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19th International Symposium, Exhibit & Workshops on

Electro- and Liquid Phase-separation Techniques

ITP 2012 Book of Abstracts (all Lecture and Poster Presentations)

Symposium Chair: Prof. Ziad El Rassi

Oklahoma State University

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LECTURE PRESENTATIONS L-101 The Critical Role of the Front End to the Mass Spectrometer for Protein and Proteome Analysis. Barry L. Karger, Barnett Institute, Northeastern University, Boston, MA, USA [PLENARY LECTURE] The revolution in mass spectrometry continues at a rapid pace. Instruments of higher resolution, faster scanning and higher sensitivity are introduced on a yearly basis. To take best advantage of these powerful systems, the front end steps (sample preparation and separation) need to be optimized. In this presentation, we will describe several approaches that provide powerful approaches to enhance MS analysis. In the first example, we demonstrate the high sensitivity of 10 µm i.d. porous layer open tubular (PLOT) columns coupled to MS for proteomics and protein characterization. Flow rates of 20 nL/min or lower lead to enhanced electrospray ionization, such that 1500 proteins can be identified with the injection of protein digests from only 50 cells. As another example, new approaches to glycan analysis of carbohydrate released from proteins using microfluidic devices coupled to MS lead to full analysis including positional and linkage isomers. The applications of these and other examples to biological studies will be shown in order to demonstrate the importance of the front end to enhance the power of mass spectrometry. L-102 The Wide World of Proteomic Technologies for Biomarker Discovery.Samir Hanash, Fred Hutchinson Cancer Research Center, Program Head/Molecular Diagnostics, Seattle, WA, USA [PLENARY LECTURE] Implementation of in-depth quantitative plasma proteomic toolset has provided an opportunity for unbiased discovery of protein marker panels indicative of risk for specific common diseases. We have implemented strategies for the discovery of risk markers for coronary heart disease (CHD), stroke, breast and lung cancer among women using plasma samples collected several years prior to diagnosis as part of cohort studies. The availability of plasmas from several hundred cases and matched controls for each disease necessitated implementation of a pooling strategy followed by expensive fractionation prior to mass spectrometry to achieve sufficient depth of analysis to identify proteins related to disease incidence. Coverage of the plasma proteome spanned no less than seven logs of protein abundance. Some of the candidate markers identified have been successfully validated using immunoassays applied to independent sets of samples that were not part of the discovery sets. Additional lycoproteomic platforms were implemented for profiling glycan structures and immunomic platforms for identifying protein antigens in circulation. The experience gained, the findings from these studies, the resulting extensive database of the plasma proteome and the challenges in developing high-throughput assays with the pre-requisite sensitivity for marker validation will be presented. L-103 New Developments in Ion Chromatography. Paul Haddad1, Greg Dicinoski1, Robert Shellie1, Emily Hilder1, Naama Karu1, Melissa Hanna-Brown2, 1Australian Centre for Research on Separation Science, University of Tasmania, Hobart, Tasmania, AUSTRALIA; 2Pfizer Inc., Kent, UK [PLENARY LECTURE] Ion chromatography (IC) is the premier technique for the separation and analysis of inorganic anions and is also highly useful for the determination of other ionic species including inorganic cations, organic acids and bases, carbohydrates, and amino acids. Despite the fact that IC is now a mature technique there have been some very significant developments in the technique over the last five years. These developments will be reviewed, with particular attention to the areas of powerful software to assist in method development, high speed separations, miniaturisation of columns, multi-dimensional IC separations, and new applications. Major advances have been made in the design of specialised software for method development in IC. Software for the simulation and optimisation of isocratic separations has been available for several years. However, most IC separations are now performed using gradient elution or using complex elution profiles combining multiple isocratic and gradient steps. There is a need for method development software for such separations. New approaches to this problem will be described, including methodology which enables isocratic retention data to be used to reliably predict retention times (and to therefore optimise separations) for any elution profile comprising isocratic steps and linear gradients. This software enables very rapid selection of optimal elution conditions for a desired group of analytes. Extension of this approach to organic anions of pharmaceutical significance will also be demonstrated. Polymeric monolithic materials can be used for IC, especially when converted into high-efficiency ion-exchangers by coating with surfactants or functionalised nanoparticles. Alternatively, short, high-efficiency packed columns can be used. These approaches make it possible to construct a two-dimensional IC system having greatly increased peak capacity when compared to conventional IC systems. Recent column developments have also included the introduction of capillary IC, which is capable of continuous operation for long periods. Capillary IC will be discussed, with a focus on method translation from conventional scale to capillary columns. New uses of suppressors will be described in which suppressors are employed for de-salting of IC eluents prior to detection using universal detection modes such as mass spectrometry, evaporative light scattering detection (ELSD) and charged aerosol detection (CAD). Studies showing how the suppression reactions affect recoveries of organic analyte ions passing through the suppressor are discussed, and new

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purpose-built suppressors are also evaluated. The use of suppressors coupled to nebulisation detectors and mass spectrometric detection will be illustrated, showing that ion-exchange separations of non-chromophoric analytes of pharmaceutical interest can be achieved under gradient elution conditions. L-104 Nanofluidics Based Single Biomolecule and Single Cell Analysis for Cancer Diagnosis and Stem Cell Therapy. Yoshinobu Baba, Nagoya University, Department of Applied Chemistry, FIRST Research Center Innovative Nanobiodevices, Synchrotron Radiation Center, Nagoya University, Nagoya, JAPAN [PLENARY LECTURE] Nanobiodevice is a piece of contrivance, equipment, machine, or component, which is created by the overlapping multidisciplinary activities associated with nanotechnology and biotechnology, intended for biological, medical, and clinical purposes. During the past decade, nanobiodevice has progressively begun to focus on the establishment of main four fields of biomedical applications of nanotechnology, including 1) diagnostic devices, 2) molecular imaging, 3) regenerative medicine, and 4) drug delivery systems. In this lecture, I will describe the development of nanobiodevices to analyze biomolecules and cells towards biomedical applications. [1-16] I developed numerous diagnostic nanodevices based on the single biomolecular separation and detection. These devices are also applicable to fast and low invasive blood marker detection of cancer with pM-fM detection sensitivity. Additionally, I developed new synthetic method of quantum dots (QDs) based on appropriate cluster confirmation by the ab initio molecular orbital calculation. QDs are applied to the development of nanobiodevice for single cancer cell diagnosis, single molecular epigenetic analysis, in vivo imaging for stem cell therapy and theranostic device for cancer diagnosis/therapy. References: [1] N. Kaji, et al., Chem. Soc. Rev., 39, 948 (2010). [2] M. Tabuchi, et al., Nature Biotech., 22, 337 (2004). [3] R. Bakalova, et al., Nature Biotech., 22, 1360 (2004). [4] M. F. Serag, et al., ACS Nano., 5, 493, (2011). [5] T. Yasui, et al., ACS Nano, 5, 7775 (2011). [6] M.F. Serag, et al., ACS Nano, 5, 9264 (2011). [7] H. Hatakeyama, et al., Biomaterials, 32, 4306 (2011). [8] H. Hatakeyama, et al., Mol. Therapy, 19, 1487 (2011). [9] K. Kitazoe, et al., Lab on a Chip, 11, 3256 (2011). [10] T. Yasui, et al., Lab on a Chip, 11, 3356 (2011). [11] T. Yasui, et al., Anal. Chem., 83, 6635 (2011). [12] K. Hirano, et al., Nucleic Acids Res., 40, 284 (2012). [13] M.F. Serag, et al., RSC Advances, 2, 398 (2012). [14] M.F. Serag, et al., Integrative Biology, 4, 127 (2012). [15] H. Yukawa, et al., Biomaterials, in press (2012). [16] S. Yamamura, et al., PLoS One, in press (2012). L-105 Forming Highly Concentric Liquid Shells with Electric Fields. Thomas B. Jones, University of Rochester, Rochester, NY, USA [KEYNOTE LECTURE] The ponderomotive force acting on uncharged but polarizable media can be used to control the static and dynamic equilibria of many liquids ranging all the way from insulating dielectric oils to cryogenic liquids to certain aqueous media. This effect, called liquid dielectrophoresis or LDEP, is widely exploited in microfluidic schemes to manipulate and control sub-microliter volumes of liquid on a substrate using specially patterned, individually addressable surface electrodes. The same ponderomotive force can be harnessed to adjust the shape of small liquid masses and also to form highly concentric double-emulsion (DE) droplets. The simple condition required to achieve the centering of one droplet within another involves the relative dielectric constants of the external and intermediate shell liquids. Many potentially useful applications for concentric DE droplets may be envisioned, one example being the formation of thin-walled foam shells for the fabrication of laser fusion targets. L-106 Dielectrophoretic Separation of Particles. Ezinwa Elele, Yueyang Shen, Boris Khusid, New Jersey Institute of Technology, Chemical, Biological, and Pharmaceutical Engineering, Newark, NJ, USA Compared to other available methods, dielectrophoresis is becoming one of the major techniques for micro- and nano-scale systems. It uses an electric field that requires no moving parts, employs a polarization force acting on a fine particle that is insensitive to its charge which is difficult to control, offers easy adaptability to electronics and, therefore, can be incorporated more favorably into micro-systems. A wide variety of dielectrophoretic microdevices have been developed to address challenges in life science and analytical chemistry, such as separating a heterogeneous population of particles into homogeneous subpopulations, manipulating and concentrating biologically relevant molecules, and distinguishing between ill and healthy cells. The majority of publications on dielectrophoresis are limited to dilute suspensions. In contrast, we will focus on experimental and theoretical study of dielectrophoretic phenomena in concentrated suspensions which are critical for concentrating particles in focused regions of a micro-system. We will show that the separation efficiency is controlled not only by the interaction of particles with an externally applied field but also by the dipole-dipole forces between the particles undergoing dielectrophoresis, as they acquire dipole moments. The main effect of the interparticle dipolar interactions is that they, if sufficiently strong, cause a suspension to undergo reversible phase transitions from a random arrangement of particles into a variety of ordered aggregation patterns. We next consider electric field induced phenomena in digital microfluidics that are based upon manipulation of discrete tiny droplets to compartmentalize samples which carry a biological or chemical content. Such devices have recently emerged as a promising alternative technology to conventional closed-channel technologies that require a network of microfabricated channels, valves, mixers, and pumps for moving fluids around on the chip. The open and scalable architecture of digital microfluidics plus readily available techniques for basic droplet

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operations (dispensing, transporting, splitting, and merging) offer the possibility of fabricating simple, easily reconfigurable and low cost devices for large-scale parallel operation of liquid samples. The current focus of the droplet-based technology is on the development of techniques to perform generic laboratory procedures inside a tiny droplet. We will discuss methods for the field and flow driven concentration and removal of particles from microdroplets. This work was supported in part by NSF ERC for Structured Organic Particulate Systems and NASA grant NNX09AK06G. L-107 Maxwell-Wagner Polarization and Frequency Dependent Injection at Aqueous Electrical Interfaces. Zachary Gagnon, Johns Hopkins University, Department of Chemical and Biomolecular Engineering, Baltimore, MD, USA A new kind of AC interfacial polarization and liquid injection mechanism, at the electrical interface created between two laminar fluid streams with different electrical properties, is reported. Using a microfluidic channel with embedded electrodes, two fluid streams - one has a greater electrical conductivity and the other a greater dielectric constant - were made to flow side-by-side without mixing. An AC field was applied across the flow channel and fluid was observed to inject across the interface. The direction of injection was frequency dependent, and is attributed to the Maxwell-Wagner interfacial polarization at the liquid-liquid electrical interface. At low AC frequency (< 1 MHz), below the interfacial charge relaxation time, the high conductivity stream is observed to inject into the high dielectric stream. Above this frequency, the direction of liquid injection reverses, and the high dielectric stream injects into the high conductivity stream. The crossover frequency between these two events is related to differences in electrical properties between the two fluids, and is well described by Maxwell-Wagner polarization mechanics. L-108 Practical Platforms for High Throughput Sample Preparation using 3D Carbon-electrode Dielectrophoresis. Rodrigo Martinez-Duarte, Ecole Polytechnique Federale de Lausanne, Lausanne, SWITZERLAND In this talk I will present the recent advances on the use of 3D carbon-electrode Dielectrophoresis (carbonDEP) for sample preparation. The aim is to develop practical platforms for a variety of applications including diagnostics, cell sorting, proteomics and food safety. Therefore, the priority is on high throughput, high efficiency and user-friendliness. I’ve previously shown that 3D electrodes penetrating the bulk of the sample improve the throughput of DEP devices by minimizing the distance from a targeted particle to the closest electrode. Moreover, the use of 3D electrodes allows for the widening of the channel cross section, particularly its height, which inherently increases the processed sample volume per unit of time. This talk begins with the low cost fabrication process of tall (>100 μm-high) carbon electrodes and their integration to robust microfluidics networks to obtain DEP chips; followed by the presentation of two user-friendly portable platforms: one based on a syringe pump and another one based in centrifugal microfluidics. At last, results obtained in collaboration are presented and include a cell viability assay, cell selection and washing to improve sensitivity of PCR, enrichment of M. smegmatis towards better drugs to treat tuberculosis, DNA concentration and on-chip electrical lysis. Current work is on the separation of melanoma cells from lymphocytes. L-109 High-Performance Affinity Microcolumns: Recent Developments and Applications. David Hage, University of Nebraska, Chemistry Department, Lincoln, NE, USA [KEYNOTE LECTURE] Affinity ligands have been used for decades for both the selective isolation and analysis of sample components in complex mixtures. The selectivity and strong binding of many biological agents that are used as affinity ligands, such as antibodies, have also made these ligands of great interest over the last decade for use in HPLC methods and, more recently, in microaffinity LC methods. This presentation will examine some recent developments in the creation and bioanalytical applications of affinity microcolumns. One set of methods that will be discussed are chromatographic-based immunoassay, in which antibodies or related agents have been used in a variety of detection formats for the rapid analysis or drugs, hormones, proteins, and other analytes. Affinity microcolumns are particularly valuable in the area of ultrafast immunoextraction, in which analytes are isolated from samples in the sub-second time domain. One unique application that will be discussed for this approach is in the analysis of free drug and drug and hormone fractions in blood. Several approaches for conducting these assays will also be examined, including a number of competitive or displacement assay formats and the use of either packed microcolumns or small affinity monoliths. Affinity microcolumns have been shown to be valuable in the studying the thermodynamics and kinetics of biological interactions. This type of application will be illustrated by using examples based on the binding of drugs with serum agents such as human serum albumin HSA, alpha 1-acid glycoprotein and lipoproteins. The benefits and limitations of using affinity microcolumns for this purpose will be examined and the use of this approach in the high-throughput screening of biological interactions will be discussed.

