O EPP-AF® é um extrato padronizado de própolis quimicamente caracterizado e com eficácia e segurança pré-clínica estabelecidas. O objetivo principal deste trabalho foi realizar um ensaio clínico de segurança para avaliar a influência do EPP-AF® na atividade da P-gp e das principais isoformas CYP, através de um teste in vivo tipo coquetel de fármacos marcadores administrados em doses subterapêuticas. Foram investigados 16 voluntários adultos sadios antes e após a exposição a 375 mg de EPP-AF® por via oral durante 15 dias. As amostras seriadas de sangue foram colhidas até 12 h após a administração do coquetel contendo midazolam (0,2 mg), cafeína (10 mg), omeprazol (2 mg), metoprolol (10 mg), losartana (2 mg) e fexofenadina (10 mg). Foram desenvolvidos e validados três métodos analíticos empregando LC-MS/MS para quantificar as concentrações plasmáticas de fexofenadina, losartana, E-3174 (método 1), omeprazol, 5-OH-omeprazol, midazolam, metoprolol, ?-OHmetoprolol (método 2) e cafeína (método 3). Os métodos não apresentaram efeito matriz ou efeito residual e mostraram-se lineares para os analitos nos intervalos de 0,05-20 ng/mL (fexofenadina); 0,03 - 5 ng/mL (losartana e E31-74); 0,1 - 50 ng/mL (omeprazol), 0,3 - 50 ng/mL (5-OH-omeprazol), 0,01 - 10 ng/mL (midazolam), 0,05 - 50 ng/mL (metoprolol e ?- OH-metoprolol) e 5 - 1000 ng/mL (cafeína). Os parâmetros farmacocinéticos dos compostos foram calculados com base nas curvas de concentração plasmática versus tempo (AUC) empregando o programa Phoenix® WinNonlin®. Os valores das razões das AUC0-t e Cmax após e antes da exposição ao EPP-AF®, apresentados como média geométrica (IC90%) foram de 0,74 (0,62 - 0,89) e 0,90 (0,76 - 1,07) para fexofenadina; 0,88 (0,80 - 0,97) e 0,86 (0,76 - 0,98) para losartana; 0,96 (0,83 - 1,11) e 0,91 (0,79 - 1,04) para E-3174; 1,18 (0,91 - 1,54) e 1,21 (0,87- 1,70) para omeprazol; 1,12 (0,95 - 1,31) e 1,22 (0,95 - 1,67) para 5-OHomeprazol; 1,14 (1,03 - 1,28) e 1,21 (1,00 - 1,46) para o midazolam; 1,04 (0,92 - 1,18) e 0,94 (0,80 - 1,12) para o metoprolol; 1,05 (0,99 - 1,12) e 0,99 (0,88 - 1,12) para ?-OH-metoprolol; 0,97 (0,77 - 1,21) e 0,87 (0,69 - 1,11) para a cafeína. Quando observadas as razões metabólicas das AUC0-t E3174/losartana, 5-OH-omeprazol/omeprazol e ?-OHmetoprolol/ metoprolol encontramos, respectivamente, 1,11 (0,98 - 1,25); 0,94 (0,81 - 1,10) e 1,01 (0,88 - 1,16), indicando que, com exceção do CYP2D6, a administração de EPP-AF® nas condições estudadas apresenta potencial para inibição das isoformas CYP2C19 e CYP3A4 e indução das enzimas CYP1A2, CYP2C9 e do transportador de efluxo P-gp, embora as suas magnitudes encontram-se abaixo dos limites definidos pelos órgãos reguladores e portanto não apresentam relevância clínica / EPP-AF® is a standardized extract of propolis chemically characterized and with established pre-clinical efficacy and safety. The main objective of this work was to perform a clinical trial to evaluate the effect of EPP-AF® on P-gp and the major CYP isoforms activity, through an in vivo assay using the cocktail approach with sub-therapeutic doses. Sixteen healthy adult volunteers were investigated before and after exposure to orally administered 375 mg/day of EPP-AF® for 15 days. Serum blood samples were collected up to 12 h after the administration of midazolam (0.2 mg), caffeine (10 mg), omeprazole (2 mg), metoprolol (10 mg), losartan (2 mg) and fexofenadine (10 mg). Three analytical methods were developed and validated applying LC-MS/MS to quantify plasma concentrations of fexofenadine, losartan, E-3174 (method 1), omeprazole, 5-OH-omeprazole, midazolam, metoprolol, ?-OH-metoprolol (method 2), and caffeine (Method 3). Neither matrix effect nor carryover effect were observed. The methods were linear for the analytes in the ranges of 0.05 - 20 ng/mL (fexofenadine); 0.03 - 5 ng/ml (losartan and E-3174); 0.1 - 50 ng/mL (omeprazole), 0.3 - 50 ng/mL (5-OH-omeprazole), 0.01 - 10 ng/mL (midazolam), 0.05 - 50 ng/mL (metoprolol and ?