Níveis não aceitáveis de resíduos de ivermectina (IVM), uma droga anti-parasitária amplamente utilizada no Brasil para controle de endectoparasitas, pode estar presente no leite para consumo humano se administrada incorretamente. O nível máximo de resíduos no leite para este composto é de 10 ng mL-1 e sua presença tem sido comprovada em pesquisas realizadas por orgãos reguladores. Com o conhecimento deste problema, um método para a detecção de ivermectina em amostras de leite foi desenvolvido utilizando a extração líquido-líquido com base em acetonitrila e hexano, seguida por derivatização com 1-metilimidazol (MI), trietilamina (TEA), ácido anidro trifluoroacético (TFAA) e ácido trifluoroacético (TFA) e cromatografia líquida com detecção fluorescente (LC-FL) para a análise. Além disso, o método proposto foi testado de acordo com parâmetros de validação estabelecidos pela ANVISA. Parâmetros, tais como seletividade, linearidade (R² 0,98), precisão (coeficiente de variação entre 0,6-19%), recuperação (90-95%) e robustez foram avaliados durante o processo de validação. Subsequentemente, foi testado em amostras de leite de vacas tratadas com uma formulação comercial de ivermectina a 1%. O método aplicado em amostras de campo, provou possuir um perfil de quantificação e de confirmação para o método concebido no presente estudo. / Non-acceptable residue levels of ivermectin (IVM), an anti-parasitic drug widely employed in Brazil for control of endectoparasites, could be present in milk for human consumption if improperly administered. The maximum residue level in milk for this compound is 10 ng mL-1 and its presence has been proved from previous government surveys. With this rising subject, a method for the detection of ivermectin in milk samples was developed using liquid-liquid extraction based in acetonitrile and hexane, followed by derivatization with 1-methylimidazole (MI), triethylamine (TEA), anhydrous trifluoacetic acid (TFAA) and trifluoracetic acid (TFA) and liquid chromatography coupled to fluorescent detection (LC-FL) for the analysis. Moreover, the proposed method was tested according to validation parameters estabilished by ANVISA. Parameters, such as selectivity, linearity (R² 0.98), precision (RSD values between 0.6 19%), recovery (90-95%) and robustness were evaluated during the validation process. Subsequently it was tested in milk samples from cows treated with a commercial ivermectin formulation at 1%. The method applied to field samples, proved a quantifiable and confirmatory profile for the method designed in this study.

O metabolismo do triptofano (Trp) se dá pela via das quinureninas (QUIN), pela via serotoninérgica (SER) e pela via das aminas traço. A primeira gera QUIN e uma variedade de outros metabólitos secundários. Quando conduzida pela enzima indolamina 2,3 dioxigenase (IDO) contribui para os fenômenos de tolerância e imune escape de células tumorais; e quando conduzida pela triptofano 2,3 dioxigenase (TDO) no fígado, participa na síntese da niacina e NAD. A via SER leva à formação do neurotransmissor serotonina (SER), que pode gerar o hormônio melatonina (MEL), respectivamente e outros metabólitos biologicamente ativos. Outra via menos estudada, a via das aminas traço, produz produtos neuroativos. Dada a abrangência e importância das rotas metabólicas do Trp, nós desenvolvemos e validamos uma metodologia bioanalítica robusta, seletiva e sensível por cromatografia líquida de alta eficiência (HPLC), acoplado espectrometria de massas (MS) para a determinação simultânea do Trp e seus 15 metabólitos. Para tanto, escolhemos para a avaliação das três vias, linhagens de glioma humano. A escolha por este tipo celular deveu-se ao grande interesse de estudos de metabolismo de Trp em células tumorais, no qual células de glioma tem sido modelo. Nos ensaios com as células de glioma acompanhamos os efeitos de um indutor e inibidores da primeira etapa de metabolização do Trp pela via das quinureninas, ou seja, IFN-γ (indutor da IDO), 1-metiltriptofano (1-MT; inibidor competitivo da IDO) e 680C91 (inibidor seletivo da TDO). Pudemos observar o impacto que a indução ou a inibição do primeiro passo teve sobre os metabólitos subsequentes e as diferenças no metabolismo das duas linhagens estudadas, A172 e T98G. A linhagem T98G só tem atividade de IDO, enquanto que a A172 tem tanto atividade IDO quanto TDO. A indução por IFN-γ mostrou que essa citocina não só atua na formação da via QUIN, mas possui um impacto modesto nas demais rotas. Observamos também que a inibição do 1-MT mostrou seu impacto nos metabólitos invdividualmente, do que a simples relação Trp-QUIN. Contudo, nosso resultados nos permitiu mostrar pela primeira vez a descrição completa dessas vias, em especial nessas linhagens celulares, podendo supor estratégias terapêuticas nessas rotas que estão relacionadas a progressão ou não tumoral. / The tryptophan metabolism (Trp) takes place by means of kynurenine (QUIN), by the serotonin pathway (SER) and by the pathway of trace amines synthesis. The first generates QUIN and a variety of other secondary metabolites. When driven by the enzyme indoleamine 2,3 dioxygenase (IDO) contributes to the phenomena of tolerance and immune escape of tumor cells; and when conducted by tryptophan 2,3 -dioxygenase (TDO) in the liver, participates in the niacin synthesis NAD. The SER pathway leads to the serotonin neurotransmitter (SER) formation, which can generate the hormone melatonin (MEL), respectively and other biologically active metabolites. Another less studied amines trace synthesis pathway produces neuroactive products. Given the scope and importance of Trp metabolic pathways,we developed and validated a robust, sensitive and selective bioanalytical method by high performance liquid chromatography (HPLC) coupled mass spectrometry (MS) for simultaneous determination of TRP and its 16 metabolites. Therefore, we chose to evaluate the three routes, glioma cell lines. The initial choice of this type of cell was due to the great interest in Trp metabolism studies in tumor cells, which glioma cells has been a model. In assays with glioma cells, we followed the effects of an inductor and inhibitors of the first stage of Trp metabolism, via the kynurenine pathway, or IFN -γ (IDO inducer) 1- methyltryptophane (1- MT; competitive IDO inhibitor) and 680C91 (selective TDO inhibitor). We could observe the first step induction or inhibition impact had over the further metabolites and the metabolism differences between the two studied strains, A172 and T98G. The T98G glioma cell has only IDO activity, while the A172 has both IDO and TDO activity as well. The IFN-γ indution showed that this cytokine not only acts in the formation of QUIN route, but has a modest impact on the others routes. Inhibition of IDO showed that the competitive inhibitor has activity in itself than a simple Trp-QUIN relationship. However, our results allow us to show the first time the complete description of these pathways, in particular, in these cell lines that can assume therapeutic strategies in these routes that are related or not with tumor progression.