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L-110 Capillary Electrophoresis and Density Functional Theory Applied to Characterization of Non-covalent Interactions of Hexaarylbenzene-based Receptor with Small Cations. Vaclav Kasicka1, Sille Ehala1, Petr Toman2, Rajendra Rathore3, Emanuel Makrlik4, 1Institute of Organic Chemistry and Biochemistry Academy of Sciences of CR, Electromigration Methods, Prague, CZECH REPUBLIC; 2Institute of Macromolecular Chemistry Academy of Sciences of CR, Prague, CZECH REPUBLIC; 3Marquette University, Department of Chemistry, Milwaukee, WI, USA; 4Czech University of Life Sciences, Faculty of Environmental Sciences, Prague, CZECH REPUBLIC In this study, capillary affinity electrophoresis (CAE) and quantum mechanical density functional theory (DFT) calculations were combined to provide quantitative and qualitative characterization of non-covalent binding interactions between a new polyaromatic hexaarylbenzene-based receptor (R), recently synthesized for potential application in sensing devices for selected ions [1], and small inorganic cations, such as ammonium and alkali metal ions, NH4+, Li+, Na+, K+, Rb+ and Cs+. The strength of the non-covalent interactions between the receptor R and the above ions was evaluated by means of stability (association) constant (Kst) of the complexes of receptor R with these ions. The stability constants Kst of the above complexes in methanolic medium were determined by CAE from the dependence of effective electrophoretic mobility of the receptor R on the concentration of ammonium and alkali metal ions in the background electrolyte (BGE) using a non-linear regression analysis. Prior to regression analysis, the effective mobilities of receptor R were corrected to reference temperature of 25°C and constant ionic strength, 25 mM, utilizing our earlier developed correction procedure [2]. The selectivity of receptor R expressed by the stability constants Kst towards alkali metal and ammonium ions was found to be increasing in the order of Na+ (Kst = −0.7) << Li+ (1.15 ± 0.09) < K+ (3.20 ± 0.22) < Cs+ (3.72 ± 0.22) < Rb+ (4.01 ± 0.21) < NH4+ (4.03 ± 0.15). The employed CAE method has proved to be a suitable and efficient tool for the quantitative evaluation of weak as well as medium strong interactions between receptor R and ammonium and alkali metal ions in methanol. The structural characteristics of the above ammonium and alkali metal ion complexes with the receptor R, such as position of the cation in the cavity of the receptor and the interatomic distances within the complexes, were estimated by quantum mechanical DFT calculations. These calculations have shown that the studied cations bind to the receptor R because they synergistically interact with the relatively polar ethereal fence and with the central benzene ring via cation-π interaction. This work was supported by GACR (projects nos. 203/08/1428, P206/12/0453), ASCR (projects nos. Research Project RVO61388963 and T400500402) and by the MSMT CR (projects nos. MSM4977751303 and MSM6383917201). [1] R. Shukla, S. V. Lindeman, R. Rathore, J. Am. Chem. Soc. 128 (2006) 5328. [2] S. Ehala, E. Makrlík, P. Toman, V. Kašička, Electrophoresis 31 (2010) 702. L-111 Capillary-based Protein Analysis: CGE for Quantitation, ACE to Study Interactions. Sabine Redweik1, Claudia Cianciulli1, Thomas Hahne1, Xi Deng1, Yuanhong Xu2, Hermann Wätzig1, 1Institute of Medicinal and Pharmaceutical Chemistry, TU Braunschweig, Braunschweig, GERMANY; 2Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Jilin, CHINA Capillary Gel Electrophoresis (CGE), also known as Capillary Electrophoresis-Sodium Dodecyl Sulfate (CE-SDS), is established in the pharmaceutical industry replacing SDS-PAGE gel electrophoresis, for example for the purity control of monoclonal antibodies. The method of an application note from Agilent [1] was used and optimized with a protein standard containing myoglobin, carbonic anhydrase, ovalbumin and bovine serum albumin (1-1.5 mg/ml). Separation voltage was -16.5 kV (-30 µA) for 30 min to analyse proteins with a maximal molecular weight (MW) of 70 kDa. It is beneficial to inject hydrodynamically instead of electrokinetically which was employed in earlier works. Both techniques were compared in long series runs (n=48). Furthermore, the use of an internal standard was investigated. The RSD% of the migration time was reduced from 0.9% to 0.25%. The RSD% of peak area was improved as well by the use of an internal standard. However, the evaluation by the 100% method avoiding the computation of the injection error into the results demonstrated RSD% for the peak areas typically between 1 and 2%. It was crucial to obtain signal-to-noise ratios greater than 100 for optimum precision [2]. Further, the use of CISS integration software proved to be favourable [3]. The optimized method was used to investigate the purity of a model antibody and the molecular weight of different reduced fragments. In comparison of CGE and SDS-PAGE the assets and drawbacks of both are discussed, plus the possibility of CGE as the second dimension in the 2D-GE. Investigations of protein properties and functionality require precise analytical tools and a thorough understanding of protein physicochemistry. The influence of ions in the surrounding solution on proteins is thus investigated by Affinity Capillary Electrophoresis (ACE). The interactions with proteins are detected by the mobility changes which occur in the presence of various ligands [4]. ACE is able to explore changes in the conformation as well as charge changes of proteins. Unspecific and specific interactions on proteins can be measured without special reagents or kits. In order to compensate for changes on the migration time, which are not due to ligand binding, the mobility ratio of an EOF-marker and the protein is used [5]. Six repeats were done with a very good precision due to the use of a special rinsing procedure [6]. The RSDs [%] of the migration times and the mobility ratios were typically below 2%, very often below 0.2%. The influence of various charged species e.g. metal ions, such as Cu2+, Mn2+ and Mg2+, pharmaceutical cations as ephedrine hydrochloride and pirenzepine dihydrochloride or anions such as glutamic acid and succinic acid were tested on the migration behavior of BSA, ß-lactoglobulin and ovalbumin.

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Furthermore the influence of phosphorylation on protein interactions was investigated at the activation site of the extracellular signal-regulated kinase 1 (ERK1). Organic cations and metal ions showed clearly different interactions with the proteins. In most cases significant interactions were found, which differed markedly from protein to protein for different ligands. Thus selective binding mechanisms are likely. This offers very valuable insights into biological functions of protein-ligand interactions in aqueous media. Y. Xu greatly acknowledges the funding from the Alexander von Humboldt Foundation References: 1. Wenz, C. (2011) Performance of commercially available gels for protein characterization by capillary gel electrophoresis with UV detection on the Agilent 7100 CE System. 2, Meyer, C.,et al. (2012) Electrophoresis, in print. 3. Schirm, B., Wätzig, H. (1998) Chromatographia 48: 331-346. 4. Busch, M. H. A. et al. (1997) J. Chromatogr. A 777: 311-328. 5. Bose, S. et al. (1997) J. Chromatogr. B 697: 77-88. 6. El-Hady, D. et al. (2010) J. Pharm. Biomed. Anal. 52: 232–241 L-112 CE-based Characterization and Receptor Affinity Determination of Nanobodies. Rob Haselberg, Gerhardus J. de Jong, Govert W. Somsen, Utrecht University, Biomolecular Analysis, Utrecht, THE NETHERLANDS The epidermal growth factor receptor (EGFR) is a recognized target for therapy of human epithelial tumors. Recently, nanobodies were introduced as a new approach to antagonize EGFR. Nanobodies are small antibody fragments derived from heavy chain-only antibodies of camelids and possess attractive characteristics, like high chemical stability and nanomolar affinity. During nanobody development and production, assessment of protein purity and receptor interaction is essential. Capillary electrophoresis (CE) provides the possibility to probe both affinity and composition of intact proteins. This presentation outlines the design and application of CE methodologies for the characterization of the nanobody EGa1 and its interaction with EGFR. To allow efficient and reproducible CE of EGa1 (also in presence of EGFR), capillaries coated with a bilayer of Polybrene-poly(vinyl sulfonic acid) (PB-PVS) or a triple layer of Polybrene-dextran sulfate-Polybrene (PB-DS-PB) were employed. EGa1 was analyzed using background electrolytes (BGEs) of low and medium pH, revealing the presence of several product-related species that contributed to about 50% of the total peak area. CE-TOF-MS was carried out to further establish the composition of the nanobody samples. Affinity CE was performed using a BGE at physiological pH including the extracellular domain of EGFR. The nanobody and its related species showed significant shifts in electrophoretic mobility upon exposure to increasing concentrations receptor. From these experiments, Kd-values could be determined, revealing 1-2 nM affinities for EGa1 and co-products towards EGFR. It is concluded that CE employing coated capillaries provides a useful platform to simultaneously probe protein purity and protein-protein interactions with low-nM affinities. L-113 Size-selective Extraction of Biological Samples with Mesoporous Adsorbents for Protein and Peptides Analysis. Hanfa Zou, Hongqiang Qin, Fangjun Wang, Ren'an Wu, Dalian Institute of Chemical Physics, Chinese Academic of Sciences, Dalian, PR CHINA [KEYNOTE LECTURE] Sample preparation has been playing an important role in the analysis of complex samples. Mesoporous materials as the promising adsorbents have gained increasing research interest in sample preparations due to their desirable characteristics of high surface area, large pore volume, tunable mesoporous channels with well defined pore-size distribution, controllable wall composition, as well as modifiable surface properties. The recent advances of mesoporous materials in sample preparation with emphases on the size selective enrichment of peptides/proteins, specific capture of post-translational peptides/proteins and enzymatic reactor for protein digestion will be presented. L-114 Selective Transport Across Supported Liquid Membranes Coupled to CE for Direct Analysis of Samples with Complex Matrices. Pavel Kuban, Isaac K. Kiplagat, Andrea Slampova, Pavla Pantuckova, Petr Bocek, Institute of Analytical Chemistry of the AS CR v.v.i., Brno, CZECH REPUBLIC Liquid phase microextraction (LPME) has gained significant attention in analysis of samples with complex matrices in recent years since it efficiently combines sample clean-up and preconcentration of target analytes into one analytical step. Additionally, LPME uses reduced amounts of organic solvents, small volumes of donor and acceptor solutions, and can easily be semi-automated. One of the perspective LPME techniques employs supported liquid membranes (SLM) and was first described by Audunsson [1]. In subsequent years, SLMs have become a basis for further development of various sample pretreatment methods (e.g. hollow fibre-liquid phase microextraction (HF-LPME), and electromembrane extraction (EME)). The above methods are based on the same fundamental principle, i.e. transport of analytes across thin SLMs. A porous inert supporting material (usually polypropylene) is impregnated with water immiscible organic solvent to form the SLM and separates two compartments filled with donor and acceptor solutions. The SLM creates a barrier that enables selective transport of analytes from donor to acceptor solution, but retains all interfering matrix components. When the extraction is finished, the acceptor solution is injected into an analytical system. This simple experimental set-up can be used for simultaneous sample clean-up and preconcentration. In this contribution, capability of selective transport across SLMs accelerated by the use of electric field will be demonstrated as a suitable pretreatment method for samples with complex matrices along with some theoretical considerations on the selective transport process. Examples will be presented for determination of endogenous concentrations of inorganic ions and

