Until now, most metabolomics protocols have been optimized towards high sample throughput and high metabolite coverage, parameters considered to be highly important for identifying influenced biological pathways and to generate as many potential biomarkers as possible. From an analytical point of view this can be troubling, as neither sample throughput nor the number of signals relates to actual quality of the detected signals/metabolites. However, a method’s selectivity for a specific signal/metabolite is often closely associated to the quality of that signal, yet this is a parameter often neglected in metabolomics. This thesis demonstrates the importance of considering selectivity when developing NMR and LC-MS metabolomics methods, and introduces a novel approach for measuring chromatographic and signal selectivity in LC-MS metabolomics. Selectivity for various sample preparations and HILIC stationary phases was compared. The choice of sample preparation affected the selectivity in both NMR and LC-MS. For the stationary phases, selectivity differences related primarily to retention differences of unwanted matrix components, e.g. inorganic salts or glycerophospholipids. Metabolites co-eluting with these matrix components often showed an incorrect quantitative signal, due to an influenced ionization efficiency and/or adduct formation. A novel approach for measuring selectivity in LC-MS metabolomics has been introduced. By dividing the intensity of each feature (a unique mass at a specific retention time) with the total intensity of the co-eluting features, a ratio representing the combined chromatographic (amount of co-elution) and signal (e.g. in-source fragmentation) selectivity is acquired. The calculated co-feature ratios have successfully been used to compare the selectivity of sample preparations and HILIC stationary phases. In conclusion, standard approaches in metabolomics research might be unwise, as each metabolomics investigation is often unique.  The methods used should be adapted for the research question at hand, primarily based on any key metabolites, as well as the type of sample to be analyzed. Increased selectivity, through proper choice of analytical methods, may reduce the risks of matrix-associated effects and thereby reduce the false positive and false negative discovery rate of any metabolomics investigation.

The conditioning effect in metabolomic studies is the phenomenon of initial variation of analytical results in the first 5-10 injections of a biological sample in chromatographic systems. The deviation manifests itself as a drift in retention time, peak area and in multivariate analysis. It is a major quality assurance problem in the metabolomic field and if not accounted for would result in high analytical variance. The aim of this study was to investigate the conditioning effect and to gain further knowledge about it. The study was carried out on UPLC of hydrophilic liquid chromatography (HILIC) type coupled to quadrupole time of flight (QTOF) MS. A systematic study was designed to investigate the effects of the age of the analytical column. An investigation into certain matrix components as a possible cause of the conditioning effect was made. Different sample preparation methods were investigated. One result showed that no conditioning could be seen and the system appeared stable from the first injection. Differences in sample composition between samples with conditioning effect and samples without conditioning effect were investigated. No correlation between conditioning effect and levels of certain matrix compounds could be found. More studies of correlation between sample composition and the amount of conditioning occurring is needed. Some samples appear to have no retention time drift but have a significant drift in peak area and in multivariate analysis. This is an indication that the conditioning effect should be analysed in more ways than one before determining if a system is stable.

Historical pharmaceutical preparations analyzed in this thesis were a senna extract more than 200 years old, an ointment "Naso-Merfen" 75 years old, and an ointment "Sulfathiazol" 42 years old. The active substances in the analyzed samples were sennosides A and B (senna extract), ephedrine and menthol (the ointment "Naso-Merfen"), and sulfathiazole (the ointment "Sulfathiazol"). The senna extract was analyzed by RP-HPLC and HPLC-MS. Separation conditions were optimized, especially for separation of the sennoside A and B enantiomers. The active substances were not detected in the sample. One degradation product and substances characteristic for senna were identified. Their presence in the historical and contemporary sample was compared. Detailed ESI− -MSn fragmentation mechanisms of sennoside A and B have been proposed. The sample of the ointment "Naso-Merfen" was analyzed by HILIC-UV, HPLC-MS, GC-MS, and AAS. Separation conditions were optimized. The active substances were quantified. Degradation products of the active substances were not detected in the sample. The sample of the ointment "Sulfathiazol" was analyzed by RP-HPLC and HPLC-MS. Separation conditions were optimized. The active substance was quantified. Degradation products of the active substance were not detected. The authenticity of...