Submitted by MARCIA ROVADOSCHI (marciar@unifra.br) on 2018-08-17T11:35:48Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_ClarissaDenardinNiederauer.pdf: 1303429 bytes, checksum: d47f59505508dd5061362119541c3f74 (MD5) / Made available in DSpace on 2018-08-17T11:35:48Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_ClarissaDenardinNiederauer.pdf: 1303429 bytes, checksum: d47f59505508dd5061362119541c3f74 (MD5) Previous issue date: 2016-07-12 / Currently the demand for aesthetic treatments has increased considerably, but often cause pain and, because of this, are used topical anesthetic prior to treatment. Because of this, the search to an agent with these characteristics is permanent, in view of the difficulty in obtaining an agent having these characteristics related to the diffusion of the drug and adequate distribution on the skin. The eugenol, the main compound of the ethanol extract of clove cloves (syzygium aromaticum), is responsible for much of the pharmacologic effects attributed to plant, including the anesthetic activity. With the purpose of obtaining a product that complies fully with the anesthetic effect topically without cause systemic adverse effect, the objective of this work was the prodution, characterization and hplc assay of a nanoemulsion containing eugenol. In the production of the nanoemulsion, the parameters evaluated were: mechanical agitation at speeds of 10,000, 20,000 and 26,000 rpm, 7.5 and 15 minutes stirring time and the active concentration of 0.50; 0.75 and 1.00%. The characterizations of the formulations were the average droplet diameter, polydispersity index, zeta potential and ph, as well as the determination of the active was performed via liquid chromatography high performance (hplc). Stability studies were performed on a 75 day period in three temperature conditions (5, 25 and 40 ° c). Best results were obtained when we used mechanical stirring with 20,000 rpm for 7.5 minutes stirring time and the concentration of 1.00% active, producing nanoemulsions having an average diameter droplet 101.17 ± 6.80 nm index a polydispersity 0.135 ± 0.027, zeta potential of -7.19 ± 0.713 and pH 4.84 ± 0.04. This formulation was stable for up to 30 days, keeping the results as stored at 5 and 25ºc. As for the determination of recoveries 72 to 106% of the assets were obtained. Thus, it is concluded that the methodology used is feasible for production nanoemulsions containing eugenol. / Atualmente a procura por tratamentos estéticos tem aumentado consideravelmente, mas muitas vezes causam dor e, devido a isso, são utilizados anestésicos tópicos previamente ao tratamento. Devido a isso, a busca a um agente com essas características é permanente, tendo em vista a dificuldade em se obter um agente com essas características relacionadas à difusão do fármaco e a distribuição adequada sobre a pele. O eugenol, principal composto do extrato etanólico do cravo-da-índia (Syzygium aromaticum), é o responsável por grande parte dos efeitos farmacológicos atribuídos a planta, entre elas a atividade anestésica. Com o proposito de obter um produto que cumpra totalmente com o efeito anestésico por via tópica, sem causa efeito adverso sistêmico, o objetivo deste trabalho foi a produção, caracterização e o doseamento por cromatografia líquida de alta eficiência (HPLC) de uma nanoemulsão contendo eugenol. Na produção da nanoemulsão, os parâmetros avaliados foram: agitação mecânica com velocidades de 10000, 20000 e 26000 rpm, tempo de agitação de 7,5 e 15 minutos e, concentração do ativo de 0,50; 0,75 e 1,00%. As caracterizações das formulações foram diâmetro médio de gotícula, índice de polidispersão, potencial zeta e pH, assim como o doseamento do ativo foram realizados via técnica de HPLC. Estudos de estabilidade foram realizados num período de 75 dias, em três condições de temperatura (5, 25 e 40 ºC). Melhores resultados foram obtidos quando se utilizou agitação mecânica com 20000 rpm, tempo de agitação de 7,5 minutos e concentração de ativo de 1,00%, produzindo nanoemulsões contendo diâmetro médio de gotícula de 101,17 ± 6,80 nm, índice de polidispersão de 0,135 ± 0,027, potencial zeta de -7,19 ± 0,713 e pH de 4,84 ± 0,04. Esta formulação foi estável por até 75 dias, mantendo os resultados quando armazenada a 5 e 25 °C. Quanto ao doseamento, recuperações de 72 a 106 % do ativo foram obtidas. Assim, conclui-se que a metodologia utilizada é viável para a produção de nanoemulsões contendo Eugenol.

Para minimizar os efeitos danosos a pele causados pelas radiações ultravioleta (UV), novos produtos cosméticos contendo filtros solares tem sido desenvolvidos e estão disponíveis no comércio. Consequentemente é necessário o desenvolvimento e validação de métodos analíticos para determinação quantitativa desses filtros solares em produtos cosméticos. O objetivo desta pesquisa foi a determinação quantitativa e simultânea dos filtros solares químicos, tais como, benzofenona-3, metoxicinamato de octila e salicilato de octila contidos em preparações cosméticas, por cromatografia líquida de alta eficiência (CLAE) e espectrofotometria no UV/visível. Paralelamente foi determinado o FPS para as amostras utilizando a espectrofotometria no uv/visível. A separação e determinação quantitativa pelo método cromatográfico foi realizada usando uma coluna LiChrospher® 100 RP-18 (5µm), fase móvel constituída de metanol-água (85:15, v/v), com vazão de 1,0mL/min e detecção no UV a 310nm. Os coeficientes de correlação e percentuais de recuperação para benzofenona-3, metoxicinamato de octila e salicilato de octila foram de 0,9999 e98,9%; 0,9995 e 99,9%;0,9998 e 98,9%, respectivamente. A média do desvio padrão relativo foi de 0,6%. A determinação espectrofotométrica só foi possível quando os filtros em estudo estão de forma isolada nas preparações cosméticas. O método espectrofotométrico foi validado a 287nm, 310nm e 306nm para benzofenona-3, metoxicinamato de octila e salicilato de octila, respectivamente; os coeficientes de correlação foram de 0,9998; 0,9999; 0,9998, respectivamente. O percentual de recuperação para a amostra comercial contendo apenas metoxicinamato de octila foi de 99,6%. A média do desvio padrão relativo foi de 0,5%. O Fator de Proteção Solar (FPS) determinado pelo método espectrofotométrico, para as preparações comerciais, não correspondeu aos mesmos valores rotulados. / In order to minimize the damaging effects to the skin caused by the ultraviolet (UV) radiation, several cosmetic products containing sunscreens has been developed and are available commercially. Consequently, it is necessary to develop and validate an analytical method for quantitative determination of sunscreen agents in cosmetic products. The aim of this research was to develop a high performance liquid chromatographic and a UV / visible spectrophotometric quantitative method for simultaneous determination of chemical sunscreens benzophenone-3, octyl methoxycinnamate and octyl salicylate contained in cosmetic product. At the same time, the Sun Protection Factor (SPF) was determined for the samples using UV / visible spectrophotometry. The separation and quantitative determination was achieved using a LiChrospher® 100 RP-18 (5µm) column, a mobile phase constituted of methanol-water (85: 15, v/v), a flow-rate of 1.0 mL/min and UV detection at 310nm. The correlation coefficient and percentage of recovery for benzophenone-3, octyl methoxycinnamate and octyl salicylate were 0.9999 and 99.05%; 0.9995 and 100.55%; 0.9998 and 98.05%, respectively. The average relative standard deviation (RSD) was 0.6%. The spectrophotometric determination was possible only when the sunscreens studied were in an isolated form, in cosmetic preparations. The spectrophotometric method was validated at 287nm, 310nm and 306nm for benzophenone-3, octyl methoxycinnamate and octyl salicylate, respectively. The correlation coefficients were 0.9998, 0.9999 and 0.9998, respectively. The percentage of recovery for commercial sample containing only octyl methoxycinnamate was 99.6%. The average relative standard deviation (RSO) was 0.5%. The Sun Protection Factor (SPF) determined by the spectrophotometric method, in the commercial preparations, did not corresponded to the labeled values.