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biomarkers in various biological matrices, such as in whole blood, blood serum/plasma, breast milk, urine, etc. Possibility for on-line combination with CE will be also discussed. Support from the Grant Agency of the Czech Republic (GA ČR, P206/10/1219) is gratefully acknowledged. References: [1] G. Audunsson, Anal. Chem. 58 (1986) 2714. L-115 No Drop Microextraction for Capillary Electrophoresis. Hye Ryeo Lee, Young Jin Koh, Jihye Kim2, Doo Soo Chung, Seoul National University, Seoul, KOREA Recently, single drop microextraction (SDME) has been demonstrated using a drop of an acceptor phase hanging at the inlet tip of a capillary for subsequent capillary electrophoresis (CE). The small surface-to-volume ratio of the drop enabled high enrichment factors to be obtained in a short extraction time. One practical issue in SDME was how to keep the drop attached to the capillary stably. Here we present novel but quite simple no drop microextraction (NDME) to solve this as well as other issues in SDME. Without forming a drop at the capillary tip, a small amount of the acceptor phase was injected into a separation capillary which had been filled with the run buffer. For headspace (HS) microextraction, the capillary was placed in the HS above the sample donor solution. Then volatile analytes in the HS were extracted into the acceptor phase in the capillary through the opening at the capillary tip. Liquid phase microextraction, either 2- or 3-phase, was also demonstrated in a similar way. After extraction, the capillary inlet was inserted in the run buffer vial and electrophoresis of the extracted analytes was carried out. Due to the lack of a hanging drop, the present scheme of NDME was extraordinarily simple and reliable. L-116 Elec troextrac tion : A New Elec tromigra tion -driven Enrichm ent Technique fo r Pep tidomics and Metabo lomics . Peter W. Lindenburg, Ubbo R. Tjaden, Jan van der Greef, Thomas Hankemeier, Leiden University, Division of Analytical Biosciences / Netherlands Metabolomics Centre, Leiden, NETHERLANDS Recently, electroextraction (EE) has emerged as a new electromigration-driven enrichment technique for peptidomics and metabolomics [1]. EE is an electromigration-based sample pretreatment technique which is fast, concentrating, reproducible, versatile and applicable to bioanalysis. EE takes place in a two-phase liquid-liquid system, consisting of an aqueous and an organic phase, where an applied electric field causes ions to be extracted very fast from the organic donor phase into the aqueous acceptor phase. As they enter the aqueous phase, the sample ions are concentrated closely after the liquid-liquid interface. With this approach, sample ions can be concentrated several orders of magnitude within a short time. In this lecture, the concept of EE is introduced and an overview is given on recent developments, focusing on various practical designs for EE. The online coupling of capillary EE (cEE) to LC-MS and the bio-analytical application of cEE-LC-MS to peptides as well as acylcarnitines in biological samples is presented. Finally, some promising approaches, concerning for example the combination of EE with CZE, chip-based EE and three-phase EE, will be treated. Special attention will be paid to large volume cEE-LC-MS. In this set-up, volumes of 100 μL and higher can be extracted and consecutively analyzed online with LC-MS. cEE takes place in a wide-bore capillary that is connected to a 2-way 10-port switching valve, which serves to couple cEE with LC-MS. Comparison of an injection using 6 minutes of large volume cEE-LC-MS with a normal LC-MS injection showed peak heights that were enhanced two-three orders of magnitude. Estimated LOD values were 10-50 nM for plasma peptides and 25-50 nM for acylcarnitines in urine. Recovery, linearity and repeatability were good to excellent. EE is a fast sample preconcentration technique that is quantitative, applicable to peptidomics and metabolomics, easy to automate, easy to hyphenate with LC-MS, and able to deal with large sample volumes (10-100 µL, with the possibility to further enlarge the volume). References [1] Lindenburg PW et al, J Chromatogr A. 2012 (3) 1249:17-24. L-117 A Recycling Immunoaffinity Microfluidic Device for Assessing Immune Function in Infants using Dried Blood Spots. Terry M. Phillips, Edward Wellner, Heather Kalish, National Institutes of Health, Immunochemistry, Bethesda, MD, USA [KEYNOTE LECTURE] A major problem in paediatric medicine is assessing the child’s immune system. Although the use of cytokines enables such an analysis to be performed, the quantity of blood required is often impossible to obtain from the subject under investigation. Dried blood spots are a useful whole blood collection device for studying biochemical parameters in children for several years, although the volume obtained from such spots (25 microliter) has restricted their use in multianalyte analyses. We have developed a recycling immunoaffinity chromatography system, based on a commercially available microfluidic device, capable of analysing 25 different analytes within a 10 microliter sample. The system is based on pumping a pre-labelled sample through an array of capillary immunoaffinity channels, each coated with a different immobilized antibody. This enables each channel to isolate and retain a single analyte, while passing the remainder of the sample onto the next channel. Following passage through the entire series of channels, the sample was collected and stored for further analysis. Detection of the bound analytes was achieved by laser-induced fluorescence (LIF), using a Bio-Rad DNA chip reader and a plastic chip holder designed to fit the reader. The array was capable of being regenerated up to 40 times, provided that the device was maintained at a cold temperature for the majority of the assay and stored at the same temperature. Each individual immunoaffinity channel was able to bind between 1.6 – 2.1 ng of analyte, depending upon the antibody immobilized, with lower limits of detection (LOD)

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in the order of 1.4–2.2 pg/mL. The inter- and intra-assay coefficients of variation (CV) for all 25 columns in the array were calculated to be 8.01+0.24 at analyte concentrations of 100pg/mL. Comparison to standard ELISA assays demonstrated r2 values in the range of 0.9003–0.9871 when analysed by least-squares linear regression. Examination of dried blood spot samples from a cohort of 2-3 day old infants divided into 3 groups with different clinically proven immune dysfunctions plus a normal, healthy control group demonstrated that the recycling device could efficiently detect the different groups using concentrations of different cytokine patterns and the findings correlated with the clinical; assessment. Further, the immobilized antibodies can be exchanged in order to measure other parameters of substituted with soluble receptors to measure bioavailability in micro samples. L-118 Reagent-Release Capillary Array-Isoelectric Focusing Device for Simple and Highly-Sensitive Bioanalysis. Hideaki Hisamoto, Osaka Prefecture University, Department of Applied Chemistry, Osaka, JAPAN We recently developed a reagent-release capillary (RRC) which releases the analytical reagents from capillary wall upon introduction of sample solution by capillary action, and the released reagents react with sample to give fluorescence response [Anal. Chem., 2007, 79, 908.]. This technique allowed the development of very simple and disposable multianalyte sensing device for use in bioanalyses by simply embedding various RRCs into PDMS microchannels. Here we prepared various RRCs for use in capillary isoelectric focusing (cIEF). In this case, covalent immobilization of poly(dimethylacrylamide) (PDMA) was carried out to suppress electro-osmotic flow, followed by physical adsorption of the mixture of carrier ampholyte (CA), specific reagents (SR), and other additives to the PDMA surface to construct a two-layer structure inside a square glass capillary. When the sample solution was introduced into the RRC, physically adsorbed CA and SR could be dissolved and released into the sample solution as in the same manner to the above described RRCs. Then, specific reaction of SR with sample molecule, mixing with CA, and exposure of the PDMA surface spontaneously occurred. Thus, simply applying electric field allowed us concentration and separation of reaction products based on the principle of IEF, which lead to very simple and highly sensitive analysis of various samples including proteins and enzyme activities. In this presentation, detection of proteins and enzyme activities will be presented [Lab Chip, 2010, 10, 3341., Proc. Micro-TAS, 2009, 2, 1204., Proc. Micro-TAS, 2011, 2025.]. For protein detection, non-selective labeling reagent of Quant-iT (Invitrogen) and selective reagents of fluorescently-labeled antibodies were immobilized as SR of RRCs. In these cases, binding with sample protein gave the fluorescent product, and separation and preconcentration by IEF was carried out by electric field application, thus, CCD image analysis allowed the highly-sensitive detection of various proteins. For enzyme activities, we focused on the use of rhodamine 110-based enzyme substrates. Since the enzyme reaction product of rhodamine 110 has positive and negative charges within one molecule, preconcentration based on the IEF principle allowed the highly-sensitive detection of enzyme activities. In each case, analysis has been done by the same operation procedure involving the simple introduction of the sample solution by capillary action, followed by application of electric field. Thus, the development of simple and highly-sensitive multi analyte sensing device by arraying these capillaries on a single microfluidic device is expected. L-119 High-throughput Screening of mAb Charge Variants Using Microchip-CZE. Bahram Fathollah, Tobias Wheeler, Lucy Sun, Rajendra Singh, Caliper, a Perkin Elmer Company, Microfluidics R&D, Alameda, CA, USA We have developed an automated, high-throughput microfluidic platform that performs multiple protein characterization assays. The current assays include (1) purity assessment by microchip CE-SDS, and (2) profiling of N-glycans, both with an analysis time of < 60 s per sample. More recently, we have developed a third assay: a high-throughput, microchip-CZE method for the characterization of mAb charge variants. In this method, mAbs with 7 < pI < 10 are fluorescently labeled while conserving net charge. Labeled sample is then drawn into a microchip through a capillary sipper and applied vacuum. Once in the microchip, the sample is electrokinetically injected and separated in a microchannel that is dynamically coated to suppress electroosmotic flow. Sufficient resolution of charge variants is achieved in < 60 s and the labeling and analysis of 96 samples requires < 2 hours. This presentation describes the separation method and the sample workflow, and demonstrates the resolution, speed, sensitivity, reproducibility, and ease-of-use of the method, for the high-throughput screening of mAb charge variants. L-120 Multiplatform Metabolomic Strategy to Study Leishmania Drug Resistance Mechanisms. Gisele A. B. Canuto1, David Rojo2, Emerson A. Castilho-Martins3, Angeles Lopez-Gonzalvez2, Luis Rivas4, Coral Barbas2, 1CEMBIO, Universidad CEU San Pablo and Institute of Chemistry University of Sao Paulo, Sao Paulo, BRAZIL; 2CEMBIO Universidad CEU San Pablo, Madrid, SPAIN; 3CEMBIO Universidad CEU San Pablo and Instituto de Biosciencias University of Sao Paulo, Sao Paulo, BRAZIL; 4Centro de Investigaciones Biologicas (CSIC), Madrid, SPAIN [KEYNOTE LECTURE] Metabolomics, has become an invaluable tool to unveil biology of pathogens, with immediate application to chemotherapy. Leishmaniasis is a parasitic disease with visceral and cutaneous manifestations that causes significant morbidity and mortality worldwide. This disease occurs in four continents and is considered endemic

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in 88 countries, 72 of them developed. Moreover, economic globalization and increased travel have extended its reach to people. Organic pentavalent antimonials [Sb(V)] have been the first-line drugs for the treatment of leishmaniasis for the last six decades, and clinical resistance to these drugs has emerged as a primary obstacle to successful treatment and control. Mechanisms of action and resistance are still under study and new therapies, including miltefosine are being introduced. However, problem of drugs commonly used is the development of resistance; therefore alternative treatments are urgently needed. It is currently accepted that there is not one single technique capable of obtaining the whole metabolic fingerprint of a biological system either due to their different physico-chemical properties or concentrations. The knowledge of the metabolic profiles of the cell’ system is very important for understanding its physiology. In this work we have explored the capability of capillary electrophoresis, liquid chromatography, and gas chromatography coupled to mass spectrometry (CE-MS, LC-MS, GC-MS) to unveil metabolic changes associated with drug resistance in leishmania through metabolic fingerprinting of susceptible vs resistant parasites. After alignment, filtering and univariate or multivariate analysis, different metabolic pathways appeared altered. The biochemical mechanisms of action of antimony are not completely clear yet, nevertheless it is supposed to kill parasites via oxidative stress. Our results have showed alterations in arginine pathway. This route is responsible to provide spermidine to glutathione pathway, in which it is used with glutathione to produce glutathionyl-spermidine and trypanothione, the main anti-oxidant defense of the parasites against host cell. According to our results, this behavior also correlates with the response to miltefosine treatment but, as far as we know, there is not any description of this relationship although it can explain the cross-resistance presented by several patients. L-121 Microfluidic Chip-Capillary Electrophoresis Devices for Metabolomics Applications. Ying-Sing Fung, Hong Kong University, Chemistry Department, HONG KONG The recently developed Microfluidic chip-Capillary Electrophoresis (Microchip-CE) devices provides a promising tool for metabolomics applications due to its capability for onsite analysis, quick turn-over for results and flexibility to handle complex and highly variable samples through on-demand sample preparation at microchip prior to high efficient CE separation and analyte-specific detection. In the present presentation, we shall discuss the design and application of microchip-CE devices as a tool to assess toxicity due to exposure to pesticides and food adulterated with toxic chemicals. The design and fabrication of microchip-CE devices to handle samples with high analyte variability and the enhancement of detection reliability via on-chip standard addition and specific detection will be discussed. Results for determining metabolites in urine and body fluids will be presented to illustrate the principle and applicability of the devices developed and fabricated for metabolomics applications. L-122 In Vivo Solid-phase Microextraction: New Tool in Metabolomics. Dajana Vuckovic1, Ines de Lannoy2, Brad Gien2, Robert Shirey3, Len Sidisky3, Janusz Pawliszyn4, 1University of Waterloo/University of Toronto, Donnelly Centre of Cellular and Biomolecular Research, Toronto, CANADA; 2NoAB BioDiscoveries Inc., Mississauga, CANADA; 3Supelco, Bellefonte, PA, USA; 4University of Waterloo, Waterloo, CANADA Sample preparation has a strong impact on the quality of metabolomics studies and biomarker discovery, but metabolism quenching step is often omitted in the studies of biological fluids such as blood. In vivo solid-phase microextraction (SPME) is a new and effective sample preparation method for untargeted LC-MS metabolomics studies which incorporates metabolism quenching step directly during the sampling process. This makes the technique ideally suited to study the changes in unstable and short-lived metabolome in blood, the type of experiment difficult to perform in untargeted fashion using traditional methods based on blood withdrawal. In this study, we compared the performance of in vivo SPME sampling of circulating mouse blood versus traditional blood withdrawal in combination with solvent precipitation, ultrafiltration and ex vivo SPME in order to study the effect of single carbamazepine dose on endogenous metabolism of mice (n=4 mice per group). All samples were analyzed using both positive and negative electrospray reversed-phase and HILIC LC-MS methods on benchtop Orbitrap system. We systematically compared in vivo SPME versus traditional methods in terms of metabolome coverage, method precision and ionization suppression. Our results show that simultaneous extraction of hundreds to thousands of metabolites is possible with newly introduced hypodermic needle SPME devices, but the overall metabolite coverage of SPME using a single coating is lower than that of solvent precipitation and ultrafiltration due to microextraction nature of the technique. The use of multiple complementary coatings further improved metabolite coverage, especially of polar metabolome. More importantly, in vivo SPME successfully captured unstable metabolites not observed by any of the traditional methods, with approximately 4-5% unique features such as carotenes and β-NAD. Whereas in vivo SPME shows excellent correlation to ultrafiltration and solvent precipitation for stable metabolites, the quantitation of unstable metabolites which could be observed by all methods (such as glutathione or adenosine) was significantly improved by the incorporation of metabolism quenching step. The repeatability of in vivo SPME sampling was suitable to study intra- and inter-animal variability in concentrations of many metabolites and to identify metabolites with rapidly changing concentrations on the timescale of the experiment. Thus, the technique is ideally suited for temporal, spatial, and longitudinal studies of the same living system and especially useful for studies of small rodents such as mice, where limited blood volume is available or precious/genetically-