-OH-metoprolol) and 5 - 1000 ng/mL (caffeine). The pharmacokinetic parameters of the compounds were calculated based on plasma concentration versus time (AUC) curves applying Phoenix® WinNonlin® software. AUC0-t and Cmax ratios after and before the EPPAF ® exposure, presented as geometric mean (CI 90%) were 0.74 (0.62 - 0.89) and 0.90 (0.76 - 1.07) for fexofenadine, 0.88 (0.80 - 0.97) and 0.86 (0.76 - 0.98) for losartan, 0.96 (0.83 - 1.11) and 0.91 (0.79 - 1.04) for E-3174, 1.18 (0.91 - 1.54) and 1.21 (0.87 - 1.70) for omeprazole; 1.12 (0.95 - 1.31) and 1.22 (0.95 - 1.67) for 5-OH-omeprazole, 1.14 (1.03 - 1.28) and 1.21 (1.00 - 1.46) for midazolam, 1.04 (0.92 - 1.18) and 0.94 (0.80 - 1.12) for metoprolol, 1.05 (0.99 - 1.12) and 0.99 (0.88 - 1.12) for ?-OH-metoprolol, 0.97 (0.77 - 1.21) and 0.87 (0.69 - 1.11) for caffeine. AUC0-t metabolic ratios of E3174/losartan, 5-OH-omeprazole/omeprazole and ?-OH-metoprolol/metoprolol we found to be, respectively, 1.11 (0.98 - 1.25), 0.94 (0.81 - 1.10 ) and 1.01 (0.88 - 1.16), indicating that, with the exception of CYP2D6, the administration of EPP-AF® under the conditions studied shows potential for CYP2C19 and CYP3A4 inhibition and CYP1A2, CYP2C9 and P-gp induction, although their magnitudes are below the limits defined by the regulatory agencies and therefore exhibit no clinical relevance

Desenvolveu-se método de alta sensibilidade e especificidade por cromatografia liquida de alta eficiência acoplada a detecção por espectrometria de massas (LC-MS/MS) para quantificação do lorazepam em plasma humano visando aplicação em estudo de bioequivalência entre duas formulações de comprimidos contendo esse fármaco. A preparação das amostras de plasma foi feita por extração líquido-líquido usando hexano:diclorometano (60:40 v/v) como solvente de extração. O padrão interno usado foi o bromazepam. A separação cromatográfica ocorreu utilizando-se coluna analítica modelo Gemini® C18 110 A (150 mm x 4,6 mm; partículas de 5µm). Mistura de metanol e tampão acetato de amônio 10 mM (80:20, v/v), acrescida de 0,1% de ácido fórmico ao final da preparação, foi usada como fase móvel. A interface entre HPLC e MS/MS foi a fonte de ionização por eletrospray (ESI) operando em modo positivo (ES+). O analito e o PI foram monitorados e quantificados através de multiple reaction monitoring (MRM). As transições monitoradas foram m/z 320,69 > 274,96 para o lorazepam e m/z 318,00 > 182,20 para o padrão interno. O método foi validado na faixa de concentração de 0,50 a 80,0 ng/ml em plasma humano. A bioequivalência entre as formulações foi determinada através dos intervalos de confiança 90 % obtidos para as razões dos parâmetros farmacocinéticos Cmax (99% - 114%), AUC0-t (93% - 105%) e AUC0-inf (96% - 107%). Concluiu-se que as duas formulações podem ser administradas de maneira intercambiável sem prejuízo da eficácia terapêutica. / A method of liquid chromatography coupled to mass spectrometric detection (LC-MS/MS) with high sensitivity and specificity was developed to quantify Lorazepam in human plasma. This method was applied in a bioequivalence study between two tablet formulations. The preparation of plasma samples were performed by liquid-liquid extraction using hexanedichloromethane (60:40 v/v) as extraction solvent. The internal standard was bromoazepam. The chromatographic separation was achieved using the Gemini® C18 110A (150 mm x 4.6mm; 5µm particles) analytical column. The mobile phase was prepared from a mixture of methanol and 10mM of ammonium acetate (80:20, v/v), and finally adding 0.1% of formic acid. The HPLC and MS/MS interface was the electrospray ionization source (ESI), operating in positive mode (ESI+). The analyte and internal standard were monitored and quantified through multiple reaction monitoring (MRM). The monitored transitions were m/z 320.69 > 274.96 for lorazepam and m/z 318.00 > 182.20 for the internal standard. The method was validated over the range 0.50 to 80.0 ng/mL in human plasma. The bioequivalence between the two formulations was determined inside the 90% confidence interval for the pharmacokinetic parameters, Cmax (99% - 114%), AUC0-t (93% - 105%) and AUC0-inf (96% - 107%). It was concluded that the two formulations can be administered in an interchangeable manner without losing the therapeutic efficiency.