Orientadores: Marco Aurélio Zezzi Arruda, Ljubica Tasic / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-23T02:17:28Z (GMT). No. of bitstreams: 1 Mataveli_LidianeRaquelVerola_D.pdf: 1935019 bytes, checksum: 059066ee53b01850e2858460ba23617e (MD5) Previous issue date: 2013 / Resumo: Este trabalho consistiu em estudos de metalômica comparativa de sementes de soja transgênica (T) e não-transgênica (NT). Para tanto, as sementes de soja T e NT foram comparadas, em um primeiro, momento levando-se em conta: concentração total dos elementos, comportamento dos elementos durante extração utilizando-se fracionamento sequencial, e bioacessibilidade dos elementos após procedimento de digestão gastrointestinal simulada (in vitro). As análises preliminares foram feitas utilizando-se ICP-MS com analisador de massas quadrupolar, e as análises posteriores utilizando espectrometria de massas de alta resolução com plasma acoplado indutivamente (HR-ICP-MS). Foram determinados 25 elementos em concentrações variando de ng g até %. Foi observado que as sementes de soja T e NT exibem diferenças estatisticamente significantes nas concentrações de Cu, Fe e Sr, sendo os dois primeiros apresentando maiores concentrações na semente T, e, o último, com maior concentração nas sementes de soja NT. Estes resultados também se refletiram nos conteúdos desses elementos em extratos aquosos e resíduos obtidos por meio de fracionamento sequencial. Ainda, os experimentos de bioacessibilidade realizados mostraram que as frações bioacessíveis de Cu, Fe, e outros elementos (Mn, S, Zn) contribuíram em maior porcentagem para a concentração total dos elementos nas sementes de soja T do que para as sementes de soja NT. Posteriormente, foi dada continuidade aos estudos utilizando cromatografia líquida bidimensional off line para as amostras de sementes de soja, sendo a primeira dimensão constituída de cromatografia de exclusão por tamanho (SEC,) e a segunda dimensão constituída de cromatografia de troca aniônica (AEX). Na primeira dimensão cromatográfica foram identificadas três frações contendo metais por meio da hifenação SEC-ICP-MS: a primeira correspondendo a massas molares entre 38,1 e 181,1 kDa, a segunda correspondendo a massas molares entre 8,2 e 17,2 kDa, e a terceira fração correspondendo a massas molares entre 0,4 e 3,8 kDa. As três frações identificadas foram separadas, liofilizadas, e separadas novamente utilizando a cromatografia de troca aniônica. Foram detectados metais em todas as frações separadas por SEC: três sub-frações da primeira fração, uma sub-fração na segunda fração e três sub-frações na terceira fração. Os eluatos foram coletados, liofilizados, digeridos e levados ao espectrômetro de massas com fonte de ionização por electrospray (ESI-MS) para identificação de proteínas. Foram identificadas 33 e, entre elas, duas proteinas previamente relacionadas a metais foram encontradas: seed lipoxygenase 1 e b-conglycinin. Após a separação na segunda dimensão cromatográfica, as sub-frações resultantes foram liofilizadas e submetidas a uma terceira dimensao de separacao, utilizando a eletroforese em gel de poliacrilamida unidimensional (SDS-PAGE). As bandas obtidas por SDS-PAGE foram recortadas e digeridas a fim de analisar os metais presentes nas mesmas, destacando os resultados obtidos para Fe, onde o mesmo foi quantificado nas bandas da sub-fração onde o pico deste elemento foi encontrado nas análises por AEX-ICP-MS. Ainda, as bandas foram digeridas tripticamente a fim de identificar as proteínas presentes, e, novamente, proteínas associadas a metais foram identificadas: chain A lipoxygenase-3 (Soybean) complex with 13(S)-hydroperoxy- 9(Z),11(E)-octadecadienoic acid; beta-amylase [Glycine max]; seed lipoxygenase-1, lipoxygenase [Glycine max], seed lipoxygenase-2 (PISUM SATIVUM) e beta-conglycinin / Abstract: This work consisted of comparative metallomics studies of transgenic (T) and nontransgenic (NT) soybean seeds. To that end, T and NT seeds were compared at first taking into account: the total concentration of the elements, the elements behavior during extraction using sequential fractionation, and bioacessibility of the elements after simulated gastrointestinal digestion procedure (in vitro). Preliminary analyzes were done using ICP-MS with quadrupole mass analyzer, and the subsequent analysis using high-resolution inductively coupled plasma mass spectrometry, (HR-ICP-MS). 25 elements were determined at concentrations ranging from 1 ng g up to %. It was observed that T and NT soybean seeds exhibit statistically significant differences in the concentrations of Cu, Fe and Sr, the first two having higher concentrations in the T seeds, and the last with the highest concentration in soybeans NT. These results were also reflected in the contents of these elements in aqueous extracts and residues obtained through sequential fractionation. Also, the contributions of bioaccessible fractions of Cu, Fe and other elements (Mn, S, Zn) to the total content of the elements in T soybean seeds were higher than those found in NT soybean seeds. Subsequently, studies were continued using bidimensional liquid chromatography, the first dimension consisting of size exclusion chromatography (SEC) and the second dimension of anion exchange chromatography (AEX). In the first chromatographic dimension three fractions containing metals were identified using hyphenation SEC-ICP-MS, the first corresponding to molar masses between 38.1 and 181.1 kDa, the second corresponding to molar masses between 8.2 and 17.2 kDa and the third fraction corresponding to molar masses between 0.4 and 3.8 kDa. The three identified fractions were separated and lyophilized, and again separated using anion exchange chromatography (AEX). Metals were found in all the separated fractions by SEC: three sub-fractions of the first fraction, a subfraction in the second fraction and three sub-fractions in the third fraction. These peaks were collected, lyophilized, digested and taken to the mass spectrometer for protein identification. 33 proteins were identified, and, among them, two proteins previously related to metals were found: seed lipoxygenase 1 and b-conglycinin. After chromatographic separation in the second dimension, the resultant subfractions were lyophilized and subjected to a third separation dimension using onedimensional polyacrylamide gel electrophoresis (SDS-PAGE). After the separation, the bands were cut out and digested to examine the metals contained therein, highlighting the results obtained for Fe, which was quantified in the bands of the sub-fraction where the peak of the element is found in the analysis by ICP-AEX - MS. Also, the bands were digested triptically to identify the proteins, and once again proteins associated to metals were identified: 3-lipoxygenase A chain (Soybean) complex with 13 (S)-Hydroperoxy-9 (Z), 11 ( E)-octadecadienoic acid, beta-amylase [Glycine max]; seed lipoxygenase-1, lipoxygenase [Glycine max] seed lipoxygenase-2 (Pisum sativum) and beta-conglycinin / Doutorado / Quimica Analitica / Doutora em Ciências