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modified strains are used. In conclusion, our results clearly show that SPME can play an important role in untargeted studies to accurately capture metabolome at the time of sampling - an important advance in the field of sample preparation for both global and targeted LC-MS metabolomics. L-123 Effect of Dietary Polyphenols on K562 Leukemia Cells: A Foodomics Approach. Alberto Valdes1, Carolina Simo1, Clara Ibanez1, Lourdes Rocamora2, Jose Antonio Ferragut2, Virginia Garcia-Canas1, Alejandro Cifuentes1, 1CSIC, Laboratory Foodomics, Madrid, SPAIN; 2UMH, Alicante, SPAIN [KEYNOTE LECTURE] The anti-proliferative effect of dietary polyphenols from rosemary on two human leukemia lines, one showing a drug-sensitive phenotype (K562), and another exhibiting a drug-resistant phenotype (K562/R), is investigated following a Foodomics strategy. To this aim, whole-transcriptome microarray together with a MS-based non-targeted analytical approach (via capillary electrophoresis-time of flight mass spectrometry, CE-TOF MS, and ultrahigh performance liquid chromatography-time of flight mass spectrometry, UPLC-TOF MS) have been employed to carry out Transcriptomics and Metabolomics analyses, respectively. Functional enrichment analysis was done using Ingenuity Pathway Analysis (IPA) software as a previous step for a reliable interpretation of transcriptomic and metabolomic profiles. Rosemary polyphenols altered the expression of ~1% of the genes covered by the whole-transcriptome microarray in both leukemia cell lines. Overall, differences in the transcriptional induction of a number of genes encoding phase II detoxifying and antioxidant genes, as well as differences in the metabolic profiles observed in the two leukemia cell lines suggest that rosemary polyphenols may exert a differential chemopreventive effect in leukemia cells with different phenotypes. IPA predictions on transcription factor analysis highlighted inhibition of Myc transcription factor function by rosemary polyphenols, which may explain the observed antiproliferative effect of rosemary extract in the leukemia cells. Metabolomics analysis suggested that rosemary polyphenols affected differently the intracellular levels of some metabolites in the two leukemia cell sublines. Integration of data obtained from Transcriptomics and Metabolomics platforms was done by overlaying datasets on canonical (defined) metabolic pathways using IPA software. This strategy enabled the identification of several differentially expressed genes in the metabolic pathways modulated by rosemary polyphenols providing more evidences on the effect of these compounds. L-124 Analysis of Secretome and Plasma using Reversed Phase Liquid Chromatography in Mass Spectrometry-based Pipeline for Enhanced Biomarker Development. Miroslava Stastna, Jennifer Van Eyk, Johns Hopkins University, School of Medicine, Baltimore, MD, USA Since the proteins secreted by the cells (secretomes) as the response to various condition alterations and pathological stimuli are likely to be secreted into blood/plasma, the pre-selection of biomarker candidate from cell secretomes with subsequent validation in plasma is a promising approach. We used a reversed phase liquid chromatography under various experimental conditions to optimize the pre-separation of protein complex mixtures of cardiac myocyte secretome and plasma prior to mass spectrometry with the goal of maximizing the proteome coverage. The various types of reversed phase columns and pH conditions were tested and evaluated in terms of number and types of proteins detected. As well, we tested the effect of various simulation/stimulation (disease model, various factors/proteins/drugs added) on cardiac myocytes protein secretion. We identified approximately 200 unique proteins, some of them were secreted exclusively during specific cardiac myocyte treatment conditions. Several proteins were found also in the plasma of patients who underwent myocardial ischemia or infarction. In conclusion, the analysis of proteins secreted by various cells is an alternative approach to the direct analysis of blood/plasma for the discovery of potential protein biomarkers and therapeutic targets. L-125 Allergomic Study of Cypress Pollen via Combinatorial Peptide Ligand Libraries. Elisa Fasoli1, Alfonsina D'Amato1, Pier Giorgio Righetti1, Youcef Shahali2, Jean-Pierre Sutra2, Norihiro Futamura3, Egisto Boschetti4, Helene Senechal2, Pascal Poncet2, 1Politecnico di Milano, Department of Chemistry, Materials and Chemical Engineering “Giulio Natta”, Milano, ITALY; 2ESPCI ParisTech, Paris, FRANCE; 3Plant Molecular Biology Laboratory, Department of Molecular and Cell Biology, Ibaraki, JAPAN; 4Bio Rad Laboratories, Gif-sur-Yvette, FRANCE The Cupressus sempervirens, (Cups) is the major cypress species growing around the Mediterranean basin. Although Cups pollen represents one of the main aeroallergens in southern Europe, only two Cups allergens have so far been identified and reported: Cup s 1 and Cup s 3. The aim of this study was to identify and characterize the largest number of allergens present in cypress pollen using an immuno-proteomic approach coupled with combinatorial peptide ligand libraries (CPLL) capture and mass spectrometry (MS) analysis. A sequential pollen protein extraction method was developed with the use of non-ionic detergent and urea followed by an extraction mimicking physiological conditions. Both extracts were then treated with CPLLs in order to visualize low-abundance species. Control extracts and CPLL eluates have then been resolved by SDS-PAGE (1D) and two-dimensional (2D) gel electrophoresis, blotted and confronted with sera from cypress allergic patients. The extracted proteins including IgE binding components were identified using MS analysis, followed by queries in UniProtKB database and an Expressed Sequence Tags (EST)-database specific of Cryptomeria japonica. More than 100 unique gene products were identified by analyzing the eluates and control loaded onto 1D SDS-PAGE. Few proteins were found in control samples and much more supplementary species were

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further identified upon CPLL treatment. Among proteins characterized in 1D, 59% are potentially allergenic in Cupressaceae because they were already described as allergens in various sources, especially a 4-EF hand Ca binding protein corresponding to the group 4 of allergens in Cupressaceae. Out of the IgE binding proteins characterized in 2D gels, 11 were already reported as allergens in various sources including the two major known allergens of Cupressaceae (group 1 and 2). Three IgE binding proteins, not previously reported as allergens, are newly described in this study.The improvement in protein extraction combined with the enrichment of low-abundance species allowed us to considerably extend the repertoire of potential Cypress pollen allergens. L-126 Microfluidic Chemical Separation Devices with Integrated Nanoelectrospray Emitters. Scott Mellors, Nick Batz, Will Black, J. Michael Ramsey, University of North Carolina-Chapel Hill, Chapel Hill, NC, USA [KEYNOTE LECTURE] We have been developing microfabricated fluidic devices for characterizing biological and chemical materials using various chemical separation strategies. The separation mechanisms used in our devices have focused on capillary electrophoresis (CE) and pressure driven chromatography (LC) as well as multidimensional separations that combining these techniques. Detection has been performed using an integrated nano-electrospray emitter coupled to commercial electrospray ionization (ESI) mass spectrometers. The electrospray emitter design includes a type of microfabricated liquid junction that allows electrical contact for applying the electrospray potential, produces a pumping source to transport ions from the CE experiment to the emitter, and can provide a make-up flow for modifying the buffer for electrospray compatibility. The integrated emitters have been shown to be as efficient as pulled capillary nano-electrospray emitters. Efficient CE separations of peptides and proteins have been demonstrated showing symmetrical peak profiles. Peak capacities of nearly 100 can be generated in one minute for proteolytic peptides. CE-ESI chips have also been configured to allow high throughput experiments to be performed for gathering both primary mass spectra and tandem mass spectra of peptides at the rate of twenty CE runs per hour with sequence coverage comparable to LC-MS experiments. Microfabricated CE-ESI devices will be described for the analysis of intact proteins, proteolytic digests, glycans, and small molecules. L-127 Unmanned Platform for Long-Range Remote Analysis of Volatile Compounds in Air Samples. Eric Tavares da Costa1, Carlos A. Neves1, Guilherme Minoru Hotta1, Denis Tadeu Rajh Vidal1, Marcelo Fagundes Barros1, Arturo A. Ayon2, Carlos D. Garcia2, Claudimir Lucio do Lago1, 1Universidade de Sao Paulo, Sao Paulo, BRAZIL; 2University of Texas San Antonio, San Antonio, TX, USA This presentation will describe a long-range remotely controlled CE system built on an all-terrain vehicle. A four-stroke engine and a set of 12-V batteries were used to provide power to a series of subsystems that include gas sensors, drivers, communication, computers, and a capillary electrophoresis module. This dedicated instrument allows air sampling using a polypropylene porous tube, coupled to a flow system that transports the sample to the inlet of a fused-silica capillary. A hybrid approach was used for the construction of the analytical subsystem combining a conventional fused-silica capillary (used for separation) and a laser machined microfluidic block, made of PMMA. A solid-state cooling approach was also integrated in the CE module to enable controlling the temperature and therefore increasing the useful range of the robot. Although ultimately intended for detection of chemical warfare agents (CWAs), the proposed system was used to analyze a series of volatile organic acids. As such, the system allowed the separation and detection of formic, acetic, and propionic acids with signal-to-noise ratios of 414, 150, and 115, respectively, after sampling by only 30 s and performing an electrokinetic injection during 2.0 s at 1.0 kV. Ref: Electrophoresis 2012 - Instrumentation Special Issue L-128 Microfluidic Liquid Chromatography Separations within the Column Dead-Volume: Conceptual Thoughts. Iulia Lazar, Virginia Tech, Blacksburg, VA, USA A microfluidic chip that enables liquid chromatography (LC) separations and matrix assisted laser desorption ionization (MALDI)-mass spectrometry (MS) detection was developed. The microfluidic device provides for the separation of a mixture into its components, and integrates a novel microchip-MS interface that facilitates the orthogonal transposition of the sample from the LC channel into an array of reservoirs for MALDI-MS detection. The device enabled the detection of peptides in the low fmol range with a dynamic range in excess of 10^3, and the detection of proteins with biomarker potential from MCF10A breast epithelial cell extracts. The ability to perform complex sample processing prior to MALDI-MS detection, and to use LC separations with large sample loading capacity, provide a clearly superior alternative to the static, low dynamic range MALDI target plates that are typically used in proteomic applications. The proposed device enables off-line sample collection, processing and storage into an array, facilitating convenient sample analysis at remotely-located MS labs, with no need for MS expertise at the collection site. The proposed LC-MALDI-MS chip design also promotes a new concept for performing sample separations within the limited dead-volume (V0) of a separation channel. The challenges associated with the practical implementation of such a separation strategy, such as preservation of chromatographic efficiency and peak capacity will be discussed. Most importantly, the challenges imposed on the pumping system, in terms of flow rate and gradient generation will be emphasized.

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L-129 Capillary Zone Electrophoresis Coupled with an Electrokinetically-pumped Electrospray Interface for Applications in Proteomics. Norman Dovichi, University of Notre Dame, Notre Dame, IN, USA [KEYNOTE LECTURE] We demonstrate the use of capillary zone electrophoresis for the analysis of a tryptic digest of a sample of intermediate protein complexity, the secreted protein fraction of Mycobacterium marinum. For electrophoretic analysis, 11 fractions were generated from the sample using reversed phase liquid chromatography; each fraction was analyzed by CZE-ESI-MS/MS, and 334 peptides corresponding to 140 proteins were identified in 165 min at 95% confidence (0% FDR). In comparison, 388 peptides corresponding to 134 proteins were identified in 180 min by triplicate UPLC-ESI-MS/MS analysis of the unfractionated peptide mixture at 95% confidence (0% FDR). 62% of peptides identified in CZE-ESI-MS/MS and 67% in UPLC-ESI-MS/MS were unique. CZE-ESI-MS/MS favored basic and hydrophilic peptides with low molecular mass. Combining the two data sets increased the number of unique peptides by 53% L-130 Capillary Electrophoresis Coupled to ESI Mass Spectrometry for the Study of Matrix Metalloproteinase. Xu Wang, Erwin Adams, Ann Van Schepdael, KU Leuven, Pharmaceutical Analysis, Leuven, BELGIUM Enzymes are major drug targets in drug discovery and development processes in the pharmaceutical and biotechnology industry. Matrix metalloproteinases (MMPs) have been considered as a potential therapeutic target in human cancer. The MMP-2 and MMP-9 have been particularly implicated in tumor invasion and metastasis formation [1]. With regard to enzyme assay, CE based enzyme assays can be divided in two categories, pre-capillary (offline) assays and in-capillary (online) assays. Although CE consumes only nanoliter volumes of sample, offline assays still need several hundreds of microliters and require a number of operations [2]. In-capillary enzyme assays integrate all reaction and analysis steps into a single instrument leading to further automation of the assays as well as extremely limited consumption of the enzyme. Mass spectrometry (MS) detection has already been used in pre-capillary assays to perform kinetic studies and identify enzyme inhibitors. However, there is no report on the use of MS as a detector for in-capillary assays. The combination of an in-capillary assay with MS would allow perform label free screening of enzyme inhibitors. In this study, a background electrolyte consisting of 20 mM ammonium acetate (pH 6.8) and a sheath liquid of water-methanol (1:1, v/v) containing 0.5% formic acid at a flow rate of 6 μl/min were selected. This system was operated in the positive ion mode with a detection limit in the nanomolar range and was successfully applied to the kinetic study of MMP-9. The results obtained were comparable with our previous study [3]. Next, the MMP inhibitory activity of several Chinese medicines can be screened with this in-capillary setup. References [1] Wang, X., Li, K., Adams, E., Van Schepdael, A. Matrix metalloproteinase inhibitors: a review on bioanalytical methods, pharmacokinetics and metabolism. Current Drug Metabolism, 2011, 12, 395-410. [2] Hai, X., Yang, B., Van Schepdael, A. Recent developments and applications of EMMA in enzymatic and derivatization reactions. Electrophoresis, 2012, 31, 211-227. [3] Hai X., Wang X., El-Attug M., Adams E., Hoogmartens J., Van Schepdael A. In-Capillary Screening of Matrix Metalloproteinase Inhibitors by Electrophoretically Mediated Microanalysis with Fluorescence Detection. Anal. Chem. 2011, 83, 425–430.. L-131 Latest Advances in CESI-MS for the Analysis of Peptide Mixtures: Application to Proteomics and Biopharmaceutical Samples. Anna Lou, Dona Neloni Wijeratne, Chitra Ratnayake, Jean-Marc Busnel, Beckman Coulter, Brea, CA, USA In this presentation, we will report some of the latest developments in the field of CESI, a technology which fully integrates the high efficiency and ultra-low flow characteristics of capillary electrophoresis (CE) with electrospray ionization mass spectrometry (ESI-MS). Additionally to profit from the intrinsic capabilities of CE, as the interface used in this platform enables the generation of a stable electrospray at flow rates below 10 nL/min, it also benefits from the nanoESI regime which favors high ionization efficiencies, resulting in higher sensitivities and reduced ion suppression of co-migrating compounds. Herein, after having reviewed some of the major fundamental aspects of CESI, the emphasis will be on the application of this technology to the analysis of complex mixtures of peptides. In this context, various examples dealing with both proteomic analysis and monoclonal antibody characterization will be reported. Through various examples, it will be demonstrated that CESI, on top of providing some unique advantages such as sensitivity and flexibility, is truly complementary to LC with an increased compatibility with small and hydrophilic peptides but also with large ones, whose behavior is close to the one of small proteins. All in all, we believe that CESI provides analytical capabilities, which makes it a very valuable platform in the field of complex peptide mixture analysis.

