3^rd International Conference and Exhibition on Advances in Chromatography & HPLC Techniques July 13-14, 2017 Berlin, Germany

Biography:

Dennis Fiorini is Associate Professor of Food Chemistry at University of Camerino. She has a Master Degree and a PhD in Chemistry and her research area deals mainly with lipid fraction of food, food volatile components and with the development of methods to extract and analyze them by means of solid-phase microextraction coupled to gas chromatography. In the last years she is focusing her research interest on olive oil and on chemical features related to its quality.

Abstract:

Polyphenols play a key role in the definition of the quality of olive oil in which they affect stability, sensorial and healthy properties (1,2). In fact EU regulation n. 432/2012 allows to acknowledge olive oil with the claim “Olive oil polyphenols contribute to the protection of blood lipids from oxidative stress” when 20 g of oil contain more than 5 mg of “hydroxytyrosol and its derivatives (e.g. oleuropein complex and tyrosol)” (3,4). However it is not specified in which way these substances should be quantified and which are exactly the substances to be quantified and since most of them are not available as commercial analytical standards, the results could differ greatly depending on the method applied, the standard used and the instrumentation available. Furthermore, the complexity of the olive oil polyphenols structures makes the chromatographic separation a difficult task, leading to obtain results not always satisfactory (5). Thus, our aim was to improve the chromatographic separation of these species by evaluating several analytical columns and elution conditions and to find a suitable quantitative method making use of instrumentation available in most of the laboratories. In fact, even if the method development was performed with the help of an ion trap mass spectrometer as detector, the quantification has been done by a diode array detector. The chromatographic column giving the best results was the Synergi Polar (250 × 4.6 mm, 4 μm), with water and methanol/isopropanol (9/1) as eluents. The quantification of secoiridoid derivatives has been done by using the oleuropein calibration curve corrected with the average response factor of tyrosol, hydroxytyrosol and oleuropein. The method allowed to quantify tyrosol, hydroxytyrosol and secoiridoid derivatives with limits of quantification of 0.32 mg kg-1, 0.17 mg kg-1 and 0.55 mg kg-1 respectively.

Biography:

Joscha Breibeck has completed his degree in Chemistry from Technical University of Munich (TUM, Germany) with a PhD in Biotechnology and Protein Biochemistry (Institute for Biological Chemistry, TUM). His projects focused the detailed biochemical and biophysical characterization of a novel class of recombinant polypeptides. During those studies and especially in viewing the polypeptides as (bio)polymers rather than proteins, he has in-depth experience with various preparative and analytical techniques, with a strong focus on chromatographic applications. Further investigations of the novel biopolymers involved their use for the hydrodynamic volume enlargement of (bio)pharmaceuticals by either genetic fusion or chemical conjugation and even as a calibration standard for mass spectrometry. He currently studies the bio scientific applicability of inorganic polyoxometalate clusters, in particular for improved protein crystallization, at the Institute for Biophysical Chemistry at University of Vienna, Austria.

Abstract:

Statement of the Problem: In size exclusion chromatography (SEC), macromolecules are sorted according to their size distribution. Therefore, SEC is also a measure for the hydrodynamic volume and yields valuable analytical information. Column calibration with well-defined polymer standards allows for estimation of apparent molecular weights. However, SEC analysis is usually considered an isolated methodology, overlooking interconnections with related methods. The present study aimed in the detailed hydrodynamic characterization of a novel recombinant biopolymer in comparison with poly(ethylene glycol) by elaborating the correlation between various techniques, including SEC, in order to obtain an integrated overall picture of hydrodynamic molecular properties. Methodology & Theoretical Orientation: In polymer sciences, a universal SEC calibration procedure is used, keeping in mind the dependence of the hydrodynamic volume on both the molecular weight and the intrinsic viscosity. Though essentially defining the solution viscosity of a macromolecule, the latter parameter is often neglected in biochemical sciences. It can be obtained from viscosity data and yields an expectation value for the hydrodynamic radius rh, assuming an ideal spherical molecule shape. Dynamic light scattering (DLS) as a shape-sensitive technique leads to diverging rh values for non-ideal, elongated molecular shapes.

