Day 2 :
- 7.Method Development
9.Applications of HPLC
11.HPLC fingerprinting in Bioinformatics and Computational Biology
Slovak Academy of Sciences, Slovakia
Dusan Berek graduated with an honour Master of Chemical Engineering from the Slovak Technical University in Bratislava. He completed his PhD Thesis at Polymer Institute, Slovak Academy of Sciences in Bratislava and Institute of Macromolecular Chemistry, Czecho Slovak Academy of Sciences in Prague. He did his DSc Thesis at Slovak Academy of Sciences and Slovak Technical University Bratislava. He is working at Polymer Institute, Slovak Academy of Sciences (PI SAS) in Bratislava since 1960. From 1992-1995 he was an elected member of the Presidium of the Slovak Academy of Sciences. He served as President of the Slovak Chemical Society, Chairman of the Czecho-Slovak and Slovak National Committee of Chemistry for the International Union of Pure and Applied Chemistry and as Member of the Board of the Federation of European Chemical Societies. He is the author of five original methods for separation of complex polymer systems; preparation and application of microporous carbon fibers. Author or co-author of two monographs on liquid chromatography and 250+ scientific papers in extenso published in refereed periodicals, and in proceedings and chapters of books, as well as 60+ patents - cited more than 2,000 times in SCI journals and monographs. He has presented over one hundred and ten invited plenary, keyand main lectures, as well as over 900 lectures short communications and poster contributions during congresses, symposia and conferences and during lecturing tours to over fourty countries. He was elected Slovak Scientist of the year 1999 and Slovak Innovator of the year 2001.
Most high-performace synthetic polymers exhibit besides dispersity in their molar mass also dispersity in another molecular characteristic, namely in chemical structure (composition) or in physical architecture of macromolecules. They are designated complex polymers. Typical examples of complex polymers are all kinds of copolymers, branched and functional polymers. Blends of macromolecules with distinct chemical structure or physical architecture are called complex polymer systems. Size exclusion chromatography (gel permeation chromatography), SEC/GPC is commonly used for assessment of molar mass averages and dispersities (distributions) of complex polymers and complex polymer systems. However, due to simultaneous dependence of size of macromolecules on all their molecular characteristics, as well as following limited separation selectivity, sample capacity, and low detector sensitivity, SEC/GPC can hardly give quantitative information on molar masses of complex polymers and complex polymer systems. For example, SEC/GPC even does not enable molecular characterization of binary polymer blends of components with fairly different molar masses, in which the content of the minor constituent is less than few percent. To characterize complex polymers and complex polymer systems, enthalpic retention mechanisms are to be coupled with entropy based size-exclusion retention mechanism. Of the presently utilized coupled methods of polymer LC the best known are liquid chromatography under critical conditions of enthalpic interactions, LC CC, eluent gradient liquid chromatography, EG LC and temperature gradient interaction chromatography, TGIC. The common drawbacks of the latter methods are limited to both sample recovery and capacity. LC CC permits only separation of two distinct sample constituents. Recently an alternative group of coupled LC metods was developed, namely liquid chromatography under limiting conditions of enthalpic interactions, LC LC. Similar to LC CC and EG LC, the molar mass effect is supressed also in LC LC. LC LC exhibits remarkable separation selectivity, as well as both high sample capacity and recovery. Moreover LC LC is highly robust, experimentally feasible and well repeatable. It was successfully applied to separation of chemically and physically similar macromolecules including low solubility polymers. The method enables reliable identification of very low (<1% and even <<1%) amounts of minor macromolecular admixtures in a polymer matrix. The basic principle of LC LC will be discussed in the contribution. For a comprehensive molecular characterization of complex polymer systems, two different retention mechanisms are to be applied in two separate chromatographic systems. This is the basis of two-dimensional polymer liquid chromatography, 2D-LC. A flexible approach called sequential two-dimensional polymer liquid chromatography, S2D-LC was recently developed. It consists of a combination of LC LC and SEC/GPC. The fractions produced by the LC LC column are in their entirety transferred into the SEC/GPC column for determination of average and disperity of sample. The principle of S2D-LC will be elucidated in detail and the typical examples of its application will be shown, especially separation and characterization of block copolymers that contain (small) amounts of their parent homopolymers.