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L-132 Field-Flow Fractionation: Improving the Method by Changing the Fluid-analyte-walls Interactions. Gary W. Slater, Tyler N. Shendruk, University of Ottawa, Physics Department, Ottawa, CANADA [KEYNOTE LECTURE] Field-Flow Fractionation (FFF) is a flexible separation method because of the wide variety of transverse external fields that can be used to create distinct concentration distributions of differing analytes. However, in order to capitalize on these differing distributions, a non-uniform flow profile is needed to carry the samples through the system with different elution times. It is generally taken for granted that the velocity of the carrier fluid obeys Poiseuille flow but we suggest three surface modifications that could be made to alter the flow profiles and retention in FFF systems in a controlled manner. One way to modify FFF would be to move the maximum flow velocity away from the mid-point of the channel by replacing one wall with a nanoengineered slip surface. Slip at the top wall enhances the performance of normal-FFF, while a large slip at the bottom wall results in a novel separation method that we call slip-FFF. Another proposal is to graft a dense polymer brush onto the accumulation wall of the microchannel to perform selective-steric-FFF. This surface modification adds two novel effects to FFF: analytes must pay an entropic cost to enter the brush and the brush has a hydrodynamic thickness that shifts the no-slip condition. Small analytes can enter the low-velocity region within the brush, while large analytes are prohibited from this region and subject to fast flows. A simple theoretical model and multi-particle collision dynamic simulations predict large improvements when analytes are smaller than the characteristic length scale of the brush. Finally, wall effects can be modified indirectly by reducing the channel height. Large analytes in microfluidic channels are perpetually near walls and so have increased friction coefficients. In this case, hydrodynamic interactions diminish the non-monotonicity of the elution order and conserve excellent resolution in the normal-mode FFF regime, as observed in experiments, simulations and numerical calculations. L-133 The Sequential Use of pH and Solvent Gradients can Double or Triple the Peak Capacity and the Resolving Power of High- or Ultra-Pressure Liquid Chromatography. Joe Foley, Adam Socia, Drexel University, Department of Chemistry, Philadelphia, PA, USA pH gradient elution followed by solvent gradient elution can be used advantageously in the context of sequential-elution liquid chromatography (SE-LC) to dramatically improve separations of ionogenic and neutral compounds via high- or ultra-pressure liquid chromatography. In SE-LC, a sample comprised of different classes of compounds is introduced into a chromatographic system that is highly retentive for all compounds because of a weak mobile phase that is initially employed. The highly-retained compounds are then subjected to a sequence of r mobile phases, each of which is designed to elute one class of compounds, the potential exception being the last mobile phase that could be more general if necessary and capable of eluting all compounds not previously eluted by any of the prior mobile phases. The result of SE-LC is the sequential elution and separation of the sample components by class and also within class, i.e., each compound within a category is also separated from other compounds in the same category. The benefits of SE-LC with r sequential elution modes are (i) an r-fold increase in peak capacity, (ii) an increase in dimensionality, (iii) a ln r-fold decrease in the disorder (entropy) of the separation, and (iv) a significant increase in the probability that all sample components will be resolved. The theory is summarized and several applications are reported, with an emphasis on an approach utilizing one or two pH gradients prior to a solvent gradient that can be employed to separate weak acids, weak bases, and neutral compounds from each other. L-134 Electromigration Dispersion due to an Interaction with a Neutral Selector. Pavel Dubsky, Vlastimil Hruska, Martin Benes, Jana Svobodova, Bohuslav Gas, Charles University in Prague, Faculty of Science, Department of Physical and Macromolecular Chemistry, Prague, CZECH REPUBLIC Electromigration dispersion (EMD) in capillary zone electrophoresis (CZE) is traditionally ascribed to an effect when an analyte considerably influences conductivity in its own zone [1]. We show for the first time herein that a parallel phenomenon may arise from an interaction of an analyte with a selector, such as utilized, e.g. in chiral CZE. Remarkably, the selector does not need to be charged, and the EMD due to an interaction with a neutral selector shall be discussed theoretically and demonstrated experimentally in this contribution. Recently we extended our linearized model of CZE [2] with a first-order nonlinear term that gives us an indication of nonlinear phenomena, namely realistic peak shapes due to EMD [3]. In this study, we inspect a special case of a complexing system consisting of a fully charged analyte interacting with a neutral selector and two intact BGE constituents in the light of the new model. It results that the EMD strongly depends on the selector concentration. Interestingly, it does not change from one limit case of no selector (EMD corresponding to the sole analyte) to the other limit of infinite selector concentration (EMD corresponding to the sole analyte-selector complex) only monotonously, but it may present an abrupt extreme giving rise to a spectrum of EMD dispersed analyte zones, which is more promoted as the complexation constant or the difference between the free and complexed analyte mobilities increase. The model outlined above was compared with an outcome from an advanced version of our simulator SIMUL, capable of solving a complete numerical solution of the (nonlinearized) model [4]. A perfect match was observed. Finally, a real systems of R-flurbiprofen as an analyte and three types of β-cyclodextrins (dimethyl-, trimethyl-, and native) were studied. Analyte peaks varying from

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almost perfectly symmetrical through highly EMD dispersed triangular shapes back to symmetrical ones were observed depending on the selector concentration, and the shapes again were in an exact agreement with theoretical predictions. This work was financially supported by the Grant Agency of the Charles University grant number 669412 and the Grant Agency of the Czech Republic, grant numbers 203/08/1428 and P206/12/P630. REFERENCES: 1. Gaš B., Štědrý M., Kenndler E., Electrophoresis 18 (1997) 2123. 2. Štědrý M., Jaroš M., Hruška V., Gaš B., Electrophoresis 25 (2004) 3071. 3. Hruška V., Riesová M., Gaš B., Electrophoresis 33 (2012) 923. 4. Hruška V., Beneš M., Svobodová J., Zusková I., Gaš B., Electrophoresis 33 (2012) in press L-135 Interfacing Microfluidics with Microelectronics via Droplet. Bingcheng Lin, Dalian Institute of Chemical Physics, Dalian, CHINA [KEYNOTE LECTURE] Microfluidics has been proved at least an academic success so far. For its further development, it is essential to introduce robustness, stringent quality control, preciseness, standardization and reproducibility from industrial sectors. Therefore, microelectronics, which has demonstrated all these advantages, will be an important technology to be introduced into microfluidics. Droplet, which has exhibited promising preformance in high-throughput chemical synthesis, DNA sequencing, drug screening, etc., is regarded as a suitable technique to interface these two technologies in between. Here we demonstrate our recent achievements in droplet technique. A microfluidic device integrated with microvalves was developed for the manipulation of droplets. The generation, sizing, fusion, fission and trapping of droplets could all be achieved by controllable turning on and off microvalves of different solutions. A novel electrical-based method was also developed that can selectively break a droplet and extract its contents into aqueous buffer in a microfluidic device. The demonstrations showed the potential application in the fields of microsphere synthesis and assembly, on-line SPE of DNA, and manipulation of single C. elegans etc. in our lab. In the ways of dealing with droplets, digital microfluidics is at least one step closer to a high-level interfacing between microfluidics and microelectronic technology, compared with traditional channel microfluidics. In digital microfluidics, electric fields are applied to manipulate droplets under electrowetting, with which precise control of the movement, position and even logistics of droplets can be simply achieved using software-driven switches. In the preliminary experiments, prototype modules of logic control and feedback control of droplets were implemented in our digital microfluidic chips. L-136 Harnessing Enzymatic Machining at the Nano- and Micro-scales to Enable Enhanced Separations. Victor Ugaz, Texas A&M University, Chemical Engineering Department, College Station, TX, USA We describe a microfluidic-based filtration method capable of performing simultaneous size-based isolation and enrichment of cells from whole blood. Instead of forcing a cell laden suspension to flow through tiny pores in a membrane filter, we are able to construct a filter oriented along the centerline of the microchannel so that it creates a barrier between the left and right hand sides. When this geometry is incorporated into a curved flow path, the resulting centrifugal forces that arise due to fluid motion act to push the cells across the centerline barrier from the inside wall to the outside wall, with only the cells smaller than the barrier gap able to pass across. High-efficiency filtration is demonstrated experimentally using whole blood (both alone and spiked with cancer cells) and mixtures of polystyrene beads, with results validated by computational simulations. We also discuss construction of this novel device design, which is enabled by harnessing specific biochemical interactions between an enzyme and a biodegradable substrate to perform precise flow-actuated micromachining. Patterning and etching occur simultaneously by co-injecting laminar streams containing an aqueous enzyme solution and a protein inhibitor, enabling the position, size, and depth of the etching zone to be precisely controlled. The resulting filtration design virtually eliminates clogging and does not impose an excessive pressure drop because the barrier is oriented parallel to the flow direction rather than perpendicular to it. Moreover, this approach is most effective at high flow rates because the magnitude of the curvature-induced transverse flow is maximized under these conditions, making it ideally suited for high-throughput processing (ml/min flow rates) of large sample volumes. L-137 A Microfluidic UPLC-MS System for Small and Large Molecule Analysis. Steven Cohen, Giuseppe Astratita, Giorgis Isaac, Angela Doneanu, Jay Johnson, Jim Murphy, Waters Corporation, Milford, MA, USA This presentation will describe novel microfluidic devices optimized for LC-MS analysis and illustrate the flexibility of this platform for analyzing a wide variety of samples including complex mixtures such as those analyzed in metabolomics and lipidomics studies. The devices are fabricated from ceramic materials that permit operation at high pressure with sub 2 micron particles and thus are capable of producing highly efficient UHPLC operation. By optimizing the channel design, and incorporating an integral mass spectrometer emitter, on-board column heating and flexible column formats, the devices are suitable for analyzing a variety of both small and large molecule samples with performance comparable to that achieved with analytical scale LC-MS analysis. One design employs devices with an internal channel diameter of 150 microns that permits no-loss direct injection of microliter sample volumes, with a sensitivity enhancement compared to 2.1 mm ID columns of 20 - 50x. Column efficiency is equivalent to analytical scale chromatography, with peak capacities exceeding 200 within a 10 minute analysis, and greater peak capacities achieved with longer gradients. For metabolomics studies, typical samples were plasma and various organ tissue extracts, and were screened for metabolites by