Findings: The discrepancy between the rh data obtained by DLS and viscometric methods was evaluated for molecular shape estimation. SEC for itself is less sensitive to molecular shape and conformation than DLS, but provided useful indications for non-ideal molecule shapes in combination with DLS. In addition, SEC data confirmed independently obtained results from microviscometry.

Conclusion & Significance: In measuring the hydrodynamic volume, SEC data holds relevant conformational information exceeding the mere molecular weight information. To access shape information, it is advisable to use complementary hydrodynamic techniques. Especially the biosciences can only benefit from changing their view on SEC from an isolated standard technique to an integrated hydrodynamic characterization tool.

Biography:

Miloš Netopilík studies in physical chemistry, Charles University, Prague (1982), Postgraduate studies in Institute of Macromolecular Chemistry, Czechoslovak Academy of Science, Prague), Ph.D., 1984, Thesis: Degradation of Polymers by Shear Stress, Magister in Luce Sparsa (Light Scattering)Magister in Luce Sparsa (Light Scattering) at Universitas Lucis Spargentis, Wyatt Technology Corporation, Santa Barbara, CA, 1998. Since 1984 Research fellow, Institute of Macromolecular Chemistry, Czechoslovak Academy of Sciences, Prague. 1998, Senior research fellow, Institute of Macromolecular Chemistry, Czechoslovak Academy of Sciences, Prague.

Abstract:

Size exclusion chromatography (SEC) is a method generally used of the separation and analysis of polymers with respect to molecular wight. The benefits of the method are exploited by additional detectors (light scattering, viscometer, UV detector) which give maximum information about the sample. The basic functioning of chromatographic separation was described by the Martin and Synge model of relatively simple mechanism of establishing an equilibrium on each of theoretical plates followed by a shift of the mobile phase by the size of the plate. The model was further developed and for SEC it applies with some constraints. The results of the chromatographic models is the band broadening function. The basic mechanism of separation is accompanied by numerous side effects and mechanisms which depend on conditions of analyzes, as flow-rate, influencing the establishing the equilibrium, the detector-cell volume, etc. The systematic experimental investigation of analyzes of polystyrene standards by size exclusion chromatography evaluated on the background of the plate-height model of separation provides the insight into the mechanism and its efficiency, a measure of reaching equilibrium. The model gives results which can be transformed into those given by the Giddings and Eyring model. The separation being improved with decreasing flow-rate is in accord with Giddings observation of transversal diffusion as a factor decreasing the broadening of the band broadening function. The efficiency of the separation is judged on the basisi of the shape of the elution curves, which depends on the experimental conditions. This suggests that it reflects rather properties of the band broadening function than the molecular weight distribution of the polymer sample. The elution curves obtained by SEC of polystyrene standards in tetrahydrofuran using pumps and UV detector Deltachrome, and one column PL gel mixed D are depicted in Figure 1. The decrease in broadness of the elution curves is obvious. The curve obtained at lowest flow-rate of 0.01 ml/ min fits the theoretical model best.

Biography:

Ana Silvia Casas obtained her Master´s Degree in Earth Sciences at the Geophysics Institute of the National Autonomous University of México (UNAM) in 2015, with the thesis: “Sulfur speciation with HPLC for of El Chichón volcano crater lake monitoring”. In the same year she was granted by the German Academic Exchange Service (DAAD, for its acronym in german) with a scholarship to pursuit her PhD in Volcanology at the Ludwig-Maximiliand University (LMU) in Munich, Germany, where she currently studies diverse volcanic phenomena.

Abstract:

To study and understand volcanism on earth (their occurrence, impacts on civilization/environment, and monitoring), are not just necessary activities for the development of risk mitigation strategies in vulnerable areas, but also very interesting research topics. To obtain reliable geochemical-data for our research, HPLC was found to be an excellent analytical tool; it allowed us to perform sulfur speciation of El Chichón volcano Crater Lake, an active volcano in South Mexico, responsible for the worst volcanic disaster in Mexico’s modern history. Our HPLC-speciation methodology adds a new element to more accurately forecasting of future periods of volcanic unrest. The next step of our research is to study gas adsorption onto ash-particles, during large volcanic eruptions. For this new research I will carry my HPLC background to characterize ash-samples before and after experiments (ash samples will be exposed to various hydrous and anhydrous volcanic gas mixtures at high temperatures (200-800°C) for different time-series (1, 3, 5, 15, 30 and 60 min). The results of these experiments will help us, to constraining more effectively the complex interactions between volcanic emissions (i.e., volcanic ash and gas) and both human infrastructure and natural systems.

