Foram desenvolvidas metodologias de cromatografia a líquido de fase reversa baseadas em injeção sequencial (SIC) e em cromatografia a líquido de alta eficiência (HPLC) para determinação de triclosan em amostras de produtos de higiene pessoal e em estudos de adsorção em argilominerais naturais e modificados, visando determinar parâmetros de adsorção de triclosan frente a alguns de seus metabólitos. A determinação de triclosan em enxaguadores bucais foi realizada por SIC com eluição isocrática usando fase móvel constituída por acetonitrila: tampão fosfato de trietilamina 70 mM pH 3,5 na proporção 70:30 (v v-1), obtendo-se limites de detecção e de quantificação de 0,22 e 0,72 mg L-1, respectivamente. Taxas de recuperação entre 96 e 98% foram obtidas da aplicação a amostras reais, sendo que os resultados obtidos pelo método proposto não apresentaram evidências de diferenças estatisticamente significativas em comparação a uma metodologia de referência baseada em HPLC com coluna empacotada. A separação de triclosan (TCS), 2-clorofenol (2-CP), 2,4-diclorofenol (2,4-DCP), 2,4,6-triclorofenol (2,4,6-TCP), 2,3,4-triclorofenol (2,3,4-TCP) e metiltriclosan (MTCS) foi estudada por SIC, obtendo-se a separação de TCS, 2-CP, 2,4-DCP e 2,4,6-TCP com duas etapas de eluição isocrática, a primeira delas com fase móvel 60:40 (v v-1) metanol: tampão acetato de amônio 20 mM (pH 5,5) seguida de eluição com fase móvel 70:30 (v v-1) metanol : tampão acetato de amônio 20 mM (pH 5,5). Nesse caso, os isômeros 2,4,6-TCP e 2,3,4-TCP coeluem. Metiltriclosan, o menos polar desses compostos, pode ser separado de TCS com etapas subseqüentes de eluição. Os métodos foram aplicados para estudar a adsorção de triclosan e seus metabólitos 2,4-DCP, 2,4,6-TCP e metiltriclosan em montmorilonita homoiônica (K+) e modificada com sal de hexadeciltrimetilamônio (HDTMA), observando-se forte adsorção de triclosan e metiltriclosan em comparação a 2-CP, 2,4-DCP e 2,4,6-TCP. A incorporação de HDTMA no argilomineral causou significativo aumento da capacidade de adsorção desses metabólitos, determinada a partir do ajuste dos dados experimentais à equação linearizada de Langmuir, observando-se que a ordem de adsorção é 2,4,6-TCP > 2,4-DCP > 2-CP / Reversed-phase liquid chromatography methodologies based on sequential injection (SIC) and high performance liquid chromatography (HPLC) have been developed for determination of triclosan in samples of personal hygiene products and in studies of adsorption on natural and modified clay minerals aiming to determine kinetic and thermodynamic parameters of adsorption of triclosan in comparison with some of its metabolites. The determination of triclosan in oral rinses with SIC was performed by isocratic elution using a mobile phase of acetonitrile : 70 mM triethylamine phosphate buffer pH 3.5 at the ratio 70:30 (v v-1), obtaining limits of detection and quantification of 0.22 and 0.72 mg L-1, respectively. Recovery rates between 96 and 98 % were obtained from the application to commercial samples, and the results obtained by the proposed method showed no evidence of statistically significant differences compared to the reference methodology based on HPLC with packed column. The separation of triclosan (TCS), 2-chlorophenol (2-CP), 2,4-dichlorophenol (2,4-DCP), 2,4,6-trichlorophenol (2,4,6-TCP), 2,3,4 trichlorophenol (2,3,4-TCP) and methyltriclosan (MTCS) was studied by SIC, resulting in the separation of TCS, 2-CP, 2,4-DCP and 2,4,6-TCP with two isocratic elution steps, the first of them with a mobile phase 60:40 (v v-1) methanol: 20 mM ammonium acetate buffer (pH 5.5) followed by elution with 70:30 (v v-1) mobile phase of methanol : 20 mM ammonium acetate buffer (pH 5.5). In this case, the isomers 2,4,6-TCP and 2,3,4-TCP coeluted. Methyltriclosan, the less polar of these compounds, can be separated from TCS with subsequent elution steps. The methods were applied to study the adsorption of triclosan and its metabolites 2-CP, 2,4-DCP, 2,4,6-TCP and methyltriclosan on homoionic montmorillonite (K+) as well as in hexadecyltrimethylammonium salt (HDTMA) modified montmorillonite, noticing a stronger adsorption of triclosan and methyltriclosan compared with 2-CP, 2,4-DCP and 2,4,6-TCP. Incorporation of HDTMA in the clay mineral caused significant increase in adsorption capacity of these metabolites. This capacity was determined by fitting the experimental data to the linearized Langmuir equation. The adsorption order was 2,4,6-TCP > 2,4-DCP > 2-CP.