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reversed phase and HILIC chromatography. Lipids were analyzed from solvent extracted samples, and amine analysis (including amino acids and biogenic amines) used deproteinized plasma and a rapid, simple one step derivatization protocol. Q-Tof and triple quadrupole mass spectrometers were used for untargeted and targeted analyses, respectively. Comparisons with analytical scale columns demonstrated equivalent results, with solvent consumption reduced by > 50 x. Intact protein separations were carried out on a wide pore C4 column, and exhibited excellent sensitivity with very high signal to noise with samples such as reduced monoclonal antibodies requiring only low nanogram amounts for analysis. L-138 New Instrumental Developments for Fast CE-MS and Hyphenation of CE-MS with Electrochemistry. Frank-Michael Matysik, Marco Grundmann, Peter Palatzky, Rebekka Scholz, Jonas Mark, Marija Cindric, University of Regensburg, Institute of Analytical Chemistry, Regensburg, GERMANY Capillary electrophoresis (CE) coupled to mass spectrometry (MS) is increasingly applied in various fields of bioanalytical research. Conventional CE-MS systems employ rather long capillaries (≥ 80 cm) with IDs of 50 or 75 µm. This contribution demonstrates that short capillaries (about 20 cm in length) with IDs down to 5 µm can be used in conjunction with a commercial sheath flow interface to perform CE-MS measurements. This configuration offers very fast separations in the time scale of seconds. Corresponding examples will be presented including catecholamine separations [1] and studies of hyaluronan oligosaccharides [2]. It will be shown, that capillaries with small IDs of less than 50 µm allow for the use of nonconventional electrolytes and can lead to very efficient CE-MS separations. In addition, a novel strategy for efficient sample injection for CE will be introduced. Very small sample volumes can be handled with the help of an injection capillary. The sample is directly injected from the injection capillary onto the inlet of the separation capillary. The second part of the presentation will introduce the concept of electrochemically assisted injection (EAI) in conjunction with CE-MS. This approach enables CE-MS studies of neutral analytes in terms of electrochemical generation of charged product species [3]. Selected examples will illustrate that EAI-CE-MS can be used as a novel means for the CE separation of neutral analytes with full compatibility with electrospray ionization (ESI) MS. In addition, the performance of ESI-MS can be considerably improved in case of hydrophobic compounds. A fully automated EAI system was constructed and optimized with the help of scanning electrochemical microscopy [4]. References: [1] Grundmann M., Matysik F.-M., Anal. Bioanal. Chem. 401 (2011) 269. [2] Grundmann M., Rothenhöfer M., Bernhardt G., Buschauer A., Matysik F.-M., Anal. Bioanal. Chem. 402 (2012) 2617. [3] Scholz R., Matysik F.-M., Analyst 136 (2011) 1562. [4] Palatzky P., Matysik F.-M., Electroanalysis 23 (2011) 269. L-139 Study on Bioconjugation of Quantum Dots by Combining Capillary Electrophoresis with Fluorescence Correlation Spectroscopy. Jicun Ren, Shanghai Jiaotong University, Department of Chemistry, Shanghai, CHINA Quantum dots (QDs) are luminescent inorganic nanomaterials (also called as semiconductor nanocrystals), and exhibit unique fascinating optical properties such as broad excitation spectra, narrow and size-dependent emission profiles, long fluorescence life time, and good photo-stability due to their quantum confinement and special components. Currently, QDs as labeling probes have been successfully used in vitro and in vivo imaging, immunoassay, DNA hybridization, and potential photodynamic therapy. The characterization of QDs bioconjugates is of great important in biological applications. In this paper, we systematically investigated the conjugation of QDs with certain biomolecules using capillary electrophoresis (CE) and fluorescence correlation spectroscopy (FCS). Commercial QDs and aqueous synthesized QDs in our lab were used as labeling probes, certain bio-macromolecules such as proteins, antibodies and enzymes, were used as mode samples, and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysulfo-succinimide (NHS) were used as linking reagents. We studied the effects of certain factors such as the isoelectric points (pIs) of bio-macromolecules and buffer pH on the bioconjugation of QDs, and found that the pIs of biomacromolecules played an important role in the conjugation reaction. By the optimization of the buffer pH, proteins with different pIs were efficiently conjugated with QDs using EDC and NHS as linking agents. Furthermore, we on-line investigated the kinetic process of QDs-bioconjugation by FCS, and found that the conjugation reaction of QDs with protein was rapid and the reaction process almost completed within 10 min. We also observed that QDs conjugates with proteins were stable for at least 5 days in phosphate buffer. In conclusion, CE coupled with FCS may be a very useful tool for study of the interaction of QDs and biomolecules in the future. Key Words: CE, Quantum Dots, Bioconjugates, Fluorescence Correlation Spectroscopy. References: 1. Xingtao Song, Liang Li, Huifeng Qian and Jicun Ren, Electrophoresis, 2006, 27, 1341-1346. 2. Xiangyi Huang, Jifang Weng, Chengxi Cao, Jicun Ren, J. Chromatogr. A, 2006, 1113, 251-254. 3. Chaoqing Dong, Rui Bi, Huifeng Qian, Jicun Ren Small, 2006, 2,534-538. 4. Jinjie Wang, Xiangyi Huang, Feng Zan, Chengxi Cao, Jicun Ren, Electrophoresis, 2012, 33, in press.

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L-140 Development of Capillary Electrophoretic Methods and Instrumentation for the Analysis of Explosives. Cameron Johns, Joseph P. Hutchinson, Michael C. Breadmore, Emily F. Hilder, Rosanne M. Guijt, Pavel N. Nesterenko, Paul R. Haddad, Greg W. Dicinoski, ACROSS, University of Tasmania, Hobart, Tasmania, AUSTRALIA The analysis of post-blast residues is vital following an explosive incident. Research in ACROSS has focused on the development of methods for inorganic (or homemade) devices and organic explosives. Ion chromatographic and capillary electrophoretic (CE) methods have been developed for complementary and confirmatory analyses. A range of inorganic cations and anions were chosen to encompass starting materials, reaction products, and species likely to be present in the environment. Eleven cations can be separated in under 7 minutes, and 16 anions in under 8 minutes. These methods have been successfully tested on witness plates and soil samples collected following deflagration of inorganic explosive devices. Separation of organic explosives can be achieved using a form of CE. Although the analytes of interest are uncharged, they can be separated under the influence of an electric field in a background electrolyte containing a pseudo stationary phase. Micellar electrokinetic chromatography (MEKC) uses the addition of a surfactant at a concentration above its critical micelle concentration to form a pseudo stationary phase. No instrumental modifications are required. Careful selection and optimization of the background electrolyte has been performed to separate a selected analyte set of 25 organic explosives. A novel CE method has also been developed for the analysis of organic peroxides. An example is triacetonetriperoxide (TATP), which has not been reported in the literature using electrophoretic methods. Such organic peroxides have generally been separated and detected using chromatographic techniques and complicated detection methods. The developed methods have been implemented on a custom built field deployable CE instrument, which is capable of running four parallel separations at the same time. This presentation will detail the CE methods and instrumentation which have been developed during this study. L-141 Versatile Capillary Electrophoresis and Capillary Liquid Chromatography for Nanodomain Interaction and Biosensor Development Studies. Marja-Liisa Riekkola1, Katriina Lipponen1, Yueqi Liu1, Geraldine Cilpa-Karhu1, Katariina Öörni2, Petri T. Kovanen2, 1University of Helsinki, Department of Chemistry, Laboratory of Analytical Chemistry, Helsinki, FINLAND; 2Wihuri Research Institute, Helsinki, FINLAND Microsystems and miniaturized techniques with special nanoscale functions are considered one of the key technologies for future progress in biochemistry, biotechnology and medicine, and especially new biomimicking tools that allow a deep understanding of the interactions under different conditions (pH, temperature, different reactions) are urgently required. From new generation biomimetic/bio-responsive biomaterials that play often an important role in the performance of versatile biomedical devices, it is beneficial that the biomaterials mimic certain characteristics of the natural human matrix, such as extracellular matrix or blood vessel. A full understanding of atherosclerosis at the molecular level requires unraveling of the changes of molecular interactions during atherogenesis. These nanoscale interactions influence the metabolism of the lipoprotein particles in the arterial wall by affecting the interactions of lipoproteins with extracellular matrix components. In addition, the nanoscale interactions are critical for the mutual interactions between individual low-density lipoprotein particles leading to either aggregation or fusion of the particles. In this talk the potential of capillary electromigration techniques and nano-liquid chromatography for the clarification of lipoprotein particle interactions in atherogenic processes will be presented. In the first techniques human lipoproteins and glycosaminoglycans of proteoglycans are exploited as biological stationary phases/ pseudo phases, and in the latter silica particles are immobilized via covalent bonding with biomolecules. The immobilization protocols developed are then also employed in the biosensor surfaces in continuous quartz crystal microbalance interaction studies. 1. K. Lipponen, P. W. Stege, G. Cilpa, J. Samuelsson, T. Fornstedt, M.-L. Riekkola, Anal. Chem. 83 (2011) 6040. 2. J. Witos, K. Meinander, L. D’Ulivo, J. Saint-Guirons, M.-L. Riekkola, Analyst 136 (2011) 3777. 3. K. Lipponen, Y. Liu, P. Wanda Stege, K. Öörni, P. T. Kovanen, M.-L. Riekkola, Anal. Biochem. 10.1016/j.ab.2012.02.017. L-142 High Throughput Verification of Recombinant Protein Production using the ISET, a MALDI Target for Integrated and Automated Proteomic Sample Preparation. Belinda Adler1, Tove Boström2, Simon Ekström1, Sophia Hober2, Thomas Laurell1, 1Lund University, Division of Nanobiotechnology, Lund, SWEDEN; 2KTH, Division of Proteomics, School of Biotechnology, Stockholm, SWEDEN Miniaturised concepts for sample decomplexing and/or affinity probing linked to MALDI MS readout holds promise of a next generation advanced MALDI analysis where affinity specific extraction and on target sample processing can be performed in an automated fashion. Inherent with this development follows the possibility of providing array based formats that can match the increasing needs for high-throughput analysis in large scale screening efforts and protein production. To meet these requirements our previous work has reported the Integrated Selective Enrichment Target - ISET - which is designed as a MALDI target that has an integrated nanocolumn running through the target plate in each MALDI-analysis position [1, 2]. The nanocolumns can be loaded with an arbitrary chromatographic matrix allowing for the design of application specific MALDI assays. We have demonstrated significantly improved analytical performance in solid phase extraction as compared to conventional solid phase pipette preparation and also reported phosphopeptide extraction from complex

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samples [3]. Critical to recent developments has been the optimisation of the design of the nanovial and specifically the configuration of the bead retention frit in the bottom of each nanovial [4]. This now enables the use of a wider range of chromatographic matrices and thus broadens the ease of application specific MALDI-target design. Based on these developments the ISET platform has now been adapted to automated sample processing where we on cobalt activated microbeads packed in the ISET enrich recombinant expressed His-tagged proteins. The proteins are subsequently digested in-situ and eluted onto the ISET backside for immediate MALDI MS or MS/MS analysis. This integrated process flow opens the route to rapid screening, verification and quality control in large-scale recombinant protein production. References: 1. Ekström S., Malm J., Lilja H., Laurell T., Marko-Varga G., Electrophoresis, 2004, 25, 3769-3777. 2. Ekström S., Wallman L., Hök D., Marko-Varga G., and Laurell T., Journal of Proteome Research, 2006, 5, 5, 1071-1081. 3. S. Ekström, L. Wallman, G. Helldin, J. Nilsson, G. Marko-Varga and T. Laurell, Journal of Mass Spectrometry, 2007, 42, 1445-1452. 4. Belinda Adler, Thomas Laurell and Simon Ekström, Electrophoresis, 2012, April 23, Accepted. L-143 Dielectrophoretic Drug Transport into Teeth. Chris S. Ivanoff, DDS, Timothy L. Hottel, DDS, MS, MBA, Franklin Garcia-Godoy, DDS, MS, Ph.D., Ph.D., University of Tennessee, College of Dentistry, Memphis, TN, USA The hypothesis that dielectrophoresis (DEP) can drive fluoride and other drugs into teeth deeper and in higher concentrations over traditional topical application was tested in in vitro studies. This drug delivery approach has never been attempted in dentistry until recently. The investigators used DEP to deliver carbamide peroxide (CP) into human tooth enamel and fluoride and casein phosphopeptide amorphous calcium phosphate (CPP-ACP) into bovine tooth enamel. The results showed a 104% increase in CP penetration into human enamel compared to the conventional diffusion delivery method and 1400% enhanced whitening effect on enamel after one twenty-minute application of a topical carbamide peroxide bleaching gel. Wavelength dispersive spectrometry showed that after one twenty-minute application of 1.23% APF gel to bovine enamel, DEP enhanced penetration and increased uptake of fluoride on average by 600% at the depth of 50 µm. The amount of fluoride delivered at 50 µm was equivalent to a life-time exposure of prophylactic fluoride, adding further clinical relevance to the development of a DEP technique as a viable delivery model for dentistry. The results imply that DEP could potentially deliver targeted doses of fluoride directly into tooth enamel and eliminate side-effects and risks thought to be associated with over-fluoridated water supplies, extend the efficacy of fluoride treatments, leading to improved remineralization and caries prevention. The long-term goal is to use dielectrophoretic technology to administer other drugs directly into teeth and make drug delivery in dentistry safer and more efficient. The microhardness of demineralized enamel remineralized in saliva was also evaluated after applying 1.23% APF gel or CCP-ACP paste with dielectrophoresis (DEP) or diffusion for 3 minutes. In this laboratory study, 25 enamel blocks of bovine incisors were soaked in demineralizing solution (10 mM HCl pH 2.5) for 10 minutes and remineralized in saliva for 1 hour, after applying 1.23% APF gel or CPP-ACP paste without fluoride with dielectrophoresis or conventional topical application for 3 minutes. The mean and percentage changes of surface microhardness in pre-demineralization, post-demineralization and remineralization stages were measured in the five groups. The average surface microhardness recovery of APF gel (12,300 ppm fluoride) applied with DEP was higher than APF gel applied topically, or CPP-ACP paste applied with DEP or topically, and water control (P< 0.05). The percentage of surface microhardness recovery for APF+DEP, APF+Diffusion, CPP-ACP+DEP, CPP-ACP+Diffusion, and water control were 88.17%, 13.62%, 7.14%, 5.45%, and 4.02%, respectively. Surface microhardness recovery of enamel with APF+DEP was 12 times or 1,200% greater than APF gel applied topically, and CPP-ACP paste either applied topically or with DEP in one 3-minute application. The study confirmed the usefulness and efficacy of fluoride in the repair of softened enamel surfaces; and further validates the exploration of DEP as a useful tool to potentially repair demineralized lesions deeper than superficial enamel layers achievable by diffusion alone. L-144 Dielectrophoretic/Electrophoretic Device for In-Situ Sample Preparation and PCR Detection of Disease Biomarkers. Michael J. Heller1, Avery Sonnenberg1, Raj Krishnan2, 1University of California San Diego, Department of Nanoengineering, La Jolla, CA, USA; 2Biological Dynamics, La Jolla, CA, USA A unique sample to answer AC/DC dielectrophoretic/electrophoretic device and methodologies have now been developed that allows the isolation of cell free circulating (cfc) DNA biomarkers and bacteria/virus, with subsequent DNA processing and in-situ PCR reactions, all carried out in the same device chamber. In initial work, we were able to demonstrate the rapid isolation, concentration and detection of high molecular weight (hmw) DNA and nanoparticles directly from high conductance buffers and whole blood. Using special microarray devices operated at 20 volts peak-to-peak and 10 kHz, a wide range of fluorescent hmw-DNA and fluorescent nanoparticles were concentrated into DEP high-field regions while blood cells are concentrated into the DEP low-field regions. A simple fluidic wash removes the blood cells while the DNA and nanoparticles remain concentrated in the DEP high-field regions where they can be detected. Hmw-DNA could be detected at 260 ng/ml, which is a detection level suitable for cell free circulating (cfc) DNA biomarker analysis. Fluorescent 40 nm nanoparticles could be detected at 9.5 x 109 particles/ml, which is a level suitable for monitoring drug delivery nanoparticles. These DEP devices were next used to isolate cfc-DNA from Chronic Lymphocytic