The environment is contaminated with a large number of compounds that may impact organisms in, e.g., surface waters, soil, sediments and also indoors. The complexity of the contaminant mixtures demands powerful separation and identification techniques. Whereas in the last part of the previous century, many priority pollutants were non-polar, fat soluble and bioaccumulating compounds, such as dioxins and polychlorinated biphenyls. The last decades showed emerging compounds of higher polarity such as many pesticides, perfluorinated compounds, personal care products and pharmaceuticals. These compounds not only or only marginally dissolve in lipids but are also found in surface waters and, consequently, threaten environmental and human health. In many cases, target analyses suffice to determine the concentrations of the aforementioned compounds. However, the increase in the number of chemicals being used has caused an ongoing flood of emerging compounds for which are no standards or which still need to be identified. Advanced detection systems such as modern mass spectrometry alone are not sufficient to elucidate the complex mixtures of chemicals present. The development of comprehensive two-dimensional gas chromatography (GCxGC) showed already to be extremely useful for the identification of emerging non- and medium-polar contaminants. Comprehensive two-dimensional liquid chromatography (LCxLC) has now shown to be extremely useful in the same way as GCxGC, but for more polar compounds. The significant enhancement of the peak capacity by using LCxLC, together with new, extensive libraries and software facilitates the rapid identification of toxic compounds, leading to a comprehensive chemical characterization of environmental water and sediment samples.
J de Boer is Director of the Institute for Environmental Studies (IVM) at the VU University in Amsterdam. He is Professor in Environmental Chemistry and Toxicology. He is Editor-in-Chief of Chemosphere. Since 2013, he is National Expert for China. His research interests are method development and analysis of contaminants and micro-plastics in the environment and indoor air. He has coordinated various European research projects and a large number of research projects for international organizations. He has published over 180 peer reviewed articles and is among the 3000 most cited scientists in the world.
Ferey Ludivine has completed her PhD from “Institut des Sciences et Industries du Vivant et de l’Environnement, AgroParisTech”. She is Associate Professor in the ChemBioMed U869 INSERM team, headed by Professor Philippe Barthélémy at the Faculty of Pharmacy of Bordeaux. Her research is based on the development of new analytical strategies using different separation techniques (HPLC, CE, microchip, etc.) hyphenated with various detectors (DAD, CAD, ELSD, LIF) and chemometric tools (Quality by Design, experimental designs etc.).
Recent pharmaceutical regulatory documents have emphasized the importance of applying Quality by Design (QbD) concept for in-depth product and process understanding to ensure product quality by the design. QbD concept has been supported by the International Conference on Harmonization guideline Q8 (R2). The goal of this present study is to demonstrate the usefulness of the QbD approach when applied to the development of separation methods in green analytical chemistry. The relevance of QbD and green analytical chemistry combination was emphasized by the case study of Active Principle Ingredient and its related substance analysis by UHPLC. Following a QbD approach, green chemistry principles were included in the analytical target profile to reduce environmental impact and minimize analyst exposure during a future routine use after the implementation of the method in pharmaceutical industry. First, a scouting phase enabled to select the stationary phase and the type of organic solvent. After applying quality risk assessment, the effects of selected critical process parameters on Critical Quality Attributes (CQAs) were evaluated through a screening design. A response surface methodology was then carried out to model CQAs as function of the retained factors and the optimal separation conditions were determined by applying desirability functions to the modeled responses. Finally, focusing at quality risk management, the design space was computed as the multidimensional subspace where the CQAs fulfilled the requirements. The method was successfully validated by the accuracy profile approach and applied to a pharmaceutical product.