Biopharmaceuticals are becoming one of the most promising drugs on the market mainly due to their successful treatment of a vast array of serious diseases, such as cancers, immune disorders, and infections. Structurally, biopharmaceuticals are proteins and it is important to mention that more than 60 % of biopharmaceuticals are glycosylated. Glycosylation is one of the most common posttranslational modifications. It is also the most demanding and the most complex posttranslational modification. The research showed that glycosylation can significantly impact on the safety, efficiency, and quality of the therapeutic glycoproteins. In the first part of the introduction of the present thesis, the development of the therapeutic glycoproteins and their classification were reviewed. Glycosylation process and nomenclature were also discussed. The second part of the introduction revealed current issues in the field of the production and the characterization of the therapeutic glycoproteins. In the context of the doctoral thesis, we introduced new approach, namely hydrophilic interaction liquid chromatography coupled to a high-resolution mass spectrometer (HILIC-HR-MS) combined with Principal Component Analysis (PCA) and classification through Soft Independent Modelling by Class Analogy (SIMCA) data treatment. Accordingly, N-glycans were first enzymatically released using peptide-N-glycosidase F (PNGase F) and reduced using sodium borohydride. Then those N-glycans were separated by HILIC and detected by HR-MS. PCA and SIMCA simplified interpretation of the MS data collected in the huge tables. PCA was applied to test whether it is possible to visualize N-glycosylation differences between samples and to help identifying within which N-glycans changes occurred. SIMCA, which is a more complex data analysis technique, was applied to build and validate a classification models. SIMCA was also applied to verify whether it is possible to use built models to classify real samples. Described approach enabled us to detect small changes in N-glycosylation of the therapeutic glycoproteins (a change of only 1% in relative glycan abundance). It was applied to assess changes in N-glycosylation of therapeutic glycoproteins. Accordingly, we tested N-glycosylation consistency between batches of infliximab, trastuzumab, and bevacizumab and monitored the N-glycosylation of bevacizumab over storage time in plastic syringes.Furthermore, we worked on the faster sample preparation technique, where online-solid-phase extraction (SPE)-LC was combined to the previously mentioned HILIC-MS-PCA/SIMCA method. Online-SPE-LC allowed us to faster the sample preparation in terms of avoiding time-consuming cleaning steps. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

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De nombreux métaux tels que, par exemple le Zn, le Fe, le Cu ou le Ni jouent un rôle essentiel dans la croissance et le développement normal des plantes car ils sont impliqués dans différents processus physiologiques pouvant être cependant perturbés par une carence ou un excès en métaux. Par conséquent, afin de réguler l'absorption des métaux, leur translocation et leur accumulation, les plantes ont développé des mécanismes divers, comme la production de métabolites de faible poids moléculaire pouvant se lier aux métaux. La connaissance de ces complexes métalliques et des processus qu'ils subissent dans les plantes peut être utilisée dans des études environnementales, nutritionnelles et toxicologiques. Cependant, cette connaissance était très limitée à cause de l'absence de méthodologie appliquée avec succès pour étudier la spéciation des traces de métaux dans les végétaux. Par conséquent, le but de cette étude fut le développement d'une méthodologie d'analyse des espèces métalliques dans des échantillons de plantes. La nouvelle approche est basée sur l'utilisation conjointe du (i) couplage HPLC – ICP MS permettant la détection de nombreuses espèces, souvent peu concentrées, contenant des métaux avec (ii) une identification en parallèle par couplage HPLC – electrospray Orbitrap MS/MS. Elle a permis l'identification (i) d’environ 60 espèces métalliques, la plupart jamais observées précédemment dans différents fluides (xylème, endosperme liquide) de Pisum sativum (petit pois) et (ii) de plusieurs complexes mixtes fer - aluminium - citrate chez Plantago almogravensis. La méthodologie développée a également été appliquée à (iii) la spéciation du sélénium dans Brassica nigra permettant l'identification de plus de 30 composés de faible poids moléculaire contenant du sélénium. / Numerous metals, such as, e.g. Zn, Fe, Cu or Ni play an essential role in normal plant growth and development as they are involved in different physiological processes that may be, however, disrupted by metal deficiency or excess. Therefore, to regulate metal uptake, translocation and accumulation plants have developed diverse mechanisms including the production of low molecular mass metal binding metabolites. The knowledge of these forms of metals and the processes they undergo in plants can be used in environmental, nutrition and toxicological studies. However, this knowledge was very limited as there was a lack of methodology that could be successfully applied to investigate trace metal speciation in plants. Therefore, the aim of this study was the development of the methodology for the analysis of metal species in complex plant samples. The novel approach is based on the use of combination of (i) HPLC – ICP MS coupling allowing the detection of numerous, often low concentrated, metal-containing species with (ii) a parallel identification using HPLC – electrospray Orbitrap MS/MS coupling. The developed approach allowed the identification of (i) ca. 60 different, mainly previously unreported metal species in saps of Pisum Sativum (green pea) and (ii) several mixed iron – aluminum – citrate complexes in Plantago almogravensis. This developed methodology was also applied to (iii) investigate selenium speciation in Brassica nigra allowing the identification of more than 30 selenium-containing low molecular mass compounds.