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Leukemia (CLL) and other cancer patient whole blood samples. Cfc-DNA could be isolated from patient whole blood samples in less than twenty minutes, with the PCR genotyping being carried out in a separate tube. We now have been able to develop AC/DC electrokinetic devices and procedures that allow all steps, including the PCR reactions, to be carried out in the same chamber (in-situ). This represents a major advancement in “sample to answer” diagnostics in that the isolated DNA/RNA does not have be removed or transported into a different component device. We have also been able to carry out the in-situ (in the same device chamber) isolation and preparation of DNA from bacteria for subsequent DNA sequencing, this included lyses of the cells, proteinase treatment, recapture of the extracted DNA and sequencing related tagmentation reactions, all done in-situ. The limitations for efficient DNA/RNA sample preparation have long plagued miniaturized lab on a chip devices and Point of Care (POC) diagnostic technologies, and significantly limited the overall assay sensitivity for many detection technologies. The ability to now rapidly isolated and detect disease related DNA biomarkers and bacteria/virus from undiluted whole blood and other samples will benefit many diagnostic applications by significantly reducing sample preparation time, cost and complexity. L-145 Pharmaceutical Analysis by using Capacitively Coupled Contactless Conductivity Detection Coupled to Capillary Electrophoresis. Mohamed N. El-Attug1, Erwin Adams2, Ann Van Schepdael2, 1Tripoli University, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Tripoli, LIBYA; 2KU Leuven, Laboratory for Pharmaceutical Analysis, Faculteit Farmaceutische Wetenschappen, Leuven, BELGIUM Capillary electrophoresis (CE) is a powerful separation technique with fast analysis speed, high separation efficiency and low reagent consumption. CE has been widely investigated in a variety of applications as Pharmaceutical Analysis. It has mainly been combined with conventional detection techniques (UV and florescence), but recently, capacitively coupled contactless conductivity detection (C4D) also has been investigated in many applications. It was proven to be a versatile mode of detection. This presentation will focus on the possible use of CE-C4D in quality control of aminoglycoside antibiotics (AGs), bioanalytical analysis of urea in bronchial fluids and analysis of other analytes as TFA which in general do not have a UV absorbing chromophore and thus pose a serious challenge for straightforward detection. It will be shown that CE-C4D can be applied for the separation of AGs like kanamycin, tobramycin and amikacin from their impurities. It is possible to obtain baseline separation in short time. The sensitivity of detection of these CE-C4D methods, which is very important for meaningful impurity analysis of drugs, and separation time, will be compared to that of existing methods. In another, bioanalytical application, the usefulness of CE-C4D for determination of amikacin and urea in bronchial fluids was with investigated the aid of urease, and subsequent determination of the ammonium formed through this enzymatic reaction. Urea concentrations were used to determine the level of dilution occurring during sampling of the bronchial fluids, which could then be applied on the amikacin levels. L-146 A Novel Synthetic Route of the Caspase 3 Sensor for Determination of Caspase 3 Based on Forster Resonance Energy Transfer. Marcela Liskova1, Karel Kleparnik1, Pavel Pazdera2, Frantisek Foret1, 1Institute of Analytical Chemistry of the ASCR v. v. i., Bioanalytical Instrumentation, Brno, CZECH REPUBLIC; 2Masaryk University, Faculty of Science, Centre for Syntheses at Sustainable Conditions and their Management, Brno, CZECH REPUBLIC We have designed the synthesis of caspase 3 sensor. The sensor is based on the principle of Förster resonance energy transfer (FRET). In this system, an energy absorbed by a chromophore (donor) is transferred nonradiatively to a fluorophore (acceptor) by dipole-dipole coupling through a linking chain. The length of the chain determines the efficiency of the transfer. In our modification, quantum dots (QDs) with high extinction coefficients and sulphorhodamine B are used as donors and acceptors, respectively. Since caspases recognize tetra-peptide sequences Asp-Glu-Val-Asp (DEVD) on their substrates and hydrolyze peptide bonds after aspartic acid residues, the sensor has the synthetic DEVD sequence near rhodamine derivative. When cleaved, the chain between QD and rhodamine is broken and therefore, the fluorescence emission of rhodamine is switched off. Thus, the red emission (610 nm) of rhodamine B is changed to the yellow-green emission (550 nm) of QD. The molecule of our sensor consists of three parts. First part is QD of a size of 3 nm with a surface ligand, O-(3-Carboxypropyl)-O′-[2-(3-mercaptopropionylamino) ethyl]-polyethylene glycol (x-PEG, molecular mass of 3000 Da). QDs with x-PEG were prepared by typical one-pot aqueous synthetic method, where cadmium chloride, trisodium citrate dihydrate, Na2teO3, x-PEG and sodium borohydride reacted only in one flask together. The second part is a modified relatively long hydrocarbon chain. In the third part, the tetra-peptide sequence N-Acetyl-Asp-Glu-Val-Asp p-nitroanilide is bind to sulphorhodamine B. New organic approaches based on piperazine and polyethylene glycol chemistries have been used to develop a cheap and simple synthesis. Products of synthesis were checked by NMR, FTIR, MS and CZE-LIF. Finally, the capability of the sensor to detect caspase 3 in apoptotic cells will be proved by fluorescence microscopy. This work was supported by The Grant Agency of the Czech Republic (P301/11/2055, P206/12/G014 and P206/11/2377), Ministry of Education, Youth and Sports (OP Education for Competitiveness-CZ. 1.07/2. 3 00/20. 0182), The Technology Agency of the Czech Republic (TA 02010672) and institutional support RVO: 68081715.

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L-147 Dielectrophoretic Protein Manipulation in a Microfluidic Device. Asuka Nakano, Fernanda Camacho-Alanis, Tzu-Chiao Chao, Alexandra Ros, Arizona State University, Department of Chemistry and Biochemistry, Tempe, AZ, USA Rapid and reliable separation and analysis of proteins is of great interest. This becomes especially challenging when only small sample volumes are available, concomitantly with low concentrations of proteins. Time critical situations such as surgeries and rapidly degrading samples pose additional challenges. Due to these challenges, conventional macro-scale separation techniques reach their limitations. While microfluidic devices require only pL-nL sample they offer several advantages such as speed, efficiency, and high throughput. Here, we propose to elucidate the capability to manipulate proteins in a rapid and reliable manner with a novel migration technique, namely dielectrophoresis (DEP). We demonstrate a detailed study of various factors influencing protein DEP, employing immunoglobulin G (IgG) under DC conditions. DEP has been extensively employed as a separation, fractionation, and pre-concentration technique for large biological objects such as cells and recently for DNA. DEP relies on a protein's polarizability in an inhomogeneous electric field. The dielectrophoretic response of proteins is provoked in a tailored microfluidic device with integrated arrays of insulating posts to create a non-uniform electric field. Unlike DNA and cells, protein DEP behavior is not well understood; therefore our detailed study of protein DEP provides novel information to eventually optimize this protein migration method for pre-concentration and other analytical techniques. First, we performed numerical simulations to predict protein migration behavior considering electroosmosis, protein diffusion, and DEP. Based on the calculated electric field, E, and ∇E2, we simulated the protein concentration profiles and found that the protein concentrates in streamlines between the rows of posts in the positive DEP case, whereas protein is depleted at the exact same regions for negative DEP. Experimentally, we investigated the DEP behavior of IgG under various buffer conditions differing in pH and conductivity. We observed streaming DEP only at pH 6.5~8 with a maximum DEP streaming enrichment of 70 %. This concentration profile was in excellent qualitative agreement with numerical simulations performed with the assumption of monomeric IgG species. Furthermore, pH dependent DEP streaming is also in agreement with the numerical simulations suggesting that the variation of protein charge and electroosmotic flow influences protein DEP streaming. Additionally, we observe micelle induced negative DEP streaming for proteins, which is also in agreement with numerical simulations. This micelle formation was further confirmed by dynamic light scattering experiments. Our study thus provides valuable information to eventually improve novel protein DEP devices for separation, pre-concentration, and fractionation. L-148 DNA Electrophoresis via Noise-Synchronized Entropic Trapping: A Versatile Tool for Enhanced Separations and Nanostructural Analysis. Nan Shi, Victor M. Ugaz, Texas A&M University, Department of Chemical Engineering, College Station, TX, USA Macromolecules confined within nanoporous surroundings experience entropic trapping (ET) when their dimensions approach the average pore size, leading to emergence of transport behavior that can be immensely beneficial (e.g., a counterintuitive trend of increasing separation efficiency with DNA size during gel electrophoresis). But the noisy uncorrelated process by which the embedded macromolecules discretely hop from pore to pore contributes additional dispersion that detrimentally impacts most practical applications. Here we show how these limitations can be overcome by imposing an oscillatory electric field at a period tuned to the activation timescale of ET in ordinary hydrogels, establishing a resonance condition that synergistically combines accelerated mobility and reduced diffusion. This effect can be exploited to induce bi-directional transport of different sized DNA fragments owing to the size-dependence of the optimal modulation period. We show this in microchip electrophoresis by achieving a state in which neighboring bands in the gel travel in opposite directions. We also show how this resonance phenomenon can be harnessed as a sensitive probe of DNA binding interactions by exploiting the fact that the conditions under which resonance emerges strongly depend on the DNA coil size. Therefore, binding interactions become visible as a distinct peak in electrophoretic mobility occurring at a conformation-dependent electric field actuation period. These measurements also enable structural information (persistence and contour lengths) to be extracted by correlating the period at maximum mobility with conformational characteristics of the DNA complex. There is no inherent lower limit on the DNA size that can be interrogated, as opposed to single-molecule studies which require large DNA (λ-phage or longer). L-149 Determination of Nitrite in Saliva using Microfluidic Paper-based Analytical Devices. Rubiane Borba1, Samir Bhakta2, Mario Junior3, Carlos D. Garcia2, Emanuel Carrilho1, 1Instituto de Quimica de Sao Carlos, Universidade de Sao Paulo, Sao Paulo, BRAZIL; 2University of Texas San Antonio, Department of Chemistry, San Antonio, TX, USA; 3Faculdade de Odontologia de Ribeirao Preto, Sao Paulo, BRAZIL The development of novel analytical strategies is critical to improve early diagnosis and follow treatment monitoring. In this regard, point-of-care platforms avoid transferring ill patients to central locations, provide fast responses, decrease the overall cost of the treatment and can significantly improve the success of the treatment. Among others, paper-based microfluidic devices are one of the most versatile and efficient platforms to design diagnostic tools. In comparison with traditional paper-based devices (such as reactive strips or