Mykotoxine sind giftige Naturstoffe, die im Rahmen des Sekundärstoffwechsels von Schimmelpilzen beim Wachstum auf pflanzlichen Substraten gebildet werden. Zu den bekanntesten Mykotoxinen zählt das Ochratoxin A (OTA), welches überwiegend in Getreide und davon abgeleiteten Erzeugnissen, aber auch in Wein und Kaffee sowie Gewürzen und Bier nachgewiesen wurde. OTA ist unter anderem immunotoxisch, nephrotoxisch und besitzt teratogene sowie kanzerogene Eigenschaften. Darüber hinaus wird OTA eine hormonelle Wirkung zugesprochen. Aufgrund der Toxizität und des relativ häufigen Vorkommens in Lebensmitteln wurden für OTA Grenzwerte festgelegt. Diese liegen für Wein, Röstkaffee und diätetische Produkte zwischen 0,5 mikrogramm /kg und 10 mikrogramm /kg. Grenzwerte für Gewürze und Bier sind geplant. Im Rahmen der Methodenentwicklung zur Bestimmung von OTA in Lebensmitteln wurden verschiedene clean-up-Techniken miteinander verglichen. Dabei wurde vor allem den kombinierten Ionentauscher/reversed phase-Säulen und der LC-MS/MS wesentliche Bedeutung beigemessen. Innerhalb der Methodenvalidierung wurden die Ergebnisse mit dem entsprechenden Standardverfahren verglichen. Es konnte gezeigt werden, dass mit oben genanntem Material in Kombination mit der LC-MS/MS sehr gute Ergebnisse erzielt werden können. Diese wurden im Rahmen der Validierung durch statistische Auswertung bestätigt. Ferner wurden im Rahmen der Methodenentwicklung, speziell bei der Bestimmung von OTA in Kaffee, Extraktionstechniken miteinander verglichen. Dabei wurde die bislang kaum beachtete ASE (Accelerated Solvent Extraction, Dionex) sowie Ultraschallextraktion mit der Schüttelextraktion aus der gültigen Norm verglichen und ihre Anwendbarkeit auf weitere Lebensmittel geprüft. In diesem Zusammenhang waren die ASE und Ultraschallextraktion der konventionellen Schüttelextraktion überlegen. Darüber hinaus wurde die Ultraschallextraktion in Kombination mit dem Ionentauscher/reversed phase clean-up erfolgreich auf Weizen und Chili angewandt. Parallel zur Methodenentwicklung wurde die Stabilität und Homogenität von OTA in Wein und Röstkaffee untersucht. Sowohl die Homogenität als auch die Stabilität des Analyten sind wichtige Faktoren bei der Entwicklung neuer Verfahren, da zusammen mit Ergebnissen der Validierung, die Messunsicherheit innerhalb neuer Verfahren eingegrenzt werden. / Mycotoxins are naturally occurring toxic substances and produced as secondary metabolites by fungi which are growing on plants predominantly. One of the most interesting mycotoxins is ochratoxin A (OTA), which was found in several foods and feed stuff like wheat and its related products, wine, coffee, spices and beer. Among other things OTA is immunotoxic, nephrotoxic and has teratogenic and cancerogenic properties. Further more OTA shows hormonal effects. Because of its toxicity and common appearance in food and feed the OTA content had to be regulated in European and or national directives. The maximum values are determined between 0.5 micrograms/kg and 10 micrograms/kg for wine, roasted coffee and dietetic products. Other maximum levels for beer and spices are intended. During the investigation of analytical methods for the determination of OTA in food stuff different clean-up techniques were compared. Thereby the mixed anion exchange/reversed phase clean-up in combination with LC-MS/MS was researched intensively. Within method validation the results were compared with the corresponding standard methods. This work shows that the mixed mode clean-up in combination with LC-MS/MS gives excellent results. These results were statistically confirmed during method validation. Further more different extraction techniques were compared during the development of analytical methods, especially for the determination of OTA in coffee. Thereby the ASE (accelerated solvent extraction, Dionex) and the ultrasonic extraction were compared with the shaking technique from current standard methods. The adaptability for other food and feed products was researched as well. In this context the new extraction techniques were superior to conventional shaking techniques. In addition the ultrasonic extraction with mixed mode clean-up and LC-MS/MS detection was applied to chilli and wheat effectively. Besides the investigation of analytical methods the stability and homogeneity of OTA in wine and roasted coffee was researched. The homogeneity and stability of OTA in matrix are very important factors during the investigation of analytical methods. In combination with the relevant results of the method validation these results can be used to determine the uncertainty of the new analytical methods.