O projeto teve por finalidade isolar e identificar fungos, avaliar o potencial toxigênico dos isolados de Aspergillus spp. e Fusarium spp., detectar e quantificar aflatoxinas B1, B2, G1, G2 e fumonisinas B1 e B2 em amostras de misturas de cereais. Foram analisadas 15 marcas de misturas de cereais, prontas para o consumo, adquiridas de supermercados e de empresas que comercializam o produto nacionalmente via internet. Foram adquiridas amostras por sete meses, totalizando 105 amostras ao final do experimento. A contagem de bolores nas amostras variou de 1,0 x 101 a 2 x 105 unidades formadoras de colônias (UFC)/g, com isolamento de sete cepas de Aspergillus flavus. As aflatoxinas B1 e G1 foram detectadas em poucas amostras e em baixos níveis, sendo que estes resultados podem ser devidos à baixa atividade de água nos produtos avaliados, a qual foi inferior a 0,63. A fumonisina B1 foi detectada em 84,8% das amostras, no entanto, a ingestão diária provável calculada para as fumonisinas esteve abaixo da recomendação do JECFA. Apenas uma amostra apresentou níveis de fumonisinas acima do limite esperado para 2016. Adicionalmente, foi observado que 21% das amostras apresentaram mais de um tipo de micotoxina, o que poderia conduzir à potencialização de efeitos tóxicos. / The project aimed to isolate and identify fungi, evaluate the toxigenic potential of isolates of Aspergillus spp. and Fusarium spp. detect and quantify aflatoxins B1, B2, G1, G2 and fumonisins B1 and B2 in samples of cereal mixtures. We analyzed 15 brands of cereal mixtures ready to eat adding up to 105 samples at the end of the experiment. Samples were acquired in supermarkets and from companies that market the product nationally by internet. The mold count in the samples ranged from 1.0 x 101 to 2 × 105 colonies forming units (CFU)/ g, with isolation of seven strains of Aspergillus flavus. Aflatoxin B1 and G1 were detected in a few samples and at low levels, what might be due to the low water activity in the product reviews, which was less than 0.63. Fumonisin B1 was detected in 84.8% of the samples, however, the probable daily intake calculated for fumonisin was bellow the JECFA recommendation. Only one sample showed fumonisin levels above the expected limit for 2016. Additionally, it was observed that 21% of the samples presented more than one type of mycotoxin, which could lead to enhancement of toxic effects.

O objetivo deste trabalho foi estudar a transferência de aflatoxinas da ração para peixes lambari (Astyanax altiparanae), avaliando a influência dos níveis de toxina na ração sobre o desempenho dos peixes. As aflatoxinas foram incorporadas à ração extrusada para peixes tropicais, previamente testada para a presença de aflatoxinas, e as concentrações foram confirmadas por cromatografia líquida de alta eficiência (CLAE). Os tratamentos foram constituídos por: Controle - ração sem toxina; A. Ração + 10 µg AFB1/kg; B. Ração + 20 µg AFB1/kg e C. Ração + 50 µg AFB1/kg. Os juvenis de lambari foram alocados em aquários com densidade de 1 peixe por litro. O experimento foi realizado por 120 dias, com três repetições. A cada 30 dias foram realizados levantamentos biométricos e avaliação do desempenho (taxa de crescimento, sobrevivência, ganho de peso). A determinação de aflatoxinas nas amostras foi realizada por CLAE em músculo e fígado. Mensalmente, dez peixes por aquário foram utilizados para compor uma amostra, totalizando 48 amostras de músculo e 48 amostras de fígado analisadas ao final do experimento. As aflatoxinas na dieta prejudicaram o desempenho dos lambaris, observando-se menor peso e tamanho dos peixes nos tratamentos com aflatoxinas em relação ao controle. Houve também menor consumo de ração pelos peixes do tratamento C no período final do experimento. De modo geral, observou-se que, principalmente a partir dos 90 dias de experimento, houve acúmulo de aflatoxinas no fígado e no músculo dos animais. A concentração de aflatoxina B1 observada no músculo dos lambaris ao final do período de exposição foi semelhante aos níveis empregados na dieta. Os resultados mostraram que o lambari é uma espécie sensível, havendo efeito das aflatoxinas sobre o desempenho e acúmulo de aflatoxinas no músculo e fígado. Assim, quando da exposição diária e prolongada de lambaris às aflatoxinas, conclui-se que os limites atuais das legislações para aflatoxinas em ração animal não garantem segurança à produção e à saúde dos consumidores. / The aim of this study was to evaluate the transfer of aflatoxins from feed to lambari fish (Astyanax altiparanae), assessing the influence of toxin levels on fish performance. Aflatoxins were incorporated into extruded feed for tropical fish, previously tested for aflatoxins, and the concentrations were confirmed by high-performance liquid chromatography (HPLC). The treatments were: Control - feed without toxin; A. Feed + 10 µg AFB1/kg; B. Feed + 20 µg AFB1/kg and C. Feed + 50 µg AFB1/kg. Juveniles of lambari fish were placed in aquariums with density of 1 fish per liter. The experiment was conducted for 120 days, with three replications. Every 30 days biometric surveys were performed, including weight and standard length. Feed intake, feed conversion, growth rate and survival were also evaluated. Determination of aflatoxins was carried out by HPLC in muscle and liver. Monthly, a pool of 10 fish were used to make a sample, adding up to 48 samples of muscle and 48 samples of liver analyzed at the end of the experiment. Aflatoxins in the diet impaired the lambari performance, with lower weight and size of fish from aflatoxins treatments compared to control. There was also lower feed intake by fish from treatment C in the final period of the experiment. In general, it was observed that, mainly from 90 days of experiment, there was accumulation of aflatoxins in the liver of lambari fish and consequently in the muscle. Aflatoxin B1 concentration observed in the muscle of lambari fish at the end of the exposure period was similar to the levels used in the diet. Results showed that lambari fish is sensitive specie, with effects of aflatoxins on performance and accumulation of aflatoxins in muscle and liver. Therefore, when daily and prolonged exposure of lambaris to aflatoxins, it is concluded that the current regulation limits for aflatoxins in animal feed do not guarantee safety production and consumer health.