La conversion de la biomasse lignocellulosique en biocarburants et molécules biosourcées produit des matrices liquides complexes thermosensibles qui couvrent une large gamme de polarités et de masses moléculaires. Les outils analytiques développés dans la littérature donnent une description partielle de ces matrices oxygénées. Pour en comprendre la réactivité et mieux guider le développement des procédés de conversion, une meilleure caractérisation est nécessaire. L’objectif de cette thèse est de démontrer l’apport d’une dimension de fractionnement pertinente en amont de techniques séparatives pour accéder à la caractérisation à l’échelle moléculaire d’échantillons ex-biomasse. Une déformulation complète et structurée par familles chimiques est visée, sans perte ni modification des composés. Deux voies de fractionnement ont été investiguées : (1) fractionnement par solubilité à l’aide de l’extraction liquide-liquide (LLE) et de la chromatographie de partage centrifuge (CPC) et (2) fractionnement par taille avec la chromatographie d’exclusion stérique (SEC). Ces techniques se veulent complémentaires à une analyse par chromatographie liquide à polarité de phase inversée avec détection par spectroscopie ultraviolet-visible et spectrométrie de masse haute résolution (RPLC-UV/HRMS). Des méthodes de fractionnement LLE, CPC et SEC ont été développées sur molécules modèles afin d’identifier les mécanismes et la sélectivité chimique mis en jeu. Des cartographies 2D inédites ont ainsi été obtenues, assurant un gain important en pouvoir résolutif et une structuration nouvelle des chromatogrammes en comparaison à l’approche RPLC-UV/HRMS. Dans un second temps, le potentiel des couplages SECxRPLC-UV/HRMS et CPCxRPLC-UV/HRMS pour la description de matrices complexes a été illustré via l’étude de deux échantillons issus d’expérimentations en unités pilotes et de compositions chimiques très différentes, représentant deux voies possibles de transformation (biochimique et thermochimique) de biomasse lignocellulosique. La complémentarité entre les approches de séparation mises au point a ainsi permis de doubler le nombre de pics détectés tout en bénéficiant de l’organisation chimique des composés. Cette aide précieuse à l’identification a été renforcée par les informations structurales délivrées via les différents modes de détection, en particulier l’HRMS. La compréhension de la structuration des cartographies 2D a été présentée et discutée afin de proposer la stratégie la plus adaptée pour déformuler complètement un échantillon en s’appuyant sur la mesure de descripteurs pertinents. Enfin, l’une des approches développée dans cette thèse a été mise en œuvre pour l’isolement sélectif et l’élucidation structurale de molécules clefs au sein d’une matrice complexe à l’aide d’expériences en fragmentation MS et spectroscopie de résonance magnétique nucléaire (RMN) / The conversion of lignocellulosic biomass into biofuels and biosourced molecules produces complex thermosensitive liquid matrices which cover a wide range of polarity and molecular weight. Analytical tools developed in the literature only give a partial description of these oxygenated matrices. To understand the reactivity of these samples and optimize the development of conversion processes, a better characterization is required. The objective of this thesis is to demonstrate the interest of a relevant fractionation step prior to separation techniques to help the molecular characterization of biomass samples. The reverse engineering proposed for the sample is desired complete and chemically controlled (without loss or sample modification). Two fractionation pathways were investigated: (1) solubility fractionation with liquid-liquid extraction (LLE) and centrifugal partition chromatography (CPC) and (2) size fractionation with size exclusion chromatography (SEC). These techniques intend to be complementary to reversed-phase liquid chromatography hyphenated to ultraviolet-visible spectroscopy detection and high resolution mass spectrometry (RPLC-UV/HRMS). LLE, CPC and SEC methods were developed on model molecules to understand mechanisms involved and control the chemical selectivity. 