Die schmerzstillende Wirkung von Opiaten wird über Opioidrezeptoren im zentralen und peripheren Nervensystem vermittelt. Die Schmerzlinderung kann jedoch mit sehr starken Nebenwirkungen einhergehen, die das Patientenwohlbefinden beeinträchtigen. Dies legt die Bedeutung von neuen Opioidanalgetika nahe, die ihre schmerzstillende Wirkung ausschließlich über Opioidrezeptoren im PNS entfalten, ohne unerwünschte zentrale Nebenwirkungen zu induzieren. Die orale Gabe von Medikamenten minimiert Unannehmlichkeiten für den Patienten, jedoch müssen die Substanzen die intestinale Barriere passieren können, um in die Blutzirkulation eintreten zu können. Die intestinale Permeabilität von zwei peripher wirksamen Opiaten (AS006 und Loperamid) wurde in Ussing-Kammer Experimenten untersucht. Um die Darmepithelpermeabilität für beide Opiate zu erhöhen, wurde der Absorptionsverstärker Chitosan verwendet. Chitosan bewirkte nach 30 Minuten bei HT29/B6 und Caco-2 Zelllinien eine Abnahme des epithelialen Widerstands in vitro. Die Permeabilität für AS006 war bei beiden Zelllinien erhöht, für Loperamid nur bei HT29/B6, jedoch nicht bei Caco-2 Zellmonolayern. Verhaltensexperimente zur Messung des antinozizeptiven Effektes von oral appliziertem Loperamid auf Entzündungsschmerz wurden an Ratten durchgeführt. Die orale Gabe von Loperamid induzierte eine Dosis-abhängige antinozizeptive Wirkung in der entzündeten Hinterpfote. Bei oraler Gabe von Loperamid in Kombination mit Chitosan wurde keine signifikante Verstärkung des maximalen antinozizeptiven Effekts von Loperamid beobachtet. Zusammenfassend ist Chitosan ein geeigneter Absorptionsverstärker für intestinale Permeabilitätsstudien von peripher wirksamen Opioidanalgetika in vitro. Die in vitro Ergebnisse haben gezeigt, dass der Effekt von Chitosan auf Loperamid möglicherweise schwächer ist als auf AS006. Dementsprechend fiel die Wirkung des Absorptionsverstärkers auf Loperamid-induzierte Analgesie im Verhaltensversuch eher gering aus. / Analgesic effects of opioids are mediated by opioid receptors that are widely distributed in the central and peripheral nervous systems (CNS and PNS, respectively). Although opioids are the most powerful analgesics, severe side effects restrict their use and affect patient convalescence. This suggests an advantage of new analgesic opioids which selectively bind to opioid receptors in the PNS. After oral administration however, peripherally restricted opioids first have to cross the intestinal epithelial barrier before absorption into the circulation and distribution to opioid receptors in peripheral tissues. Here, the transport across intestinal epithelia of two opioid ligands (AS006 and loperamide) that selectively activate peripheral opioid receptors without entering the CNS were investigated. To increase the intestinal passage of these drugs, the absorption enhancer chitosan was used. Chitosan significantly decreased the transepithelial resistance of HT29/B6 and Caco-2 cell monolayers after 30 min in vitro. The permeability values for AS006 increased from < 0.3 × 10-6 cm/s up to 10 × 10-6 cm/s in the presence of chitosan. In contrast, HT29/B6 monolayers showed moderate loperamide permeability in the presence of chitosan, and chitosan had no effect on the permeability of loperamide using Caco-2 monolayers. Oral administration of loperamide induced a dose-depended elevation of paw pressure thresholds in inflamed paws that lasted for 60 min. Oral administration of loperamide combined with chitosan slightly but nonsignificantly enhanced the antinociceptive effect of loperamide. In conclusion, chitosan is a suitable absorption enhancer for in vitro intestinal permeability studies. Future in vivo experiments might investigate different formulations and application schedules, and further address the effects of chitosan on the antinociceptive efficacy of hydrophilic opioids.

Hepcidin-25, ein 2000 entdecktes Peptidhormon, das eine Schlüsselrolle im Eisenstoffwechsel spielt, hat das Verständnis von Eisenerkrankungen revolutioniert. In dieser Studie wurde LC gekoppelt mit einem Triple-Quadrupol MS in einer schnellen und robusten Methode zur Quantifizierung von Hepcidin-25 in menschlichem Serum verwendet, welche letztlich in Routine-Laboratorien genutzt werden soll. Zu diesem Zweck wurden zwei Probenvorbereitungsstrategien und zwei komplementäre LC Bedingungen untersucht, wobei eine saure mobile Phase (0,1% TFA) mit einem neuartigen Ansatz unter der Verwendung einer basischen mobilen Phase (0,1% NH3) verglichen wurde. In einem laborinternen Vergleich beider LC-MS/MS Methoden wurde Hepcidin-25 in humanen Proben unter Verwendung der gleichen Kalibrierstandards quantifiziert und eine sehr gute Korrelation der Ergebnisse ermittelt. Hierbei wurde die Analysestrategie mit saurer mobiler Phase als hochsensitiv (LOQ von 0,5 μg/L) und präzise (CV <15%) befunden und als Kandidat einer Referenzmethoden für die Hepcidin-25 Quantifizierung in realen Proben empfohlen. Einer der neuartigen Aspekte der Methodik war die Verwendung von Amino- und Fluor-silanisierten Autosampler-Fläschchen, um die Adsorption des 25 Reste umfassenden Peptids anOberflächen zu reduzieren. Darüber hinaus wurde diese LC-MS/MS-Methode in einer internationalen Ringversuchsstudie eingesetzt, bei der ein sekundäres Referenzmaterial als Kalibrierstandard verwendet wurde, und gemäß des International Consortium for Harmonization of Clinical Laboratory Results (ICHCLR) als optimal bewertet. In dieser Arbeit wurde die Bildung von Hepcidin-Komplexen mit Kupfer(II) untersucht. Die erste Umkehrphasen-chromatographische Trennung von Hepcidin-25/Cu(II) und Hepcidin-25 (Kupfer "frei") wurde unter Verwendung von mobilen Phasen mit 0,1% NH3 erreicht. LC-MS/MS und FTICR-MS wurden für die Charakterisierung der gebildeten Hepcidin-25-Cu(II)-Spezies bei pH-Werten von 11 bzw. 7,4 verwendet. / Hepcidin-25, a key iron-regulatory peptide hormone discovered in 2000, has revolutionized the understanding of iron-related pathology. This study applied LC-MS/MS, using the triple quadrupole mass spectrometer, in a rapid and robust analytical strategy for the quantification of hepcidin-25 in human serum, to be implemented in routine laboratories. For this purpose, two sample preparation strategies and two complementary LC conditions were investigated, where the use of acidic mobile phases (0.1% TFA) was compared with a novel approach involving solvents at high pH (0.1% NH3). The application of these LC-MS/MS methods to human samples in an intra-laboratory comparison, using the same hepcidin-25 calibrators, yielded a very good correlation of the results. The LC-MS/MS employing trifluoroacetic acid-based mobile phases was selected as a highly sensitive (LOQ of 0.5 µg/L) and precise (CV<15%) method and was recommended as a reference method candidate for hepcidin-25 quantification in real samples. One of the novel aspects of the methodology was the use of amino- and fluoro-silanized autosampler vials to reduce the interaction of the 25-residue peptide to laboratory glassware surfaces. Moreover, this LC-MS/MS method was used for an international round robin study, applying a secondary reference material as a calibrator and its performance was found to be in the optimal range as defined by the International Consortium for Harmonization of Clinical Laboratory Results (ICHCLR). In this work, the formation of hepcidin-25 complexes with copper(II) was investigated. The first reversed-phase chromatographic separation of hepcidin-25/Cu2+ and hepcidin-25 (copper “free”) was achieved by applying mobile phases containing 0.1% NH3. LC-MS/MS and FTICR-MS were applied for the characterization of the formed hepcidin-25-Cu(II) species at pH values of 11 and 7.4 respectively. A new species corresponding to hepcidin-25 complexed with two copper ions was identified at high pH.