Biography:

Dusan Berek works at the Polymer Institute, Slovak Academy of Sciences in Bratislava. He has served as the Elected Member of the Presidium of the Slovak Academy of Sciences, President of the Slovak Chemical Society, Chairman of the Czecho-Slovak and Slovak National Committee of Chemistry for IUPAC. He is a corresponding member of the Central European Academy of Sciences and member of the Learned Society of the Slovak Academy of Sciences. He has authored two monographs and more than 300 scientific papers in reputed journals, proceedings and book chapters. He has more than 60 patents. He has presented over 120 invited plenary, key and main lectures, as well as over 900 regular lectures and poster contributions in symposia and conferences.

Abstract:

Block copolymers represent an important group of materials with extensive applications in science, medicine and technology. In the block copolymer, two-, three- or even several chemically distinct polymer chains are mutually connected with a chemical bond, whereas the length and polarity of particular blocks can differ remarkably. Molecular characterization of block copolymers involves determination of chemical composition of particular blocks, their molar mass, both averages and distributions, as well as presence and amount of parent homopolymers. Determination of molar mass of block copolymers and assessment of parent homopolymers, represent an analytical challenge. Most synthesists employ gel permeation chromatography, GPC (size exclusion chromatography) for determination of molar mass of precursors, as well as for a rather rough assessment of total molar mass of block copolymers. However, GPC produces only molar mass estimates of block copolymers because size of their macromolecules in solution depends on properties of both kinds of chains. Special problem is determination of amount and characteristics of parent homopolymers, which are present in most block copolymers, and which constitute highly undesired, expensive ballast. Due to low separation selectivity and detector sensitivity of GPC, parent homopolymers usually cannot be efficiently separated from the block copolymers to even trace their presence. We will discuss principles and applications the alternative liquid chromatography methods for comprehensive molecular characterization of block copolymers, namely liquid chromatography under critical conditions, LC CC, liquid chromatography under limiting conditions of enthalpic interactions, LC LC and sequential two-dimensional polymer liquid chromatography, S2D LC. LC CC is rather frequently used for separation of one parent homopolymer from a block copolymer and for estimation of molar mass of blocks created in the second stage of synthesis. However, LC CC exhibits numerous drawbacks such as low experimental robustness and therefore limited repeatability of measurements, restricted sample both recovery and capacity, as well as extensive band broadening. LC LC is rather robust and experimentally feasible. Its separation selectivity is very high and sample recovery is reasonable. LC LC can easily and efficiently discriminate both parent homopolymers from diblock copolymers. The base-line separated peaks of all sample constituents obtained with help of LC LC can be one-by-one on-line forwarded into the GPC column for molar mass average and distribution determination. Parent homopolymers present in the block copolymer at very low concentrations below 1% of can be traced and characterized, by this new approach, called S2D-LC.

Biography:

Feng Zhang has her research interests in drug safety and efficacy determination. For Traditional Chinese Medicine (TCM), she has suggested a comprehensive strategy to understand the active marker compounds, therapeutic mechanisms and synergistic properties of TCM, by using LC TOF/MS, LC-MS and LC-UV methods, metabolomics technology, as well as some conventional pharmacological analyses. For chemical drugs, she promotes better standardization of analytical practices in LC-MS based therapeutic drug monitoring analyses in clinical application. It is the key issue that makes the way of LC-MS into clinical routine laboratories.

Abstract:

Statement of the Problem: Multiple myeloma (MM), a malignant neoplastic serum-cell disorder, has been a serious threat to human health. The determination of 6 commonly used drug concentrations, including thalidomide (THD), lenalidomide (LND), cyclophosphamide (CTX), bortezomib (BTZ), dexamethasone (DXM) and adriamycin (ADM), in MM patients was of great clinical interest. Drug pharmacokinetic monitoring was important to ensure safety and efficacy during chemotherapy, but assays of one or two of these drugs in bio-fluids had been published.

Methodology & Theoretical Orientation: We reported a method for the rapid and simultaneous measurement of the above therapeutics by liquid chromatography-tandem mass spectroscopy (LC–MS/MS) method with solid phase extraction. Analysis was performed on a Waters XBridge®BEH C18 column (2.5 μm, 2.1 mmx50 mm) with formic acid aqueous solution and acetonitrile as the mobile phase at flow rate 0.3 mL/min.
 