2D contour plots were obtained, improving the resolving power and structuring chromatograms in comparison with RPLC-UV/HRMS. Then, SECxRPLC-UV/MS and CPCxRPLC-UV/MS hyphenations were applied to describe two complex samples from different substrates produced on experimental pilot units from two possible conversion pathways of lignocellulosic biomass (biochemical and thermochemical). The complementarity of separation modes allows to double the number of peaks detected, benefiting from the chemical organization of compounds. This constitute a support to identification also enhanced by multi-detection which provide additional structural information on compound detected, especially HRMS. Chemical organization in 2D contour plots were presented and discussed to propose the most adapted strategy to fully fractionate a sample based on the measurement of relevant descriptors. Finally, one of the fractionation approach developed in this thesis was used to isolate and structurally elucidate key molecules of a complex sample through MS fragmentation experiments and nuclear magnetic resonance spectroscopy (NMR)

(EN) In the first part of this work, analytical methods for determination of impurities of active pharmaceutical ingredients (API) in combined pharmaceutical dosage forms were developed and validated. Development of the methods covered both the optimization of sample preparation procedure and chromatographic conditions. The methods were validated according to International Conference on Harmonization guideline and both of them were confirmed to be able to analyze stability samples. Impurities in paracetamol, codeine phosphate hemihydrate and pitophenone hydrochloride in the presence of fourth API fenpiverinium bromide were separated by using ion-pair reversed phase chromatography with gradient elution. Symmetry C18, 250 x 4,6 mm, 5 µm heated to 35 řC was used as a separation column. A diode array detector was used. The detection wavelengths were set as follows: 220 nm for paracetamol impurity K, 245 nm for paracetamol and its other impurities and 285 nm for codeine, pitophenone and their impurities. Impurities in valsartan, amlodipine besylate and hydrochlorothiazide were separated by reversed phase UHPLC method with gradient elution. Chromatographic column Zorbax Eclipse C8 RRHD, 100 x 3,0 mm, 1,8 µm heated to 30 řC and spectrophotometric detection were used. The detection wavelengths were set as...

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Rigid poly[hydroxyethyl acrylate-co-poly(ethylene glycol) diacrylate] (Poly(HEA-co-PEGDA) monoliths were synthesized inside 75-µm i.d. capillaries by one-step UV-initiated copolymerization using methanol and ethyl ether as porogens. The optimized monolithic column was evaluated for hydrophobic interaction chromatography (HIC) of standard proteins. Six proteins were separated within 20 min with high resolution using a 20 min elution gradient, resulting in a peak capacity of 54. The performance of this monolithic column for HIC was comparable or superior to the performance of columns packed with small particles. Monoliths synthesized solely from PEGDA were also found to show excellent performance in HIC of proteins. Continuing efforts showed that rigid monoliths could be synthesized from PEGDA or poly(ethylene glycol) dimethacrylates (PEGDMA) containing different ethylene glycol chain lengths for HIC of proteins. Effects of PEG chain length, bi-porogen ratio and reaction temperature on monolith morphology and back pressure were investigated. Monoliths prepared from PEGDA 258 were found to provide the best chromatographic performance with respect to peak capacity and resolution. An optimized PEGDA 258 monolithic column was able to separate proteins using a 20-min elution gradient with a peak capacity of 62. The preparation of these in situ polymerized single-monomer monolithic columns was highly reproducible. The single-monomer synthesis approach clearly improves column-to-column reproducibility.The highly crosslinked monolith networks resulting from single crosslinking monomers were found to enhance the surface area of the monolith and concentrations of mesopores. Thus, monolithic columns were developed from four additional crosslinking monomers, i.e., bisphenol A dimethacrylate (BADMA), bisphenol A ethoxylate diacrylate (BAEDA, EO/phenol = 2 or 4) and pentaerythritol diacrylate monostearate (PDAM) for RPLC of small molecules. Gradient elution of alkyl benzenes and alkyl parabens was achieved with high resolution using all monolithic columns. Porogen selection for the BADMA and PDAM was investigated in detail with the intention of obtaining data that could possiblly lead to a rational method for porogen selection.

No description available.

No description available.

Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul - FAPERGS / The human parathyroid hormone (hPTH) is a polypeptide secreted by the parathyroid glands that is essential for the maintenance of the calcium ion homeostasis in the blood. The recombinant DNA technology has enabled the expression of hPTH gene in Escherichia coli, and thus the large-scale production of recombinant human parathyroid hormone (rhPTH 1-34), teriparatide, which contain the active amino-terminal fragment of the full length hPTH. The rhPTH is clinically used to treat osteoporosis at high risk of fractures in postmenopausal women, men with osteoporosis primary or hypogonadal and adults with glucocorticoid-induced osteoporosis (GIO). Cappilary zone electrophoresis (CZE) method was developed and validated for the assessment of rhPTH in biopharmaceutical formulations. The analysis for CZE method was performed on a fused-silica capillary (effective length, 40 cm; 50 μm i.d.), using electrolyte solution consisted of 50 mM dihydrogen phosphate solution at pH 3.0. The capillary was maintained at 25º C, the applied voltage was 20 kV. Injections were performed using a pressure mode at 50 mbar for 45 s, with detection by photodiode array detector set at 200 nm. Separation was obtained with a migration time of 5.3 min, and was linear over the concentration range of 0.25-250 μg mL-1 (r2 = 0.9992). The limits of detection and quantitation were 0.12 and 0.40 μg/mL, respectively. Specificity and stability-indicating capability were established in degradation studies, which also showed that there was no interference of the excipients. The accuracy was 100.28% with bias lower than 0.85%. Moreover, the in vitro cytotoxicity test of acidic, photolytic and thermal degradated forms showed significant differences (p<0.05) compared to intact molecule. The cell proliferation and alkaline phosphatase activity bioassays in UMR-106 cells were developed and applied to assess the biological activity of rhPTH in biopharmaceutical formulations The results of content/potency were correlated to those of the validated reversed-phase liquid chromatography (RP-LC), size-exclusion liquid chromatography (SE-LC) and CZE methods, showing significant correlation (p> 0.05) Thus, the application of the validated physico-chemical methods together with in vitro bioassays, was suggested to improve quality control of rhPTH biotechnology-derived product and to support studies of biosimilars. / O hormônio da paratireóide humano (hPTH) é um polipeptídeo produzido e secretado pelas glândulas paratireóides, e é fundamental para a manutenção da homeostase dos íons cálcio no sangue. A tecnologia do DNA recombinante possibilitou a expressão do gene do hPTH em Escherichia coli, e a produção em grande escala do hormônio da paratireóide humano recombinante (rhPTH 1-34), também denominado Teriparatida, o qual apresenta a sequência de aminoácidos responsável pela porção biologicamente ativa do paratôrmonio natural. O rhPTH é clinicamente indicado para o tratamento da osteoporose de alto risco de fraturas em mulheres pós-menopausa, de homens com osteoporose primária ou hipogonadal, e da osteoporose associada à terapia sistêmica com glicocorticóides. Neste trabalho foi desenvolvido e validado método por eletroforese capilar de zona (ECZ) para a avaliação de rhPTH em produtos biofarmacêuticos. No método por ECZ, utilizou-se capilar de sílica fundida (40 cm de comprimento efetivo x 50 μm d.i.) e solução eletrolítica composta de fosfato de sódio dihidrogenado 50 mM, pH 3,0. O capilar foi mantido a temperatura de 25ºC, e a tensão aplicada foi de 20 kV. O tempo de injeção foi de 45 s, com pressão de 50 mBar, e detecção por arranjo de diodos (DAD), em 200 nm. A separação eletroforética foi obtida com tempo de migração de 5,3 min, sendo linear na faixa de concentração de 0,25-250 μg/mL (r2 = 0,9992). Os limites de detecção e quantificação foram de 0,12 e 0,40 μg/mL, respectivamente. A especificidade foi avaliada através de análises com os excipientes da formulação biofarmacêutica e estudos de degradação, demonstrando a seletividade do método. A exatidão foi 100,28% com bias inferior a 0,85%. Além disso, realizou-se o teste de citotoxicidade in vitro das formas degradadas, apresentando, para as amostras submetidas às condições ácida, fotolítica e térmica, diferença significativa (p< 0,05) em relação à molécula íntegra. Os bioensaios de proliferação celular e da atividade da fosfatase alcalina em células UMR-106 foram desenvolvidos e aplicados para avaliação da atividade biológica de rhPTH em formulações biofarmacêuticas. Os resultados de teor/potência foram correlacionados com os métodos já validados por cromatografia líquida em fase reversa (CL-FR), cromatografia líquida por exclusão molecular (CL-EM) e ECZ, apresentando correlação significativa (p> 0,05). Assim, sugere-se que o métodos físico-químicos validados sejam aplicados paralelamente aos bioensaios in vitro para aprimorar o controle da qualidade do produto biotecnológico de rhPTH, e para avaliação da biossimilaridade de rhPTH.

