME), Stir Bar Sorptive Extraction (SBSE) and Dispersive Liquid-Liquid Microextraction (DLLME) techniques have been widely used for extraction of pesticides in water. In this review, the principles of these sample preparation techniques associated with the analysis by Liquid Chromatography with Diode Array Detection (LC-DAD) or Mass Spectrometry (LC-MS) are described and an overview of several applications were presented and discussed.