Atualmente, a demanda e a necessidade de metodologias analíticas e bioanalíticas que promovam análise rápida e seletiva tem impulsionado o constante desenvolvimento de sorventes para preparo de amostra e fases estacionárias para cromatografia líquida de alta eficiência (HPLC). Desta maneira, esta tese tem por objetivo desenvolver materiais utilizando precursores de sílica com aplicação em preparo de amostra e suportes cromatográficos para cromatografia líquida de ultra alta eficiência (UHPLC). Foram sintetizadas e caracterizadas duas fases extratoras as quais tiveram sua aplicabilidade demonstrada na extração de fármacos em fluidos biológicos empregando a técnica de extração em fase sólida (SPE). A primeira metodologia desenvolvida foi a síntese de partículas de sílica empregando precursores de baixo custo (silicato de sódio) pelo método sil-gel. Após a funcionalização com grupamento C18, e caracterização da fase extratora, o material sintetizado foi aplicado em SPE off-line para a determinação de fluoxetina e seu metabólito, norfluoxetina, em plasma humano por HPLC-UV. O método desenvolvido foi validado e aplicado em amostra de plasma de pacientes sob tratamento de fluoxetina. Além disso, o sorvente desenvolvido apresentou resultados similares quando comparado com fases extratoras comerciais. O segundo método desenvolvido utilizou a técnica de impressão molecular (MIP) a qual tem se tornado uma fase extratora atrativa devido a sua maior seletividade em relação as fases típicas empregadas em SPE. Ao mesmo tempo, o uso de SPE online com MIP (MISPE) é uma alternativa atraente no preparo de amostra, pois é simples, diminui o tempo total da análise, necessita de pequena quantidade de amostra, e pode ser automatizado. Assim, MIP amino-funcionalizado pelo processo sol-gel utilizando ibuprofeno como template foi sintetizado. Para comparação, o polímero não impresso (NIP) foi preparado nas mesmas condições, sem a presença do template. As análises por cromatografia líquida foram realizadas utilizando uma coluna de extração MIP e uma coluna analítica em fase reversa configuradas para operar no modo SPE online por column switching. O método desenvolvido, MISPE-HPLC-UV, foi validado e aplicado na extração seletiva de cinco anti-inflamatórios não esteroidais (NSAIDs) em urina humana com um tempo total de análise de 22 minutos. Além dos materiais voltados para preparo de amostra, foi avaliado a síntese e caracterização de suporte cromatográfico baseado em sílica com diâmetro de partícula abaixo de 2 µm com aplicabilidade em análises rápidas em cromatografia (UHPLC). Nesta vertente, partículas de sílica esférica não porosa (0,9 µm) foram sintetizadas pelo método sol-gel com DPR abaixo de 10 %. As partículas foram funcionalizadas com o grupamento C18 (ODS) e submetidas posteriormente ao processo de endcapping (TMS) para operar em modo reverso. Métodos físico-químicos de caracterização foram utilizados para determinar a morfologia, área superficial e quantidade de carbono na superfície do material. Adicionalmente, as técnicas de infravermelho e ressonância magnética nuclear forneceram informações sobre a estrutura da sílica e das ligações após a funcionalização. A fase estacionária foi empacotada em uma coluna (40 mm x 0,25 mm) e a sua aplicabilidade foi avaliada em cromatografia líquida capilar (cLC). Apesar de não alcançar a eficiência ótima da separação cromatográfica devido a limitação instrumental, a fase estacionária sub 1 µm apresenta potencial para separações rápidas sem perda de eficiência. / Currently, the demand and need for bioanalytical and analytical methodologies that promotes rapid and selective analysis has stimulated the development of sorbents for sample preparation and stationary phases for high performance liquid chromatography (HPLC). Therefore, this thesis aims to develop materials using silica precursors with application in sample preparation and chromatographic supports for ultra high performance liquid chromatography (UHPLC). Thus, in this study two sorbents were synthesized and characterized and their applicability was demonstrated in the extraction of drugs in biological fluids employing the solid phase extraction technique (SPE). The first method developed was the synthesis of silica particles employing low cost precursors (sodium silicate) using the sil-gel method. After functionalization with C18 group and characterization of the extraction phase, the synthesized material was applied to SPE off-line for the determination of fluoxetine and its metabolite, norfluoxetine, in human plasma by HPLC-UV. The method was validated and applied to plasma samples from patients treated with fluoxetine. Furthermore, the labmade sorbent presented similar results when compared with commercial ones. The second method developed used the molecular imprinting technique (MIP) that has become an attractive sorbent due to its greater selectivity over the typical phases employed in SPE. At the same time, the use of online SPE with MIP (MISPE) is an attractive alternative in sample preparation because it is simple, reduces the total time of analysis, needs little amount of sample and can be automated. Thus, MIP amino-functionalized by the sol-gel method using ibuprofen as template was synthesized. For comparison, the non-imprinted polymer (NIP) was prepared under the same conditions without the presence of template. LC analysis was performed using a MIP extraction column and a reverse phase analytical column in order to perform column switching on-line sample separation. The developed method, MISPE-HPLC-UV, was validated and applied to the selective extraction of five anti-inflammatory drugs (NSAIDs) in human urine with a total analysis time of 22 minutes. Moreover it was evaluated the synthesis and characterization of chromatographic support based on silica with particle diameter under 2 µm with applicability in fast LC analysis (UHPLC). In this instance, spherical particles of non-porous silica (0.9 µm) were synthesized by sol-gel method with RSD below 10 %. The particles were functionalized with the C18 group and submitted to the endcapping process with TMS to operate in reverse phase mode. Physico-chemical methods were used to determine the morphology, surface area and amount of carbon in the silica surface. Additionally, the infrared and nuclear magnetic resonance techniques provided information about the structure of silica and bonds after the functionalization step. The stationary phase was packed into a column (40 mm x 0.25 mm), and its applicability was evaluated in capillary liquid chromatography (cLC). Although optimum chromatographic efficiency was not achieved separation due to instrumental limitations, the sub 1 µm stationary phase has potential for rapid separations without loss of efficiency.

