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

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.