No / Nanoscale reversed-phase liquid chromatography (LC) combined with electrospray ionization-tandem mass spectrometry (ESI-MS/MS) has been used as a method for the direct identification of multiple cytochrome P450 (P450) isoforms found in male and female rat liver. In this targeted proteomic approach, rat liver microsomes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by in-gel tryptic digestion of the proteins present in the 48- to 62-kDa bands. The resultant peptides were extracted and analyzed by LC-ESI-MS/MS. P450 identifications were made by searching the MS/MS data against a rat protein database containing 21,576 entries including 47 P450s using Sequest software (Thermo Electron, Hemel Hempstead, UK). Twenty-four P450 isoforms from the subfamilies 1A, 2A, 2B, 2C, 2D, 2E, 3A, 4A, 4F, CYP17, and CYP19 were positively identified in rat liver.

No / Surfactants are commonly used as cleansing agents and yet there are concerns that they may also have a role in skin irritation. The lack of suitable methods for the quantitative and qualitative analysis of surfactant deposition on skin has hindered the in-depth investigation of such effects. Here, we report the application of reversed-phase liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) assays for two surfactants commonly used in consumer products, namely sodium lauryl ether sulfate (SLES) and laurylamidopropyl betaine (LAPB), to a baseline study aiming to assess deposition levels on human skin. The linearity of the assays was established at 3-20 ng, with coefficient of variation below 5%. The detection limits were 100 pg for LAPB and 1 ng for SLES; quantitation limits were 500 pg for LAPB and 2.5 ng for SLES. The baseline study was conducted using a panel of 40 healthy volunteers. Skin extract samples were taken in triplicate from forearms, using ethanol. SLES was detected on most volunteers, with 75% of them having SLES deposits in the range of 100-600 ng/cm(2). LAPB was detected on the skin of all volunteers with 85% of them having deposit levels within the concentration range of 1-100 ng/cm(2). These results demonstrate the extent to which commonly used surfactants remain on the skin during the day. The analytical methods reported here can be applied to the investigation of surfactants in relation to general skin condition and to the development and optimisation of new consumer wash products. / EPSRC-DTA award / School Life Sciences

Botulinum toxin type A (BTTA) is a polypeptide with 1296 amino acids and its used in some pathologies like hyperhidrosis and migraine, but the cosmetic area is the most important application. BTTA is produced from broth-culture synthesized by the Clostridium botulinum as a single peptide with 150 kDa. Reversed-phase liquid chromatography (RP-LC) method was developed and validated for the assessment of botulinum toxin type A in biopharmaceutical formulations. A RP-LC method was carried out on a Zorbax 300 SB C18 column (150 mm x 4.6 mm i.d.), maintained at 45 ºC. The mobile phase A consisted of 0.05 M sodium sulphate buffer, pH 2.8, and the mobile phase B was acetonitrile, run isocratically at flow rate 0.3 mL/min. Cromatographic separation was obtained with retention time of 11.4 min, and was linear over the concentration range of 0.2-100 IU/mL (r2 = 0.9999) with photodiode array (PDA) detection at 214 nm. The limits of detection and quantitation were 0.04 and 0.16 IU/mL, respectively. Specificity was established in degradation studies, which also showed that there was no interference of the excipients. Equally, the accuracy was 100.31% with bias lower than 0.80%. The method was correlated with in vivo and in vitro bioassays. Moreover, the in vitro cytotoxicity test of related proteins and higher molecular weight forms showed significant differences (p <0.05) compared to intact molecule. The validated methods were applied for the determination of BTTA in biopharmaceutical-derived products, giving mean values of the estimated content/potencies 1.16% lower compared to the in vivo bioassay. It is concluded that represents a contribution to establish new alternatives to monitor stability, quality control and thereby assure efficacy of the biotechnology-derived product. / A toxina botulínica tipo A (BTTA) é um polipeptídio constituído de 1296 aminoácidos e é recomendada para patologias, como hiperhidrose e enxaqueca, mas é especialmente usada como cosmético. A toxina botulínica é produzida através do processo de fermentação bacteriana e após sucessivas etapas de purificação resulta em uma proteína com 150 kDa responsável pelo seu efeito biológico. No presente trabalho foi desenvolvido e validado método por cromatografia líquida em fase reversa (CL-FR) para a avaliação de potência de BTTA em formulações de produtos biofarmacêuticos. No método foi utilizada coluna Zorbax 300 SB C18 (150 mm x 4,6 mm d.i.), mantida a 45 ºC. A fase móvel A foi constituída por tampão sulfato de sódio 0,05 M, pH 2,8, e a fase móvel B por acetonitrila, eluídas em vazão isocrática de 0,3 mL/min. O método utilizou detector de arranjo de diodos (DAD) a 214 nm. A separação cromatográfica foi obtida em 11,4 min, sendo linear na faixa de concentração de 0,2-100 UI/mL (r2 = 0,9999). Os limites de detecção e quantificação foram 0,04 e 0,16 UI/mL, respectivamente. A especificidade foi avaliada em estudos de degradação, que também demonstraram que não houve interferência dos excipientes. A exatidão foi 100,31 com bias inferior a 0,80. O método validado foi correlacionado com bioensaios in vivo e in vitro. Além disso, realizou-se o teste de citotoxicidade in vitro das formas degradadas, as quais apresentaram diferença significativa em relação a forma intacta (p <0,05). O método proposto foi aplicado para avaliação da potência de BTTA em formulações biofarmacêuticas, e os resultados foram comparados com o bioensaio in vivo, observando-se diferença das médias de teor/potência de 1,16% inferior para o método cromatográfico. Contribuíu-se assim para estabelecer procedimentos que aprimoram a caracterização ou o controle da qualidade, garantindo a segurança e eficácia do produto biotecnológico.

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)

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.

High Performance Liquid Chromatography (HPLC) is a widely used tech-nique both for detecting and purifying substances in academy and in the industry. In order to facilitate the use of, and knowledge in HPLC, character-ization of stationary phases is of utmost importance. Tailor made characteri-zation methods and workflows are steadily increasing the speed and accura-cy in which new separation systems and methods are developed. In the field fundamental separation science and of preparative chromatography there is always the need for faster and more accurate methods of adsorption isotherm determination. Some of that demand are met with the steadily increase of computational power, but the practical aspects on models and methods must also be further developed. These nonlinear characterization methods will not only give models capable of describing the adsorption isotherm but also actual values of local adsorption energies and monolayer saturation capacity of an individual interaction sites etc.The studies presented in this thesis use modern alkali stable stationary phas-es as a model phase, which will give an insight in hybrid materials and their separation mechanism. This thesis will include an update and expansion in using the Elution by Characteristic Points (ECP) method for determination of adsorption isotherms. The precision is even further increased due to the ability to use slope data as well as an increase in usability by assigning a set of guidance rules to be applied when determine adsorption isotherms having inflection points. This thesis will further provide the reader with information about stationary phase characterization and the power of using existing tech-niques; combine them with each other, and also what the expansion of meth-ods can revile in terms of precision and increased usability. A more holistic view of what benefits that comes with combining a non-linear characteriza-tion of a stationary phase with more common linear characterization meth-ods are presented.