In the past two decades, the identification of the structures involved in the mechanisms of cancer development have led to a paradigm shift in cancer therapy. Since 2001 orally administered small molecules such as kinase inhibitors are fundamental drugs in the personalized and targeted therapy of hematologic and oncologic neoplasms. However, side effects, therapeutic success is often accompanied by relevant restrictions, which may result in disease progression, a low quality of life, or treatment discontinuation. In addition, for some kinase inhibitors, therapy failure during treatment after an initially good response has been reported. It is known that the response to drug therapy varies considerably in patients. The same dosage may be effective in some patients while ineffective or toxic in others. Interindividual variabilities in drug exposure, and consequently sub- or supratherapeutic serum concentrations may be the cause. Factors such as drug-drug- or food-drug-interactions with drug-metabolizing enzymes and lifestyle (e.g., smoking, weight) are known to cause interindividual differences. In general, oral administration is predetermined to cause large interindividual variability in drug exposure because absorption in the gastrointestinal tract depends on food intake and the intragastric pH. For the recently approved kinase inhibitors afatinib, axitinib, bosutinib, cabozantinib, dabrafenib, lenvatinib, nilotinib, osimertinib, ruxolitinib, and trametinib, interindividual variability despite the same dosage has been described. So far, knowledge on the serum exposure and the therapeutically relevant serum concentrations from clinical routine is scarce. It is unclear whether side effects correlate with achieved serum concentrations and if therapy failure correlates with subtherapeutic serum concentrations. Innovation and paradigm shift are not only observed in cancer therapy but also in the handling of complications following cancer therapy. In patients undergoing allogeneic stem cell transplantation (allo-HSCT), cytomegalovirus (CMV) reactivation and disease are associated with increased transplant related mortality. Letermovir is a novel anti-CMV drug, which is used for prophylaxis in adult CMV-seropositive allo-HSCT recipients. Letermovir represents a scientific breakthrough, as the side effect profile compares favorably with the known highly toxic anti-CMV agents. However, letermovir is the victim and perpetrator in many clinically relevant drug-drug-interactions. As known from other anti-viral agents, inter- or intraindividual variability in systemic exposure may lead to therapy failure or the development of drug resistance. In vitro and clinical observations suggest that letermovir has a low genetic barrier to resistance. So far, knowledge on the serum exposure and the therapeutically relevant serum concentrations of letermovir from clinical routine is scarce. The objective of this work was to develop bioanalytical methods which are then used for the quantification of the systemic exposure of the ten kinase inhibitors and the anti-CMV agent letermovir in clinical routine. The aim was to gain first insights into the achieved serum concentration and the extent of interindividual variability in treated patient in a real-life setting. First, a high-performance-liquid chromatography (HPLC) method coupled with tandem-mass spectrometry (LC-MS/MS) for the quantification of the ten kinase inhibitors was developed and validated according to the relevant U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) guidelines. Expected cabozantinib, dabrafenib, nilotinib and osimertinib serum concentrations were quantifiable using the less sensitive but widely available diode array detector (DAD). Therefore, a cost-efficient alternative using HPLC coupled with DAD was developed and validated accordingly. The HPLC-DAD method was cross-validated using the gold standard LC-MS/MS and patient samples. For all four kinase inhibitors over 67% of patient samples per compound met FDA and EMA limits for reanalysis and EMA limits for cross-validation. The results indicate that the measured concentrations are comparable when using two different detectors. The developed LC-MS/MS method was applied to measure patient serum samples. Patients receiving the commonly used kinase inhibitors cabozantinib, dabrafenib, nilotinib, osimertinib, ruxolitinib and trametinib showed a high interindividual variability in serum concentrations for the same kinase inhibitor at the same dosage. The number of patients receiving afatinib (one) and osimertinib (two) was not sufficient to draw definite conclusions. Within this work, more detailed investigations were conducted in melanoma patients treated with dabrafenib and trametinib and in patients who received ruxolitinib after allo-HSCT for the treatment of Graft-versus-Host-Disease (GvHD). In melanoma patients, the median of the individual mean trough levels was 45.0 ng/mL (range: 20-155 ng/mL, dosage: 150 mg twice daily). For trametinib, the median of the mean trough levels was 11.2 ng/mL (range: 6.22-15.9 ng/mL, dosage: 2 mg once daily). Dose adjustments because of undesirable side effects in clinical routine were common. The observed data do not support a general monitoring of dabrafenib and trametinib in clinical routine. However, they provide the evidence for associations between toxicity and exposure, supporting the existing literature. Therefore, measuring exposure may assist decision-making regarding dose adjustments in clinical routine. In GvHD patients, a high degree of inter individual variability in median serum trough levels was observed: 39.9 ng/mL (range: 5.6-99.8 ng/mL, dosage: 10 mg twice daily). Patients not requiring dose reduction due to side effects had a significantly higher median of individual mean trough serum concentrations at the same daily dose (39.8 ng/mL versus 60.6 ng/ml). The study showed that a serum concentration