O benzoato de emamectina, um antiparasitário da classe das avermectinas, é amplamente utilizado em diversos países para o tratamento de infecções causadas por helmintos intestinais e ectoparasitas, os quais estão relacionados a grandes prejuízos na produção aquícola. Apesar de sua eficácia ser conhecida na aquicultura, seu uso não está regulamentado no Brasil. Diante da demanda nacional por uma maior variedade de medicamentos veterinários regulamentados para uso em espécies aquícolas, o presente projeto visou estimar um período de carência para o antiparasitário benzoato de emamectina, a partir de um estudo experimental de depleção residual desse fármaco em tilápia (filé com pele em proporção natural), a espécie de maior importância comercial para a produção aquícola no Brasil. Para tanto, fez-se necessárias as seguintes etapas: (i) incorporar o benzoato de emamectina na ração através da técnica de revestimento polimérico em equipamento de leito fluidizado; (ii) desenvolver e validar métodos analíticos para a determinação do benzoato de emamectina na ração e em filé de tilápia empregando cromatografia líquida de alta eficiência acoplada à espectrometria de massas (LC- MS/MS); e (iii) realizar um ensaio com tilápias para avaliar a depleção residual de emamectina B1a no filé. O revestimento da ração com 1,5% de etilcelulose permitiu incorporar o benzoato de emaectina na ração sem alterar a estrutura física da mesma, o que torna o processo vantajoso, visto que a ração mantém suas características nutricionais e de flutuabilidade. Além disso, o processo foi homogêneo (CV de 2,1%) e não apresentou taxa de lixiviação quantificável do fármaco para a água, contribuindo para uma maior segurança em relação à dose de tratamento a ser administrada e em relação ao risco potencial ao meio ambiente. A emamectina B1a foi extraída da ração por processo sólido-líquido e do filé de tilápia pelo método QuEChERS modificado. Os métodos foram avaliados mediante os seguintes parâmetros: seletividade, curva analítica, linearidade, precisão, exatidão, limite de detecção (LD) e de quantificação (LQ). Todos os parâmetros avaliados se encontraram conformes às recomendações dos guias de validação tomados como referências, qualificando os métodos como adequados para os objetivos propostos no presente trabalho. Para avaliar a depleção de emamectina B1a no filé de tilápia, os peixes receberam o fármaco via ração em dose de 50 ?g/kgPV/dia durante sete dias consecutivos. As curvas de depleção residual de emamectina B1a se ajustaram ao modelo exponencial de primeira ordem. Tomando como referência o limite máximo de resíduo de 100 ?g/kg recomendado pelo Codex Alimentarius, não houve a necessidade de se propor um período de carência mínimo para o abate dos peixes, considerando as condições experimentais empregadas. / Emamectin benzoate, an antiparasitic of the class of avermectins, it is widely used in several countries for the treatment of infections caused by intestinal helminths and ectoparasites, which are related to great losses in aquaculture production. Although its known effectiveness for aquaculture, its use is not regulated in Brazil. The national demand for a greater variety of regulated veterinary drugs for use in aquaculture species,the current project that aimed to evaluate a withdrawal period for the antiparasitic emamectin benzoate, based on an experimental study of residual depletion of this drug in tilapia ( fillet with skin in natural proportion) the species of major commercial importance for aquaculture production in Brazil. To do so, it was necessary to: (i) incorporate emamectin benzoate into the feed through the polymer coating technique in fluidized bed equipment; (ii) to develop and validate the analytical methods for the determination of emamectin benzoate in the feed and in tilapia fillet using high-performance liquid chromatography coupled to mass spectrometry (LC-MS / MS) and (iii) perform a tilapia test to evaluate the depletion of emamectin B1a in fillet. The feed coating with 1.5% ethylcellulose allowed to incorporate the emaectin benzoate into the feed without altering the physical structure, which makes the process advantageous, since the feed retains its nutritional and buoyancy characteristics. In addition, the process was homogeneous (CV 2,1%) and did not present quantifiable leaching rate of the drug into the water, contributing to a greater safety in relation to the dose of treatment to be administered and in relation to potential risk to the environment. Emamectin B1a was extracted from the feed by solid-liquid process and from tilapia fillet by the modified QuEChERS method. The methods were evaluated using the following parameters: selectivity, analytical curve, linearity, precision, accuracy, limit of detection (LD) and quantification (LQ). All the evaluated parameters were in accordance with the recommendations of the validation guides taken as references, qualifying the methods as adequate for the objectives proposed in the present work. To evaluate the depletion of emamectin B1a tilapia fillet, the fish received the drug via feed at a dose of 50 ?g / kgPV / day for seven consecutive days. The residual depletion curves of emamectin B1a were fitted to the first order exponential model. Taking into account the maximum residue limit of 100 ?g / kg recommended by the Codex Alimentarius, there was no need to propose a minimum withdrawal period for the slaughtering of fish, considering the experimental conditions used.