Opole University, Poland
Anna Poliwoda has completed her PhD from Opole University (Poland). Currently, she works as researcher at the Division of Analytical and Ecological Chemistry of the Opole University. In the years 2001-2002, she was a recipient of grants from Socrates-Erasmus and Swedish Natural Science Research Council in the Faculty of Analytical Chemistry, Lund University (Sweden) – research on analysis of peptides in biological samples. She has co-authored 25 scientific papers, including several chapters in monographs.
One of the sources of naturally occurring bioactive alkaloids with hallucinogenic properties are mushrooms of the genius such as Amanita, Psylocybe and Pholiotyna. These mushrooms produce substances (the structural analogues of tryptamine (indole) or isoxazoles derivatives) that caused the user to have a heightened state of awareness of sensory input (audio, visual, etc.). Nowadays, they are generally illegal to use in many countries, but their recreational use, especially by young people, has become an increasing problem in Europe. The twentieth-century technological achievements (such as internet) make it incredibly easy to purchase hallucinogenic mushrooms without any limitations. The main danger of fungal hallucinogens is not their toxicity, but their unpredictability actions. Therefore, their occurring and concentration level in various types of samples (e.g. mushrooms, biological fluids) must be monitored. In this presentation, the chromatographic and electrophoretic methodologies used in recent years in the analysis of fungal hallucinogenic alkaloids such as psilocybin, psilocin, ibotenic acid and muscimol in various complex sample matrices (i.e. body fluids, hallucinogenic mushrooms, etc.) will be described. The review will focused on comparison of applied analytical methods, taking into account both selectivity and efficiency of separation procedures, the used detection mode, method automation, requirements of application of sample clean-up step or derivatization. Additionally, the methods considering the analysis of fungal hallucinogens that have been developed in our laboratory will be presented too.
Université de Montpellier, France
Gaëlle Coussot is an Associate Professor at the Faculty of Pharmacy of Montpellier, France. She obtained her PhD in Analytical Chemistry in 2003. She then joined for 15 months the MD Anderson Center Cancer (Houston, Texas) for a Post-doctoral position in proteomic analyses. Currently, her researches focus on the development of bioanalytical methodologies using electrophoretic, chromatographic techniques and immunoassays to characterize and/or quantify proteins and others biopharmaceuticals. Research fields include Quality Control of biopharmaceuticals and study of antibodies resistance to particular environmental constraints. She has published 1 patent and 14 papers in international analytical and biochemical journals.
Monoclonal antibodies (mAb) represent the largest class of therapeutic molecules entering clinical studies. Due to their inherent structural complexity, the quality control (QC) of such type of molecules, necessary for their development and commercialization, represents a challenging analytical task. Common approaches in mAb QC are based on mAb reduction or proteolysis to produce a mixture of mAb fragments that are further analyzed by separation methods. These cleavage steps are usually performed off line (i.e. prior to the separation step in a distinct reactor). This is an important limitation in terms of time, reactant consumption and cross contamination by the possible formation of endogenous compounds that may further impair the quality assessment of the mAb drug. To overcome these limitations, we have first developed a fully integrated bio-analytical miniaturized methodology called D-PES (Diffusion–mediated Proteolysis and Electrophoretic Separation) for the QC of polymer-drug conjugates. With the D-PES methodology, both cleavages and separation steps are performed in-line, silica capillary being used both as nano-reactor and separation support. The methodology is based on transverse diffusion of laminar flow profile (TDLFP) mixing of reactant nano-volumes (proteolytic buffer (PB), substrate (S), enzyme (E) or reducing agent (R)) inside the capillary. Principles and results of these rapid and low operating costs microanalyses will be presented for mAb drugs. Separation optimization and mAb cleavage conditions (choice of background electrolyte, PB, ionic strength, pH…) will be discussed to demonstrate the robustness of the D-PES methodology.