of 21.1 ng/mL before next dose intake was associated with experiencing at least three side effects which should be evaluated in future studies. The study in GvHD patients showed that ruxolitinib, a predominantly Cytochrome P450 (CYP) 3A4 substrate, is commonly co-administered with posaconazole, a CYP3A4 inhibitor. Depending on the regulatory agency, the recommendations regarding dose adjustment were different (no (FDA) vs. 50% (EMA) dose adjustment). The magnitude of drug-drug-interaction in our clinical situation was investigated by developing a physiologically-based pharmacokinetic model using PK-Sim. The model predicted an increase in ruxolitinib maximum plasma concentration and area under the concentration-time curve by 20.5% and 59%, respectively, due to the concomitant posaconazole administration. The observed data do not support a general dose reduction by 50%, as suggested by the EMA. However, given the observed high degree of variability in ruxolitinib exposure some patients may be at risk for ruxolitinib overexposure because of the described interaction. Therefore, standard dosing should be applied with caution and ruxolitinib exposure should be monitored to identify patients at risk for overexposure and associated toxicity. For the quantification of letermovir serum concentrations a HPLC-DAD method was developed, validated, and applied to patient samples. A high degree of interindividual variability was observed. The analysis of trough concentrations revealed a median letermovir concentration of 2,603 ng/mL ( range: 175–12,281 ng/mL). The data represent first findings on letermovir exposure in routine clinical practice and remain to be placed in a clinical context. The developed methods and the results of this work provide the basis for future clinical investigations regarding dose individualization for the investigated kinase inhibitors and letermovir. Given the observed variability between patients at the same dosage, the methods are helpful to identify patients at risk for over- or underdosing at an early stage during therapy. Measuring drug exposure and establishing safe and effective therapeutic ranges may assist decision-making regarding dose adjustments in clinical routine. / In den letzten zwei Jahrzehnten hat die Identifizierung von Strukturen, die an den Mechanismen der Krebsentstehung beteiligt sind, zu einem Paradigmenwechsel in der Krebstherapie geführt. Seit 2001 sind oral verabreichte „small molecules“ wie Kinase-Inhibitoren grundlegende Bestandteile der personalisierten und gezielten Therapie von hämatologischen und onkologischen Neoplasien. Aufgrund ihrer Nebenwirkungen ist der therapeutische Erfolg jedoch oft mit erheblichen Einschränkungen verbunden, die unter anderem zum Fortschreiten der Krankheit, zu einer geringen Lebensqualität oder zum Abbruch der Behandlung führen können. Darüber hinaus wird bei einigen Kinase-Inhibitoren über Therapieversagen während der Behandlung nach anfänglich gutem Ansprechen berichtet. Es ist bekannt, dass das Ansprechen auf eine Arzneimitteltherapie bei Patienten sehr unterschiedlich ausfällt. Die gleiche Dosierung kann bei einigen Patienten wirksam, bei anderen dagegen unwirksam oder toxisch sein. Interindividuelle Schwankungen in der Serum- oder Plasmakonzentration und folglich sub- oder supratherapeutischen Serumkonzentrationen können die Ursache sein. Faktoren wie Interaktionen zwischen Enzymen des Metabolismus und Arzneimitteln oder Nahrungsmitteln, und der Lebensstil (beispielsweise Raucherstatus und Gewicht) können zu interindividuellen Unterschieden in der Arzneimittelexposition führen. Im Allgemeinen ist bei oraler Verabreichung eine große interindividuelle Variabilität der Arzneimittelexposition zu erwarten, da die Absorption im Gastrointestinaltrakt von der Nahrungsaufnahme und dem pH-Wert im Magen abhängt. Für die kürzlich zugelassenen Kinase-Inhibitoren Afatinib, Axitinib, Bosutinib, Cabozantinib, Dabrafenib, Lenvatinib, Nilotinib, Osimertinib, Ruxolitinib und Trametinib wurde eine Variabilität der Serumspiegel zwischen Patienten bei gleicher Dosierung beschrieben. Bislang gibt es nur wenige Erkenntnisse über die Serumexposition und die therapeutisch relevanten Serumkonzentrationen aus der klinischen Routine. Es ist unklar, ob Nebenwirkungen mit den erreichten Serumkonzentrationen korrelieren, und ob ein Therapieversagen mit subtherapeutischen Serumkonzentrationen zusammenhängt. Innovation und Paradigmenwechsel sind nicht nur in der Krebstherapie zu beobachten, sondern auch bei der Behandlung von Komplikationen nach einer Krebstherapie. Bei Patienten, die sich einer allogenen Stammzelltransplantation (allo-HSCT) unterziehen, sind die Reaktivierung des Cytomegalievirus (CMV) und die Erkrankung mit einer erhöhten transplantationsbedingten Sterblichkeit verbunden. Letermovir ist ein neues Anti-CMV-Medikament, das zur Prophylaxe bei erwachsenen CMV-seropositiven allo-HSCT-Empfängern eingesetzt wird. Letermovir stellt einen wissenschaftlichen Durchbruch dar, da das Nebenwirkungsprofil im Vergleich zu den bekannten hochtoxischen Anti-CMV-Wirkstoffen günstig ist. Letermovir ist jedoch Opfer und Verursacher vieler klinisch relevanten Arzneimittelwechselwirkungen. Wie von anderen antiviralen Arzneimitteln bekannt, können inter- oder intraindividuelle Schwankungen der systemischen Exposition zu Therapieversagen oder der Entwicklung von Arzneimittelresistenzen führen. In-vitro- und klinische Beobachtungen deuten darauf hin, dass Letermovir eine niedrige genetische Barriere für Resistenzen aufweist. Bislang gibt es jedoch nur wenige Daten zur Serumexposition und die therapeutisch relevanten Serumkonzentrationen von Letermovir aus der klinischen Routine. Im Rahmen dieser Arbeit wurden bioanalytische Methoden entwickelt, die dann zur Quantifizierung der systemischen Exposition der zehn genannten Kinase-Inhibitoren und des Anti-CMV-Wirkstoffes Letermovir in der klinischen Routine eingesetzt wurden. Ziel