O aumento no uso abusivo e nas apreensões de comprimidos contendo 1-(3-clorofenil)piperazina (mCPP) têm sido observado na Europa desde o final do século 20. A mCPP promove efeitos semelhantes a metilenodioximetanfetamina (ecstasy) e surgiu como uma alternativa menos neurotóxica. Os principais efeitos descritos pelos usuários são sensação de bem-estar, euforia e empatia. Os efeitos adversos observados em casos de intoxicação aguda são a ansiedade, confusão, insônia, ataques de pânico, estados convulsivos, taquicardia e até mesmo a morte. A mCPP frequentemente tem seu uso associado com a privação de sono dos usuários em ambientes noturnos (festas e danceterias). Além disso, o fármaco provoca insônia no usuário, agravando ainda mais as consequências ao sono do indivíduo. O sono REM, em humanos, ou chamado de sono paradoxal nos animais, é uma fase importante do sono, por ser ela a fase de retorno da homeostasia comportamental e bioquímica. O objetivo deste trabalho foi avaliar os efeitos comportamentais dos isômeros da clorofenilpiperazina e desenvolver um método analítico para identificar e quantificar a mCPP em amostras de plasma de camundongos submetidos à privação de sono paradoxal (PSP) por 24 e 48 horas. A ferramenta analítica empregada para identificar e quantificar a mCPP foi a cromatografia líquida acoplada à espectrometria de massas (LC-MS/MS). As análises comportamentais de ansiedade e atividade locomotora dos camundongos utilizaram os testes do Labirinto em Cruz Elevado e o teste do Campo Aberto, respectivamente. Os resultados mostraram que a associação da PSP com o uso da mCPP acarretou mudanças comportamentais que voltaram ao nível homeostásico somente após 48 horas de rebote de sono. Além disso, observou-se um aumento significativo na concentração circulante de mCPP nos animais PSP por 48 horas em relação ao grupo controle. Por fim, concluiu-se que a privação de sono paradoxal associada com a administração da mCPP produziu graves consequências comportamentais em camundongos e que a concentração do fármaco encontrado no plasma foi maior nos animais submetidos à privação de sono paradoxal. / The increase on abusive use and seizures of tablets containing 1-(3-chlorophenyl)piperazine (mCPP) have been seen in Europe since the late 20th century. The mCPP promotes effects similar to methylenedioxymethamphetamine (ecstasy) and emerged as a less neurotoxic alternative. The main effects described by users are sense of well-being, euphoria and empathy. The adverse events observed in acute poisoning cases are anxiety, confusion, insomnia, panic attacks, convulsive states, tachycardia and even death. mCPP is often associated with sleep deprivation by their users and on night scenery (parties and discos). In addition, the drug causes insomnia on user, further aggravating the consequences to the individual sleep. REM sleep in humans or referred as to paradoxical sleep, in animals, is an important sleep phase, because it was the phase which promote the return of behavioral and biochemical homeostasis. The aim of this study was to evaluate the behavioral effects of the isomers of chlorophenylpiperazine and develop an analytical method to identify and quantify the mCPP in plasma samples from mice subjected to paradoxical sleep deprivation (PSD) for 24 and 48 hours. The analytical tool used to identify and quantify the mCPP was liquid chromatography coupled to mass spectrometry (LC-MS/MS). The behavioral analysis of anxiety and locomotor activity of mice used the Elevated Plus Maze and Open Field tests, respectively. The results showed that association of PSD with the use of mCPP led to behavioral changes that back to the homeostatic level only after 48 hours of rebound sleep. Furthermore, there was a significant increase in circulating concentration of mCPP in animals paradoxical sleep deprived for 48 hours compared to control group. Finally, it is concluded that paradoxical sleep deprivation associated with administration of mCPP produced severe behavioral effects in mice and concentration of drug found in plasma was greater in animals submitted to paradoxical sleep deprivation than control group.