Polymer brushes are extremely versatile materials, as monomer choice allows the user to design a material with the desired physiochemical properties. Given the wide variety in monomer functionality, polymers can be fine-tuned for a specific application. In this work, polymer brushes bound to a silica support are designed and utilized to enhance performance of protein extraction and chromatographic separations. <br> The effectiveness of an analytical method is strongly affected by matrix composition, however, the presence of species other than the target analyte is usually unavoidable. An excellent technique will be able to identify and/or quantify the analyte even when its concentration is low compared with interfering molecules. Protein analysis is particularly challenging, since many proteins of clinical and scientific significance are present in complicated matrices such as plasma or cell lysates. <br>A common method to specifically separate a protein from a complicated matrix is solid phase extraction. In this method, a species (such as an antibody) with high specificity towards the target is immobilized onto a solid substrate (commonly beads or small particles for greater surface area). Next, the target is collected onto the surface, bound by the species. The solid substrate is rinsed of the liquid matrix, before elution of the target. Only the active species should interact with the analyte, and the surface should be otherwise inactive. However, nonspecific interactions lead to binding/adsorption of undesirable compounds. Therefore, an optimal substrate for protein extraction must be 1) easily and completely removable from the liquid phase, 2) have a high concentration of active sites for specific binding, and 3) exhibit low nonspecific binding. As part of this work, commercial magnetic particles were coated with a nonporous silica layer that tolerates the acid bath and silane coating necessary to attach a polymer layer. On the silane coating, a polymer layer was covalently bound; this layer contains epoxide active groups for immobilizing antibodies. These antibodies bind to the target molecule with high specificity, and low nonspecific binding. Obtained particles were evaluated for protein extraction, where antibodies as well as specifically engineered drug compounds were successfully bound to the particle surface.<br>Glycosylation influences several physiopathological processes in proteins. Glycans can act as receptors, modify protein solubility, and participate in folding conformation. Altered glycosylation is a common feature in tumorous cells. As such, many modifications in glycoproteins have been related to cancer, including increased branching of N-glycans or augmented units of sialic acid. Therefore, characterization of glycoproteins is important not only as a diagnostic tool, but also to monitor patients’ response to treatment. Furthermore, it is important in the growing field of monoclonal antibodies as drug carriers. <br>Among different methods used for glycosylation analysis, Hydrophilic Interaction Liquid Chromatography (HILIC) has showed important advantages over time-consuming digestion-MS based techniques. An adequate HILIC stationary phase can be used to rapidly differentiate glycoforms present in a sample. In the second part of this work, a polymer brush based bonded phase was developed as a HILIC stationary phase. The new polymer improved the separation of a model glycoprotein compared with a commercial HILIC column, while also exhibiting enhanced stability over a previous bonded phase synthetized in our group.<br><br>

The objective of this research is to develop and apply a HILIC UHPLC stationary phase that allows for separation of intact glycoproteins. In Chapter 1 I give an overview of the problems of current glycosylation profiling with regards to biotherapeutics, and my strategy to separate the intact glycoprotein with HILIC. Chapter 2 describes the methods used to produce the nonporous packing material and stationary phase. In Chapter 3 I describe previous work in developing a HILIC polyacrylamide stationary phase, and further improvements I have made. Chapter 4 describes development of an assay in collaboration with Genentech of therapeutic mAb glycosylation. In Chapter 5, I show HILIC-MS of digested ribonuclease B as a beginning step to analyze glycosylated biomarkers.