Findings: All analytes showed good correlation coefficients (r>0.996), and LLOQ of THD, LND, CTX, BTZ, DXM and ADM were 4, 2, 2, 2, 2 and 2 ng/mL, respectively. The inter- and intra-day precisions and stability were expressed as variation coefficients within 15% and relative error less than 15%. Dilution effect, carryover and incurred sample reanalysis were in the acceptable levels.

Conclusion & Significance: Validation method was investigated according to the 2015 edition Chinese Pharmacopoeia guidelines, as US FDA (2013, revision 1) required. The descried method was successfully applied to determine the clinical incurred serum samples from MM patients, and the tested results were also provided to the doctors for consideration. The LC MS/MS based assay described in this article may improve future clinical studies evaluating common therapeutics for MM treatment.

Biography:

Tserendorj Tugsuu has completed her PhD in 2013 from National University of Mongolia (NUM), her dissertation subject was a Chemical study of some products after cracking processes for petroleum derived atmospheric residue. She has published more than 15 papers in scientific journals and has participated in more than 9 research projects as a Project Coordinator and as an Investigator. She has worked at the Petroleum Chemistry Laboratory, Center of Chemistry and Technology of New Materials, NUM as a Researcher from 1997 to 2002, now she has been working in the Department of Chemical and Biological Engineering, School of Engineering and Applied Science, NUM, as an Associate Professor since 2005. She has done her research work at the Ohtsuka Laboratory, Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Japan as a Research Fellow.

Abstract:

The World Health Organization classifies benzene as a human carcinogen because of its known effect on increasing risk of cancer as it is keeps accumulating in the body. Gasoline is a common product which has benzene. At present, benzene contained in gasoline should be less than 5%, volume as regulated by MNS 0217:2006 standard. In European Union, Russia and China the standard for gasoline the benzene content should not exceed 1% as regulated by EN 228:2012, GOST32513-2013, GB 17930-2013 standard. In Mongolian case, many type of petroleum products and other natural solvents, which contain benzene, are used in daily life. To predefine content of C6 hydrocarbons in product because to prevent the arising benzene from reforming processing, and can calculate and monitor benzene content that may arise from these process. Within the research, to improve “to determine benzene content” standard MNS GOST 29040:2008 which is valid in Mongolia, in addition, to determine benzene content can be safety data sheet for those employees who are working in those condition. The repeatability of the determination of benzene content is lower than 0.014-0.08% with high injection fault. Compared to other researches, the research determined benzene content 0-10% with higher interval and limit of detection of 0.28%. During the determination of hexane and cyclohexane, pole column was more suitable and repeatability was 0.009-0.013. Hexane sensor range was 0.14%, cyclohexane was 0.16%, and determined with limit of 0-10% range using direct method. The total content of petroleum C6 hydrocarbons cannot be determined by above mentioned two columns. The benzene content was 0.08% in gasoline fraction of Zuunbayan crude oil and 0.13-0.14% in gasoline fraction of Tamsagbulag crude oil. Because benzene fraction output is low, benzene content is lower in Zuunbayan field's petroleum. The calculated content of benzene, which is based on C6 hydrocarbons content in Tamsagbulag crude oil, was 0.76%. Therefore, it can be used as base data in petroleum processing in Mongolia. The imported products which are light petroleum have 0.62% and pyrolysis gasoline has 8.9% benzene content. Therefore, a pyrolysis gasoline does not meet Mongolian standard with 8.9% benzene content.

Biography:

Martina Hermannova is currently working at Contipro (pharmaceutical company in the Czech Republic), where she is the Leader of 2 research groups focusing on Analytical Science, Department of Pharmacokinetics and Hyaluronan Fragments. She is also a member of Contipro Scientific Board. She has almost 15-years of experience in polymer separation and characterization by SEC-MALLS. In recent years, her research work focuses on elucidation of structural changes in vitro and in vivo.