O albendazol (ABZ) é um antiparasitário amplamente utilizado na medicina veterinária para tratar infecções causadas por parasitas helmínticos, devido ao seu baixo custo de aquisição, alta eficácia e facilidade de administração. Apesar, de sua eficácia ser mundialmente conhecida, seu uso não é regulamentado em espécies aquícolas. Diante da demanda nacional por uma maior variedade de medicamentos veterinários regulamentados para uso na aquicultura, o presente projeto visou estimar o período de carência para o ABZ. Para tanto, foi necessário: (i) incorporar o ABZ na ração através da técnica de revestimento polimérico em equipamento de leito fluidizado; (ii) desenvolver e avaliar os métodos analíticos para a determinação do ABZ na ração e do ABZ e seus metabólitos em filé de pacu empregando cromatografia líquida de alta eficiência acoplada à espectrometria de massas (LC-MS/MS) e (iii) realizar um ensaio com pacu para avaliar a depleção do ABZ e seus metabólitos no filé desses peixes. O revestimento da ração com 1,5% de etilcelulose permitiu incorporar o ABZ na ração sem alterar a estrutura física da mesma, o que torna o processo vantajoso, visto que, a ração mantém suas características de flutuabilidade. Além disso, o processo foi homogêneo e diminuiu a lixiviação do fármaco para a água, garantindo a segurança da dose administrada e do meio ambiente. O ABZ foi isolado da ração por extração líquido-líquido. O ABZ e seus metabólitos foram extraídos do filé de pacu pelo método de QuEChERS modificado. Para os dois métodos desenvolvidos, a separação cromatográfica foi realizada em coluna de fase reversa octadecil híbrida. Os métodos foram avaliados mediante os seguintes parâmetros: seletividade, curva analítica, linearidade, precisão e exatidão. Para o método do filé de peixe avaliou-se também o CC? e o CC?. Todos os parâmetros avaliados se encontram de acordo com os guias de validação, o que torna os métodos adequados para os objetivos propostos no presente trabalho. Para avaliar a depleção do ABZ e seus metabólitos no filé de pacu, os peixes receberam o fármaco via ração em dose única de 10 mg de ABZ (kg peso vivo)-1. As curvas de depleção residual do ABZ e seus metabólitos se ajustaram ao modelo exponencial de primeira ordem. Tomando como referência as recomendações da EMA, o período de carência foi estimado em sete dias. / Albendazole (ABZ) is an antiparasitic widely used in veterinary medicine to treat infections caused by parasites anthelmintic, due to its low cost, high efficiency and ease of administration. Regardless of its world widely known effectiveness, its use is not regulated in aquaculture species. Faced with national demand for a greater variety of regulated veterinary drugs for use in aquaculture, this project aimed to estimate the withdrawal time for the ABZ. Therefore, it was necessary to (i) incorporate ABZ the feed through the polymeric coating by spouted bed technique; (Ii) develop and evaluate analytical methods for determining the ABZ in feed and ABZ and its metabolites in pacu fillet using high-performance liquid chromatography coupled to mass spectrometry (LC-MS / MS) and (iii) conduct a test with pacu to evaluate the depletion of ABZ and its metabolites in the fillet of these fish. The ration with 1.5% coating of ethylcellulose allowed incorporate ABZ the feed without altering the physical structure of the same, which makes the process advantageous, since the feed maintains its buoyancy characteristics. Furthermore, the process was homogeneous and reduced leaching of the drug into the water, ensuring the safety of the administered dose and the environment. The ABZ ration was isolated by liquid-liquid extraction. The ABZ and its metabolites were extracted from pacu fillet the modified QuEChERS method. For both developed methods, chromatographic separation was performed in a hybrid octadecyl reverse phase column. The methods were evaluated by the following parameters: selectivity, analytical curve, linearity, precision and accuracy. For fish fillet method also assessed whether it CC? and CC?. All parameters are according to the validation guides, which makes the appropriate methods for the objectives proposed in this paper. To evaluate the depletion of ABZ and its metabolites in pacu fillet, the fish were dosed via single dose in feed 10 mg ABZ (kg body weight)-1. The curves of residual depletion ABZ and its metabolites adjusted to the exponential model of the first order. With reference to the recommendations of the EMA, the withdrawal time was estimated at seven days

O sequenciamento genético do DNA humano permitiu maior compreensão da funcionalidade dos seres vivos e principalmente a causa de muitas doenças. Entretanto, os estudos em genética têm se limitado a resolverem os problemas da ciência, e atualmente, a solução para o avanço nessa área tem se atribuído à proteômica. Dessa forma, a pesquisa em química analítica intensificou-se na busca de estratégias melhores para a caracterização de proteomas, em três aspectos principais: preparo de amostra, desenvolvimento da instrumentação analítica e bioinformática. Verifica-se a possibilidade da aplicação de muitas técnicas, atualmente, destaca-se a análise de peptídeos (proteômica shotgun) por cromatografia líquida multidimensional acoplada à espectrometria de massas sequencial (LC/LC-MS/MS), devido à possibilidade de automatização, minimização dos problemas e resultados satisfatórios na análise de amostras biológicas complexas. Portanto, neste trabalho desenvolveu-se um método LC/LC-MS/MS (modalidade on-line column switching) o qual se constitui de coluna trocadora catiônica (homemade), trap de aprisionamento e limpeza, coluna capilar hidrofóbica e separação e detecção por espectrometria de massas sequencial automatizada. Com a proposta de uma instrumentação analítica aperfeiçoada, realizamos a confecção de um trap com partículas de elevada retenção dos peptídeos, o que permite ótima recuperação de amostra. Por se tratar de uma técnica de elevada complexidade instrumental, devido a difícil compatibilidade entre as dimensões cromatográficas, possíveis perda de analito no processo e elevado tempo de análise, propomos