SerpinA12 (vaspin) is thought to be mainly expressed in adipose tissue and has multiple beneficial effects on metabolic, inflammatory and atherogenic processes related to obesity. KLK7 (kallikrein 7) is the only known protease target of vaspin to date and is inhibited with a moderate inhibition rate. In the crystal structure, the cleavage site (P1-P1′) of the vaspin reactive centre loop is fairly rigid compared with the flexible residues before P2, possibly supported by an ionic interaction of P1′ glutamate (Glu379) with an arginine residue (Arg302) of the β-sheet C. A P1′ glutamate seems highly unusual and unfavourable for the protease KLK7. We characterized vaspin mutants to investigate the roles of these two residues in protease inhibition and recognition by vaspin. Reactive centre loop mutations changing the P1′ residue or altering the reactive centre loop conformation significantly increased inhibition parameters, whereas removal of the positive charge within β-sheet C impeded the serpin–protease interaction. Arg302 is a crucial contact to enable vaspin recognition by KLK7 and it supports moderate inhibition of the serpin despite the presence of the detrimental P1′ Glu379, which clearly represents a major limiting factor for vaspin-inhibitory activity. We also show that the vaspin-inhibition rate for KLK7 can be modestly increased by heparin and demonstrate that vaspin is a heparin-binding serpin. Noteworthily, we observed vaspin as a remarkably thermostable serpin and found that Glu379 and Arg302 influence heat-induced polymerization. These structural and functional results reveal the mechanistic basis of how reactive centre loop sequence and exosite interaction in vaspin enable KLK7 recognition and regulate protease inhibition as well as stability of this adipose tissue-derived serpin.

L’objectif de ce travail de thèse était d’étudier l’effet de la stérilisation par électrons accélérés sur les copolymères d’oléfines cycliques (COC), utilisés comme conditionnement de produits pharmaceutiques, ainsi que son impact sur les interactions avec des formulations pharmaceutiques. Grâce à la méthodologie analytique adoptée qui a fait appel à différentes techniques de caractérisation, telles que la chromatographie d’exclusion stérique, la chromatographie liquide haute performance à polarité de phases inversée, la spectroscopie infra rouge à transformée de Fourier, la microscopie à force atomique et les mesures d’angles de contact, nous avons pu mettre en évidence différents types de modifications dans le volume et sur la surface du matériau après stérilisation ainsi qu’après vieillissement. La modification principale du dans la masse du matériau, observée à la dose réglementaire de stérilisation (25 kGy), est la scission des chaînes du polymère, qui s’accompagne de la création de composés de faible masse molaire, donc de migrants potentiels risquant d’influencer la sécurité d’emploi des COC. En effet, certains de ces composés ont été retrouvés avec une concentration relativement importante dans les solutions de mise en contact avec les COC stérilisés, et notamment en solution aqueuse. Toutefois, l’étude préliminaire de toxicité a montré l’absence de cytotoxicité des extractibles obtenus à la dose de la stérilisation.Les modifications relatives à la surface des COC radio-stérilisés sont, quant à elles, de deux natures : physique avec une augmentation de la rugosité de surface et chimique avec la formation de produits d’oxydation polaires ; ces deux types de modifications conduisent à l’augmentation de la mouillabilité de surface. Cependant dans certains cas, notamment après vieillissement, ces modifications sont relativement faibles, même à des doses supérieures à celle préconisée pour la stérilisation, ce qui peut être corrélé à l’absence de l’effet de l’irradiation sur le comportement des COC vis-à-vis des solutions médicamenteuses. En effet, aucune variation de la sorption des principes actifs choisis n’a été montrée entre les COC irradiés et non irradiés vieillis. / The aim of this work was to study the effect of electron beam radio-sterilization on cyclo olefins copolymers (COC) used as pharmaceutical storage materials, as well as to investigate its impact on the interaction with pharmaceuticals formulations. Due to the analytical methodology used which dealt with different techniques of characterization such as size exclusion chromatography, reversed phase high performance liquid chromatography, Fourier transformed infrared spectroscopy, atomic force microscopy and contact angle measurements, we have been able to put into evidence different kinds of modifications both in the bulk and on the surface of the sterilized material and also after ageing.The principal modification of material’s bulk, observed at the recommended dose for sterilization (25 kGy), was polymer chains scissions, accompanied with creation of low molecular weight compounds, that are potentials migrants that risk to affect the safe use of COC. Indeed, some of these compounds have been found with a relatively important concentration in the solutions where sterilized COC was stored, especially in aqueous solutions. However, the preliminary study of toxicity has shown the absence of cytotoxicity of the extractables obtained at the sterilization dose.Surface modifications of radio-sterilized COC are of two types: a physical one, with an increase of the surface’s roughness and a chemical one with the formation of polar oxidation products; these two modifications result in an increase of surface’s wettability that may be important. However, in some cases such as for aged samples, these modifications are relatively weak even at doses higher than the one recommended for sterilization, which can explain the absence of the effect of radiation on the behavior of COC towards drug solutions. Indeed, no variation of drug sorption has been observed between aged COC irradiated and none irradiated.