Abstract:

The physicochemical properties and biological functions of hyaluronan (HA) are closely related to its molecular weight (MW) and molecular weight distribution (MWD). Therefore, it is crucially important to provide a reliable characterization of these parameters for proper use of HA and its degradation products in both chemical and clinical fields. In this study, we present a novel method for the preparation of HA fragments of defined size with narrow molecular weight distribution. The HA fractionation was performed using an anion-exchange chromatography and is applicable either after enzymatic or chemical hydrolysis of polymeric HA. Isolated fractions with a molecular weight ranging from 3000–420,000 g mol-1 were analyzed by size exclusion chromatography with multi-angle laser light scattering (SEC-MALLS). Hundred-milligram scale HA fragments were obtained from 5 g hyaluronan starting material. Independently on weight-average molecular weight (Mw), the polydispersity index (PDI) of the HA fractions was less than 1.23. The fractionation methodology can be easily up-scaled and is applicable on any negatively charged polymers. We have also found that PDI is insufficient to characterize almost monodisperse fractions and for proper material characterization we proposed a new characteristic termed “distribution angle ΘD”, calculated from the slope of the cumulative molecular weight distribution curve. Compared to PDI, the distribution angle reflects more efficiently changes in size distribution and thus is highly recommended to be used along with Mw determination of any polymer. Apart from that, SEC separation conditions were exhaustively optimized with the great emphasis laid on the separation efficiency.

Biography:

Sena Caglar Andac has completed her PhD from Istanbul University and Post doctoral studies from Medical Center of Munich University, Laboratory of BioSeparation. She is now working as an Associate Professor at Istanbul University, Faculty of Pharmacy. She has her research works published in liquid chromatography mass spectrometry for biological fluid analysis of drugs; on-line solid phase extraction coupled liquid chromatography, determination of drugs and degradation studies by high performance liquid chromatography, spectrophotometry and spectrofluorimetry.

Abstract:

Analysis of drugs and metabolites in biological fluids is essential for bioanalysis. An optimal and effective sample preparation method plays the most important role as the depletion of the matrix in biological fluids is the biggest issue for a trouble-free analysis. It is impossible to inject the biofluid directly to the chromatographic system with traditional methods due to possible matrix effect and clogging issues. Liquid–liquid extraction (LLE), protein precipitation and solid-phase extraction (SPE) are the most common and offline/manual sample preparation methods to deplete macromolecules (i.e., proteins) present in the biological fluid prior to liquid chromatographic analysis. To speed-up the clean-up process, fully automated on-line techniques that combine sample preparation with separation could be a remarkable alternative. This could be achieved by the hyphenation of SPE with LC via a switching valve resulting online SPE-LC. This method allows direct repetitive injection of biological sample to a single SPE column. Use of restricted access materials (RAM) as SPE-column packing materials enables the depletion of high molecular weight matrix while the small analyte molecules are retaining; this fractionation is mostly based on 2D chromatography combination of size exclusion chromatography with reversed phase chromatography. Coupling SPE column with LC leads to complete automation improving the analytical quality due to enhanced reproducibility, elimination of human errors and the possibility of multiple step elutions for clean-up of complex samples, reducing the cost and analysis time required.

Biography:

Shao-Ting Wang has his expertise in development of novel mass spectrometry methodologies for drug analysis and endogenous biomarker detection. He is the In-charge of the Center of Mass Spectrometry in Renmin Hospital of Wuhan University since 2015 and gained abundant experience on applying chromatography and mass spectrometry techniques in clinical field. After systematic evaluation, reliable laboratory developed tests have been carried out in the center, including newborn screening based on tandem mass spectrometry, therapeutic drug monitoring of anti-schizophrenic and anti-epileptic drugs, and detection of fat soluble
vitamins. These contributions significantly promoted the popularization of the chromatography and mass spectrometry techniques for clinical usage in Hubei
province of China.

Abstract:

Statement of the Problem: Chinese schizophrenia cases meets a 132% increase from 1990 to 2010 (3.09 vs. 7.16 million). During treatment, therapeutic drug monitoring (TDM) is highly required to compensate individual variation and realize rational medication. Although the AGNP Consensus Guidelines present excellent directions on “therapeutic reference range” for all the frequently used drugs, before introducing it for Chinese patients, significant ethno-cultural variation between Chinese and Caucasians should be carefully considered. Unfortunately, one could find few related data at present time. To fill the gap, the purpose of this study is to establish a highly applicable UPLC-MS/MS method for simultaneous TDM of five anti-schizophrenic drugs and briefly discuss the applicability of AGNP Guidelines for Chinese patients.
 