uma nova abordagem de otimização, por meio de estudos quimiométricos. Assim, neste trabalho, foram avaliados doze parâmetros instrumentais e destes houve uma simplificação de apenas dois fatores, responsáveis por 95% da resposta ótima do método. Este fato permitiu valores da cobertura da proteína de BSA (82,54%), número de peptídeos (65) e score (2134,05) superiores aos reportados na literatura, os quais apresentam tempos de análise maiores. Este estudo fornece informações do comportamento químico dos peptídeos em relação ao método proposto, por meio de uma superfície de resposta e equação matemática que pode contribuir para a aplicação em diferentes proteomas. / The genetic sequence of human DNA has helped the comprehension of life and principally the cause of various diseases. However, genetic studies have limited to resolve science problems and currently solutions to advance in this field have been attributed to proteomics. Thus, the analytical chemistry has intensified on the search for a better strategy to proteomic characterization in three principal aspects: sample preparation, development of analytical instrumentation, and bioinformatics. Proteomics involves the application of many techniques, currently; the peptide analyses (shotgun proteomics) by multidimensional liquid chromatography coupled to tandem mass spectrometry (LC/LC-MS/MS) is the state of the art. The main reasons are because it allows full system automation, less problems in repeatability, and adequate results in analysis of highly complex biological samples. Therefore, this dissertation developed the method LC/LC-MS/MS (modality on-line column switching), this has a cation exchange column (homemade), trap column to clean, hydrophobic capillary column and separation and detection by tandem mass spectrometry. We have proposed an improved analytical instrumentation, with a homemade trap that particles have high retention of peptide, which permit great recuperation of samples. Because it is an instrumental technique difficult, such as, obtain compatibility between the chromatography dimensions, can lose samples in the process and long time analysis, we have proposed a new optimization approach with chemometric analysis of data. On that, were evaluated twelve instrumental parameters and there was a simplification only two factors, these were responsible for 95% of greater response of method. As a result, the coverage of BSA protein was 82,54%, number of peptides 65 and score of 2134,05 values of high significance if compare from that there are in literature that presented greater time analysis. This work describes information about chemistry of peptides to method proposed through a surface of response and math equation that can contribute to different proteomes.

Les sulfonamides sont parmi les antibiotiques les plus couramment utilisés en élevage. Ils peuvent en effet, si leur utilisation n’est pas conduite de manière raisonnable, être une source de nombreux risques pour la santé publique. Au Liban, il n’existe pas à l’heure actuelle de règlementation fixant les limites de résidus des sulfonamides dans les tissus d'origine animale. En outre, aucune investigation sur la présence des résidus des sulfonamides sous leurs formes actives ou métabolisée dans les denrées animales n’a été menée. L'analyse d'une matrice complexe telle que la viande a nécessité la mise en œuvre d’une préparation d'échantillon rigoureuse afin obtenir une analyse reproductible, et suffisamment sensible pour atteindre les limites de détection requises. Cette thèse décrit le développement de deux méthodes analytiques pour la détermination de sulfonamides et de leurs métabolites à l’état de traces dans les tissus d’origine animale (bœuf, volaille, porc…). Elles sont basées sur une étape d'extraction utilisant la méthode QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) suivie d'une analyse par HPLC couplée à un analyseur triple quadripôle MS/MS ou LTQ-Orbitrap Velos. Les performances analytiques de ces méthodes ont été évaluées et comparées. Les méthodes d’analyse ont été validées suivant les recommandations de la décision de l’UE (2002/657/EC) et lors d’une étude de validation inter-laboratoires organisée par FAPAS (Food Analysis Performance Assessment Scheme). Au vu des performances obtenues, une étude de contrôle a été réalisée sur les résidus des sulfonamides et de leurs métabolites dans plus de 300 échantillons de différents tissus d’origine animale dérivant de poulets de chair, bœufs, brebis et porcs collectés dans différentes régions d’élevage libanaises / Sulfonamides are amongst the most commonly used veterinary antibiotics. The uncontrolled exposure to sulfonamides upon consumption of meat products can be harmful to human health. In Lebanon, there are no current regulations that specify the safe levels of sulfonamides in animal products. In addition, no studies describing the presence of sulfonamides residues in their active or metabolized form in animal tissues exist. The analysis of residues at trace levels in complex matrices such as meat required the implementation of a rigorous sample preparation and analytical protocol that yields reproducible results at very low concentration levels. This thesis describes the development of two analytical methods for the determination of sulfonamides and their metabolites in animal tissues (e.g. poultry, sheep, pork) at trace levels. They are both based on an extraction step using QuEChERS extraction (Quick, Easy, Cheap, Effective, Rugged and Safe) followed by HPLC. In terms of mass analysers the use of LTQ-Orbitrap Velos and triple quadrupole MS/MS was optimized and the figures of merit were compared. The analytical methods were validated according to the EU decision (2002/657 / EC) criteria and by the participation in an inter-laboratory validation study organized by FAPAS (Food Analysis Performance Assessment Scheme). The methods were applied to carry out a monitoring study to detect and quantify residues of sulfonamides and their metabolites in more than 300 different samples of animal tissues derived from poultry, beef, sheep and pork collected from different regions of Lebanon..