war es, erste Erkenntnisse über die erreichten Serumkonzentrationen und das Ausmaß der interindividuellen Variabilität bei behandelten Patienten in einer realen Umgebung zu gewinnen. Zunächst wurde eine Hochleistungs-Flüssigchromatographie-Methode (HPLC) gekoppelt mit einem Tandem-Massenspektrometer (LC-MS/MS) zur Quantifizierung der zehn Kinase-Inhibitoren entwickelt und gemäß den einschlägigen Richtlinien der US-amerikanischen Food and Drug Administration (FDA) und der Europäischen Arzneimittelagentur (EMA) validiert. Die erwarteten Cabozantinib-, Dabrafenib-, Nilotinib- und Osimertinib-Serumkonzentrationen waren mit dem weniger empfindlichen, aber weit verbreiteten Diodenarray-Detektor (DAD) quantifizierbar. Daher wurde eine kosteneffiziente, alternative Methode unter Verwendung von HPLC gekoppelt mit DAD entwickelt und entsprechend validiert. Die HPLC-DAD-Methode wurde mit dem Goldstandard LC-MS/MS und Patientenproben kreuzvalidiert. Bei allen vier Kinase-Inhibitoren erfüllten pro Substanz mehr als 67 % der Patientenproben die FDA- und EMA-Grenzwerte für die Reanalyse und die EMA-Grenzwerte für die Kreuzvalidierung. Die Ergebnisse zeigen, dass die gemessenen Konzentrationen mithilfe der unterschiedlichen Detektoren vergleichbar sind. Mit der LC-MS/MS-Methode wurden Serumproben von Patienten gemessen. Patienten, die mit den häufig verwendeten Kinase-Inhibitoren Cabozantinib, Dabrafenib, Nilotinib, Osimertinib, Ruxolitinib und Trametinib behandelt wurden, zeigten bei gleicher Dosierung eine hohe interindividuelle Variabilität der Serumkonzentrationen. Die Anzahl der Patienten, die Afatinib (einer) und Osimertinib (zwei) erhielten, reichte nicht aus, um eindeutige Schlussfolgerungen zu ziehen. Im Rahmen der Arbeit wurden detailliertere Untersuchungen bei Patienten mit Melanom, die mit Dabrafenib und Trametinib behandelt wurden, sowie bei Patienten, die Ruxolitinib nach einer allo-HSCT im Rahmen Behandlung der Graft-versus-Host-Disease (GvHD) erhielten, durchgeführt. Bei Melanompatienten lag der Median der individuellen mittleren Talspiegel für Dabrafenib bei 45,0 ng/mL (Spannweite: 20-155 ng/mL, Dosierung: 150 mg zweimal täglich). Für Trametinib lag der Median der mittleren Talspiegel bei 11,2 ng/mL (Spannweite: 6,22-15,9 ng/mL, Dosierung: 2 mg einmal täglich). Dosisanpassungen aufgrund von unerwünschten Nebenwirkungen in der klinischen Routine waren häufig. Die beobachteten Daten sprechen nicht für eine generelle Überwachung der Dabrafenib- und Trametinib-Spiegel in der klinischen Routine. Sie weisen jedoch darauf hin, dass es zwischen Toxizität und Exposition einen Zusammenhang gibt, und stützen damit die bestehende Literatur. Daher kann die Messung der Exposition bei Entscheidungen in Bezug auf die Dosisanpassung hilfreich sein. Bei GvHD-Patienten wurde ein hohes Maß an interindividueller Variabilität der mittleren Serum-Talspiegel beobachtet: 39,9 ng/mL (Spannweite: 5,6-99,8 ng/mL, Dosierung: zweimal täglich 10 mg). Bei Patienten, die keine Dosisreduzierung aufgrund von Nebenwirkungen benötigten, war der Median der individuellen mittleren Serum-Talspiegel bei gleicher Tagesdosis signifikant höher (39,8 ng/mL versus 60,6 ng/mL). Eine Serumkonzentration von 21,1 ng/mL vor der nächsten Dosiseinnahme konnte mit dem Auftreten von mindestens drei Nebenwirkungen jeglichen Grades assoziiert werden. Dieser Wert sollte in zukünftigen Studien evaluiert werden. Die Studie an GvHD-Patienten zeigte, dass Ruxolitinib, ein vorwiegend über Cytochrome P450 (CYP) 3A4 verstoffwechseltes Substrat, häufig zusammen mit Posaconazol, einem CYP3A4-Inhibitor, verabreicht wird. Je nach Zulassungsbehörde waren die Empfehlungen zur Dosisanpassung unterschiedlich (keine laut FDA bzw. 50 % Dosisanpassung laut EMA). Das Ausmaß der Arzneimittelwechselwirkung, in der im Rahmen dieser Arbeit durchgeführten klinischen Studie, wurde durch die Entwicklung eines physiologisch basierten pharmakokinetischen Modells mit PK-Sim untersucht. Mithilfe des Modells wurde ein Anstieg der maximalen Plasmakonzentration von Ruxolitinib und der Fläche unter der Konzentrations-Zeit-Kurve um 20,5 % bzw. 59 % aufgrund der gleichzeitigen Verabreichung von Posaconazol vorausgesagt. Die Daten sprechen nicht für eine allgemeine Dosisreduktion um 50 %, wie von der EMA vorgeschlagen. Angesichts der beobachteten großen Variabilität der Ruxolitinib-Exposition besteht jedoch bei einigen Patienten, aufgrund der beschriebenen Wechselwirkung, möglicherweise ein Risiko für eine Ruxolitinib-Überdosierung. Daher sollte die Standarddosierung mit Vorsicht angewendet und die Ruxolitinib-Exposition überwacht werden, um Patienten zu identifizieren, bei denen ein Risiko für eine Überexposition und eine damit verbundene Toxizität besteht. Zur Quantifizierung der Letermovir-Serumkonzentrationen wurde eine HPLC-DAD-Methode entwickelt, entsprechend den Richtlinien der EMA und FDA validiert und auf Patientenproben angewendet. Es wurde ein hohes Maß an interindividueller Variabilität beobachtet. Die Analyse des Talspiegel ergab eine mediane Letermovir-Konzentration von 2.603 ng/mL (Spannweite: 175-12.281 ng/mL). Die Daten stellen erste Erkenntnisse zur Letermovir-Exposition in der klinischen Routine dar und bleiben in den klinischen Kontext einzuordnen. Die entwickelten Methoden und die Ergebnisse dieser Arbeit bilden die Grundlage für zukünftige klinische Untersuchungen zur Dosisindividualisierung für die untersuchten Kinase-Inhibitoren und Letermovir. Angesichts der beobachteten Variabilität zwischen den Patienten bei gleicher Dosierung, sind die hier beschriebenen Methoden hilfreich, um Patienten mit dem Risiko für eine Über- oder Unterdosierung frühzeitig zu erkennen. Die Messung der Serumkonzentration und die Festlegung sicherer und wirksamer therapeutischer Bereiche können die Entscheidungsfindung in Bezug auf die Dosisanpassung in der klinischen Routine unterstützen.

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