Methodology & Theoretical Orientation: An UPLC-MS/MS method for routine therapeutic drug monitoring of aripiprazole, amisulpride, olanzapine, paliperidone and ziprasidone was developed and carefully evaluated. The TDM data from 253 clinical samples was collected and analyzed to investigate applicability of the AGNP therapeutic reference range for Chinese patients.

Findings: Good consistency for olanzapine, aripiprazole, paliperidone and ziprasidone was observed. While for amisulpride, the plasma concentration level (445.2±231.5 ng/mL) was higher than the recommended range (100-320 ng/mL).

Conclusion & Significance: The developed UPLC-MS/MS method is highly suitable for routine TDM usage. And there is necessity of reconstructing a Chinese-specific therapeutic reference range for amisulpride treatment, which would be quite helpful to improve the medication efficiency and safety for Chinese patients.

Biography:

Ahmad Aqel received his BSc and MSc degrees in 2005 and 2008, respectively, from Hashemite University, Jordan, and received his PhD degree in 2012 from King Saud University, KSA. Currently, he is an Assistant Professor of Analytical Chemistry at King Saud University. He has worked as a Researcher at King Abdullah Institute for Nanotechnology (2008-2013), Assistant Researcher at Hashemite University (2008), and as a Teaching Assistant at Hashemite University (2005-2007). Currently, he is working on a variety of separation and chromatographic topics ranging from preparation and development of packing materials for chromatographic columns to extraction and pre-concentration of various organic and inorganic samples. He is the co author of more than 50 scientific contributions; 21 original papers, 2 review articles, 1 patent, 2 book chapters and 27 presentations in local and international conference proceedings.

Abstract:

Capillary liquid chromatography has become one of the most important developments in separation technology. According to the literature, it’s widely accepted that capillary liquid chromatography performed using columns with an internal diameter less than 500 μm. This technique carried out using fused silica capillary columns and prepared with a variety of different stationary phases. However, the successful development of this technique is closely related to the technical challenges associated with the columns manufacturing. Monolithic media have rapidly become popular and attracted increasing interest as separation phases. They consist of a single rigid piece of porous material that possesses a unique bimodal pore structure distribution with micrometer sized macropores and nanometer sized mesopores. Unfortunately, unmodified monolith is lake of small mesopores that comes at the cost of surface area. The better specific surface area for methacrylate polymers is within 10 m2/g. Although, the large macropores provides advantages in the separations of large molecules such as proteins, it does not provide the sufficient interaction sites for separation of small molecules especially with isocratic modes. However, several approaches have been proposed to enhance the separation efficiency of the monolithic columns. In this work, small amounts of micro/nanoparticles such as carbon nanotubes, metal organic frameworks and sporopollenin have been incorporated into the porous polymer monolithic capillary columns under specific conditions to enhance the separation efficiency of small molecules. Porous and hydrodynamic properties and the morphology of the prepared columns were thoroughly characterized. The columns were evaluated by separation mixtures of different compounds such as phenols, aromatics, ketones and drugs. The combination of both monoliths and capillary liquid chromatography systems offer several advantages that include fast and sensitive analysis, in addition to the consumption of much smaller amounts of solvents, samples and stationary phase materials, which will reflect positively on the environment and cost.

Biography:

Dr.rer.nat. Hans-Jürgen Rieger studied Chemistry at the Freie Universität in Berlin and completed his studies with a thesis in Physical Chemistry in 1989. He was PostDoc at the Institute of Organic Chemistry at the University of Freiburg until 1993. He is working with the Molnár-Institute since 1999, specializing in software development. He is responsible for product management.  He is also teaching DryLab courses worldwide.

Abstract:

In UHPLC method development, our goal is to achieve a satisfactory separation in less than one day. To do this, we use the software DryLab. We are starting with 2 gradient runs with a difference of factor 3 in their slope, tG1 and tG2. We run these 2 runs also at 2 temperatures T1 and T2 of 30°C difference. The resulting tG-T-model is the most successful design in UHPLC. To create a “Cube”, we repeat our tG-T-model for polar compounds at 3 different pH values. For neutral substances, we vary 3 different organic eluents (MeOH, ACN and 50:50) to obtain selectivity changes (peak movements), which help us to find the best separation. The next step is Peak Tracking, which means to align a peak in a peak table in a horizontal line. Here we use peak areas to locate a peak in up to 12 runs. Further support in identification is the use of molecular masses and different UVwavelengths. After peak tracking is finished, the model (the “Cube”) is calculated based on the so called “critical resolution” with more than 106 different chromatograms. The best separation can be found with one mouse click. However one separation is not telling you, how robust your method will be. Therefore we calculate the influence of tolerance limits for each parameter by producing for 6 factors at 3 levels (3^6=729) virtual experiments and calculate the success rate from them. This enables us to avoid “Out of specification” results, which are objects of a tedious “Change management” procedure, involving regulatory oversight and high costs. Finally we write a Knowledge Management Document, which includes the details of the above process, the input parameters, the input experiments, the graphical output of the Design Space and all necessary elements for a commercial authorization of the product by the regulatory agencies like the FDA, etc.

Biography:

Cenk A Andac works as an Assistant Professor in the School of Pharmacy at Istinye University. He has completed his Master’s degree and PhD work from the Faculty of Pharmacy at the University of Wisconsin-Madison, WI, USA (UW-Pharmacy, USA). He has been involved in teaching drug actions and delivery, and pharmaceutical biochemistry and biotechnology courses at UW Pharmacy, USA for four and half years. He has also taught Medical Pharmacology courses as an Assistant Professor for three years in Medical School of Turkey. His current researches are development of novel anticancer agents inhibiting G-coupled receptors in cancer stem cells; development of novel aminoglycoside antibiotics; determination of 3D structures of biological and synthetic compounds by NMR techniques; computer-assisted drug development by AMBER, CHARMM and quantum mechanics; and pharmacokinetics and pharmacodynamics properties of drug-receptor interactions. He currently holds a patent for a potentially active anti-cancer agent against breast cancer.

Abstract:

A specific H-bond between U33 and the phosphate group between C36 and G34 in the anticodon loop region of t-RNA renders a very compact anticodon loop structure, hence called a closed-loop structure. It is known that the closed loop structure is in exchange with an open-loop structure in which the aforementioned specific H-bond in the anticodon loop region is broken. In order to shed light on to the dynamics of the open-loop structure of the transfer RNA, a 17-mer RNA hairpin molecule, representing the open-loop structure anticodon region of a transfer RNA, with the sequence 5'GGGAGUXAGCGGCUCCC 3' (X=3-N-methyl uridine) was synthesized using the dimethoxytrityl, tert-butyldimethylsilyl and 2-cyanoethyl diisopropylphosphoamidite. The crude product was then purified by reversed phase HPLC using an 8 mm C-18 Radial-Pak column (by Waters Assoc., Inc) on a Beckman (Fullerton, CA, USA) System Gold HPLC instrument with ultra violet detection at 254 nm. Optimal conditions for the separation of the 17-mer RNA were provided by using an isocratic elution system based on 0.05 M ammonium acetate (NH4OAc) solution in RNase-free dd.H2O (buffered at pH=7.0). The retention time was 9 min and the elution speed was 0.7 ml/min. The purified RNA hairpin was further studied by NMR and it was found out that two major conformationally different structures of the RNA exist in slow exchange with concentrations ~51% and ~43% on NMR time scale. AMBER molecular dynamics and cluster analysis studies indicate reveal two open-loop structures of the RNA hairpin.

Biography:

Zeynep Altintas has her expertise in the fields of biosensors, biomimetic materials and diagnostics. She is the Head of Biosensors and Receptor Development Group in Technical University of Berlin. She has worked at the Cranfield University as a Faculty Member as well as in other institutes as Visiting Professor and Researcher. She pioneered nanoMIPs-based SPR sensors for the detection and removal of pharmaceuticals, toxins and viruses using a novel solid phase synthesis method. Her research on virus imprinting area creates new pathways for virus sensing and removal by providing strong alternatives to natural antibodies. Her works have received several awards from international organizations in recent years. She is serving as an Expert Reviewer for EU and Wisconsin Groundwater Coordinating Council (USA) founded projects in addition to acting as the reviewer for several important journals in her areas of expertise.

Abstract:

Statement of the Problem: The removal of pharmaceutical compounds from water sources has an immense impact on public health, since the contamination arising from their presence in drinking water leads to gain tolerance in human body and can significantly decrease the effective treatment later on. The use of rationally designed affinity materials in separation columns and membrane filters may help to solve this problem; therefore, pharmaceuticals specific molecularly imprinted polymers nanoparticles (MIPNPs) were synthesized and applied onto the polyvinylidene fluoride (PVDF) membranes previously subjected to the plasma treatment.

Methodology & Theoretical Orientation: Computationally designed diclofenac-, metoprolol- and vancomycin-MIPs were applied onto the membranes and scanning electron microscopy was employed to visualize MIPNPs on the membrane. After functionalization of the membranes with target-specific MIPs the molecularly imprinted membranes (MIMs) affinity against their targets was evaluated using solid phase extraction (SPE) technique coupled with high performance liquid chromatography (HPLC). MIMs were used as filters to load the target solutions and evaluate the amount of pharmaceuticals in filtrate. Furthermore, a comparative study was performed by comparing the efficiency of MIMs functionalized either by adsorption or covalent immobilization.

Findings: The capacity analysis of MIPNPs by SPE–HPLC revealed 100%, 96.3%, and 50.1% uptake of loaded solution of metoprolol, diclofenac and vancomycin, respectively. MIMs showed 99.6% uptake with a capacity of 60.39 ng cm2 for metoprolol; 94.7% uptake with a capacity of 45.09 ng cm2 for diclofenac; and 42.6% uptake with a capacity of 16.9 ng cm2 for vancomycin. HPLC detection limits of targets were found as 3.7, 7.5 and 15 ng mL−1 for diclofenac, metoprolol and vancomycin, respectively. A small scale pilot test was also conducted which indicates the promising future applications of the developed MIMs for high volume of filtrates especially in the case of the plasma-treated PVDF membranes prepared by covalent immobilization of the MIPs.

Conclusion & Significance: MIPNPs were successfully incorporated into SPE columns and PVDF membrane. Nanostructured polymeric membrane is capable of capturing targets from water which demonstrates a new approach for water purification of pharmaceuticals.

Biography:

Dagmar Heinova has her experience in enzymology with a special focus to proteolytic enzymes and lactate dehydrogenase isoenzymes. She developed a colorimetric method for determination of pepsin activity which was patented. She succeeded in separating bird LDH isoenzymes based on experience in her research. Results of her research are applied in the education of students at the University of Veterinary Medicine and Pharmacy in Kosice in the field of Biochemistry.

Abstract:

Statement of the Problem: Lactate dehydrogenase (EC 1.1.1.27, LDH) is an enzyme ubiquitously distributed in cells of vertebrates, plants, and bacteria. Structurally, it is a tetramer of four units which in animals exists in five electrophoretically distinguishable forms known as isoenzymes. Electrophoretic techniques routinely applied in separating mammalian LDH isoenzymes use a buffer system of pH 8.6 at which their electrophoretic migration depends on the migration of the two pure types, i.e., H4 and M4 forms. The more the two homotetramers differ in charge, the more separable are the hybrids by electrophoresis. In the case of bird LDHs, the two pure types migrate close together towards the anode at pH 8.6 producing only one diffuse enzymatic zone.

Methodology & Theoretical Orientation: For the separation of bird LDHs, we chose isoelectric focusing technique in a gradient of pH 3 to 9. Separation conditions were used as follows: 2000 V, 2.5 mA, 3.5 W, 15ºC, 20 min  separation time. Gels were stained with nitro blue tetrazolium technique in 0.1 M Gly-NaCl-NaOH buffer pH 8.3 at 37ºC for 30 min.

Findings: Using above described methodology we achieved good and clear resolution of all five forms of the enzyme of bird origin with their localization being in the pH region of 6.2 to 8.1 (chicken LDH isoenzymes), 5.4 to 7.7 (pheasant LDHs), and 5.3 to 8.3 (turkey lactate dehydrogenase isoenzymes). Mammalian molecules of LDH were more acidic and widespread with the pI values being in the range of 4.5 to 9.0.

Conclusion & Significance: Using IEF technique it was possible to compare the pattern of LDH isoenzymes in serum and tissues of mammals and birds as well as to observe the pattern of the enzymes in some tissues of chicken embryo.