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ELISA), paper-microfluidics are faster, require smaller samples, are far more efficient and can perform multiple and complex biochemical reactions without cross-contamination or external power. Moreover, instead of giving a negative/positive response, paper microfluidic devices can provide a visual reading that is proportional to the concentration of the target marker. Considering the potential impact that this technology could have in oral health (e.g. periodontitis), the objective of this work was the development of microfluidic paper-based analytical devices (μPAD) to identify and quantify levels of nitrite in saliva. Devices were fabricated by wax printing and allowed the detection using a colorimetric reaction (Griess’s method). Results related to the design, characterization, and application of these devices will be discussed along with a general overview of the advantages of this emerging technology. L-150 Microfabricated Sensors with Amalgam Working Electrodes. Petra Juskova1, Hana Cernocka2, Veronika Ostatna2, Emil Palecek2, Frantisek Foret1, 1Institute of Analytical Chemistry ASCR v.v. i., Brno, CZECH REPUBLIC; 2Institute of Biophysics ASCR v. v. i., Brno, CZECH REPUBLIC Rapid and sensitive characterization of protein forms and modifications is one of the challenges in current proteomic research. Development of new sensitive and selective bioanalytical tools to address this issue and systems amenable to automation and parallelization with reduced requirements for the sample amount are of key importance. Electrochemistry with its sensitivity, relative simplicity and low power requirement represents one of the promising methods for protein analysis with high miniaturization potential. Practically all proteins produce electrocatalytic chronopotentiometric peak H at the mercury containing electrodes with sensitivities down to subnanomolar concentrations [1]. Recently we have developed fabrication process for preparation of a microarray of the amalgam working electrodes with proven ability to protein analysis [2]. Electrodes were prepared in microarray format enabling parallel operation; however, manipulation with the external connections to the reference and the counter electrodes was still complicated. Here we present the fabrication process for improved version of the electrode array integrating amalgam working electrodes with the counter and reference electrodes into one sensor. This simplified operation with the whole system, requiring only deposition of a small sample amount (microliters) onto the sensor surface. Performance of the newly developed system for universal and/or selective biological specimen detection was tested for protein analysis using constant current stripping analysis. This work was supported by The Grant Agency of the Czech Republic (P301/11/2055, P206/12/G014) and institutional support RVO: 68081715. References [1] Palecek, E; Ostatna, V. Electroanalysis, 2007, 19, 2383-2403. [2] Juskova, P; Ostatna, V; Palecek, E; Foret, F. Analytical Chemistry, 2010, 82, 2690-2695 L-151 Studies of Interaction of Ionic Liquids with DNA and Protein Based on Capillary Electrophoresis. Xinying Zhao, Feng Qu, Fan Chen, Aiqin Luo, Beijing Institute of Technology, School of Life Science, Beijing, CHINA Capillary electrophoresis (CE) with the advantages of fast, high resolution, low sample consumption and smart separation modes is one of the powerful techniques for characterization of interaction. Ionic liquids (ILs), with “green” characteristics and large liquid range, high thermal and chemical stability, strong solubility power and designable cation and anion feature, are applied increasingly in biocatalytic transformation, analytical and separation process in recent years. In this work, the interaction of ILs on ds DNA (calf thymus DNA), random ss-DNA (80 nt) pool and aptamer (Cytochrome C aptamer, 57 nt) were investigated by capillary zone electrophoresis (CZE), which hinted the conformation change of DNA. The interaction strength is dsDNA > ssDNA > aptamer. Meanwhile, the interaction of ILs on myoglobin (Mb), bovine serum albumin (BSA), hemoglobin (Hb) and transferring (Trf) were compared by affinity capillary electrophoresis (ACE) based on the mobility ratio and Kb, which showed that the interaction strength reduced in the turn of Trf, Hb, BSA and Mb. The interaction of thrombin (Thr), BSA, cytochrome c (Cyt C) with ss-DNA or aptamer were discussed also. Finally, the effect of ILs on protein and random ss-DNA pool mixture was explored. As well known, CE is one of the most powerful methods for protein aptamer sieving (known as CE-SELEX), the knowledge of ILs effects on DNA, protein and their mixture may benefit the understanding of interaction and improve the protein aptamer sieving. ACKNOWLEDGEMENT The National Natural Science Foundation of China (No.21175011) and the National Basic Research Program of China (973 Program, No.2012CB910603). L-152 Validation and Optimization of a Liquid-Phase Microextraction Method Based on the Solidification of Floating Organic Micro Drop for Trace Analysis of BTEX in the Water Samples. Mohamadreza Tajbakhsh1, Masoumeh Helalizadeh2, Hakim Faraji3, 1Iranian Research Institute of Plant Protection, Tehran, IRAN; 2Islamic Azad University, Young researchers Club, Varamin-Pishva Branch, Varamin, IRAN; 3Islamic Azad University, Varamin-Pishva Branch, Chemistry Department, Varamin, IRAN Liquid-phase microextraction (LPME) based on solidification of a floating organic micro drop followed by high performance liquid chromatography (photo diode array detector) to pre-concentrate and determine benzene, toluene, ethylbenzene and xylenes (BTEX) in the aqueous samples has been investigated. The effects of type and volume of extraction solvent, stirring rate, extraction time, temperature and ionic strength in LPME of BTEX were evaluated. The results confirm that the proposed procedure provides an improved accuracy, linear range, limit of detection, limit of quantification and is very effective for analyzing the BTEX compounds in water

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samples. Under the optimized conditions, pre-concentration factor of 149–285 and extraction efficiency of 31-61% were obtained. Repeatability (1.59-5.83%) and intermediate precision (2.05-7.14%) were in acceptable range. The relative recovery obtained for each analyte in different water samples was higher than 82.3% at three fortification levels with the relative standard deviation of less than 7.5%. Keywords: Validation; LPME; Solidification of floating drop; BTEX; Water samples L-201 Extended-Nano Fluidic Systems on Chip for Analysis. Takehiko Kitamori, The University of Tokyo, Graduate School of Engineering, Tokyo, JAPAN [PLENARY LECTURE] Micro and extended-nano (EN) fluidic device technology has brought evolutional progress in chemistry and medical biology although it is still on the way of development for practical use. At least in our research group, micro fluidic device technology has already gone into practical phase. Design, fabrication, surface modification (functionalization, in terms of MEMS), fluid control, and operation methods have almost established. Those technologies were published in some books as well as in many published original papers and reviews (lists are available in our website http://park.itc.u-tokyo.ac.jp/kitamori/) . A part of them are commercially available (http://www.i-mt.co.jp/). In contrast to micro fluidics, extended-nano (EN) fluidics is still in launching stage. However, in terms of the sample volume, analytical instrumentation technology is getting new tools to reduce the volume evolutionally, that is the micro and EN fluidic device providing nano, pico, femt and atto litter orders. The size region of 101-102 nm, that is EN space fluidics, is still unexplored area in science and technology. In our group, we fabricated EN channels into glass microchips, and succeeded in fluidic control and chemical processing such as chromatographic separation. Actually, we applied the extended-nano channels and spaces to pico-litter immunoassay and atto-litter chromatography. Pico-litter is smaller than the volume of a living cell, and therefore, single cell analysis keeping the cell alive will be available. The atto-litter chromatography is really challenging, and some examples will be introduced in this talk. One of the critical issues of micro/EN fluidic technology is detection. Especially, optical detection of non-fluorescent analytes is quite difficult, because optical pass length of channels is extremely short. Therefore, we developed the thermal lens micro-detection TLM method, and it was proved to be a very powerful detector even at zmol regions. However, optical pass length is a couple of orders shorter in EN, and it was still very difficult to apply. Therefore, we got an idea of introducing differential interference contrast DIC optical configuration into TLM. DIC-TLM enabled to detect optical absorption in EN optical pass length, and we could obtain chromatogram of non-fluorescent molecules. Principle of DIC is based on wave optics, and EN optical pass length was still sufficiently long for detecting phase shift induced by TL effect in EN channels. The combination of EN fluidics and DIC-TLM will be a powerful tool for the next generation EN fluidic chemical and biological devices. L-202 Isotachophoresis for Extraction and Rapid Hybridization of Nucleic Acids. Crystal M. Han, Moran Bercovici, Lewis A. Marshall, Giancarlo Garcia-Schwarz, Alex Persat, Joe C. Liao, Juan G. Santiago, Stanford University, Mechanical Engineering Department, Stanford, CA, USA [PLENARY LECTURE] Microfluidics research is growing in complexity and variety, even as the technology matures and is commercialized. Yet there remains a glaring weakness common to most microfluidic systems: The lack of automated sample preparation and its integration with downstream assays. This is particularly true for selective extraction and preconcentration of nucleic acids from complex biological samples such as blood, urine, and cultured cells. This talk will summarize research at Stanford toward meeting this unmet challenge. We are developing on-chip isotachophoresis (ITP) processes which selectively focus target analytes from a complex mixture, while rejecting unwanted molecules which may inhibit hybridization or amplification. We will present extraction of various target nucleic acids including DNA from blood and bacterial rRNA from urine. We will also present examples of integrating ITP sample preparation with downstream assays. We have integrated RNA extraction with sequence-specific quantitation using molecular beacons and alternately by combining fluorescent DNA probes with functionalized, photopatterned hydrogels. We use ITP to purify and preconcentrate target and probe molecules by >10,000x into order 10 pl reaction volumes. We show specific and sensitive detection of target sequences in order 2 minutes with little or no off-chip sample preparation, and without target amplification. We have demonstrated profiling of microRNA from total RNA, and quantitation of bacterial 16S rRNA from infected urine lysate. L-203 Advancements in Microfluidic Acoustophoretic Cell Separation Utilizing 2-Dimensional Ultrasonic Standing Wave Pre-focusing. Thomas Laurell, Lund University, Division of Nanobiotechnology, Lund, SWEDEN [KEYNOTE LECTURE] Microchip acoustophoresis has in recent years become a viable strategy for advanced cell manipulation in life science applications, including cell separation, buffer switching, valving, affinity bead extraction, cell interaction studies [1-3]. Cells undergoing acoustophoresis experience a low mechanical stress and display unaffected viability [4]. This consequently opens the route to a wide range of bioanalytical and clinical applications in cell separation and analysis. We have previously reported Free Flow Acoustophoresis – FFA -as a means of continuous flow based separation of multiplex particle or cell samples [1]. Although initial data demonstrated the power of FFA based separation, significant carry over between different bead or cell populations were evident.

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This was induced by the Poiseuille flow profile which yielded different retention time in the acoustophoretic separation zone and hence a severe band broadening and overlap of the different target species. By reconfiguring the acoustofluidc design of our microsystem to include a step of 2-dimensional acoustic pre-focusing we can now align the cells/particles in a confined flow velocity domain prior to entering the critical acoustophoresis zone. This in turn reduces the band broadening and provides significantly improved FFA conditions. Based on these developments we have recently developed an acoustofluidic microsystem that enables label free extraction of tumor cells from blood samples at recoveries of 97% and a purity of 98% at flow rates of ≈70 uL/min. Two dimensional acoustic pre-focusing has also enabled us to develop an acoustic standing wave based microfluidic FACS for rare event cell sorting, where target cells/particles are detected by means of fluorescence and subsequently switched by acoustophoresis to a stream line that ends up in the desired rare event collection output. Acoustophoresis offers a relative simple mode of non-contact manipulation and separation of cell and particles in microfluidic systems. By implementing 2-dimensional acoustic prefocusing we demonstrate that a vastly improved performance is accomplished in several acoustophoresis applications, which now opens the route to more delicate on-chip sample preparation and separation steps. References: 1. Petersson F. T., Åberg L. B., Swärd-Nilsson A.-M. S. and Laurell T., Analytical Chemistry, 2007, 79, 14, 5117-5123. 2. Persson J., Augustsson P., Laurell T. and Ohlin M., FEBS J, 2008, 275, 5657-5666. 3. Petersson F., Nilsson A., Jönsson H., and Laurell T., Anal. Chem, 2005, 77, 5, 1216-1221. 4. J. Dykes, A. Lenshof, I. Åstrand, T. Laurell, S. Scheding, PLOS One, 2011, 6, 8, e23074 L-204 Thermally Responsive Phospholipid Preparations for Fluid Steering and Separation in Microfluidics. Lisa Holland, Xingwei Wu, Ted Langan, Brandon Durney, West Virginia University, Chemistry Department, Morgantown, WV, USA Phospholipid preparations comprised of dimyristoylphosphatidylcholine (DMPC) and dihexanoyl-phosphatidylcholine (DHPC) spontaneously assembly to form disks or wormlike micelles. In bulk solution, these assemblies possess thermally responsive viscosities. For example, a 7 °C change in temperature can produce a ~370-fold increase in viscosity. We utilize these materials to enhance capillary electrophoresis separations of biomolecules. The material produces separations with theoretical plate counts as high as 640,000 for separations based on hydrodynamic volume and 2,600,000 for sieving. In addition to serving as effective separation additives, phospholipid preparations are utilized to steer fluids in microfluidic chips and support hydrodynamic delivery of sample across the double T injection region in a chip. The fact that the phospholipids are fully integrated as a valving material as well as a separation medium is demonstrated through the separation of biopolymers. The properties and separation performance of the material are reported. Innovative separation and detection strategies are presented and the promise of a wide range of applications is discussed. L-205 Microfluidic Separation Devices using Periodic Stationary Media. German Drazer, Johns Hopkins University, Chemical and Biomolecular Engineering, Baltimore, MD, USA We discuss the separation of suspended particles in microfluidic devices. A typical system in our studies consists of suspended particles moving either through a periodic array of obstacles or on top of a periodic pattern fabricated in the bottom surface of a channel. In all cases, we investigate how to take advantage of the repetitive effects present in periodic systems to promote and amplify the separation of suspended particles. We present analytical and experimental results that show the potential for separation of these systems. L-206 Single-Stream Free-Flow Isoelectric Focusing with pH Gradients Induced by Water Splitting in Bipolar Membranes-Integrated Microfluidic Devices. Li-Jing (Larry) Cheng, Hsueh-Chia Chang, University of Notre Dame, Notre Dame, IN, USA In this abstract we report a novel microfluidic technology to generate stable pH gradients, using integrated bipolar membrane (BPM) components, for free-flow isoelectric focusing in a single-stream format. Microfluidic free-flow isoelectric focusing (FF-IEF) is a versatile and rapid preparative separation and analysis techniques in chemistry and biology. However, stable pH gradient formation is one of the practical challenges. Several techniques have been developed to produce pH gradients, including introduction of acid and base solutions and electrolysis of water. These methods, however, require either multiple input streams for specialized sheath flow or additional setup to prevent the effect of bubble generation. We have developed a simple, single-stream microfluidic pH actuation technique that directly dopes the upstream analyte flow with proton and hydroxide ions and forms a stable pH gradient in a downstream separation channel. The excess H+ and OH- ions are generated by electric field-enhanced water dissociation with two sets of BPM. Under a reverse voltage of 4.5 V at which the anode connects to the anion exchange side, the high electric field at the BPM junction directly splits water molecules into H+ and OH- ions. Without any electron-transfer reaction to generate gas, the process produces H+ and OH- ions more efficiently than electrolysis. Furthermore, the injection of H+/OH- ions does not alter the total analyte volume and hence allows for pH adjustment without diluting analyte concentration. The microfluidic pH actuation was designed to offer efficient ion injection while insensitive to flow rate. Separation of a mixture of fluorescent pI markers has been successfully demonstrated by using the FF-IEF device under a flow rate of 10 µl/min and a separation electric field of 180 V/cm. The simple, high-speed FF-IEF separation

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makes the device applicable to proteomic analysis and flexible for further integration to downstream analysis tools, such as mass spectroscopy. L-207 Recent Developments in Capillary Isoelectric Focusing Separations. Gyula Vigh1, Nino Kokiashvili2, Ming-chin Li1, 1Texas A&M University, Chemistry Department, College Station, TX, USA; 2Iv. Javakishvili Tbilisi State University, Tbilisi, GEORGIA [KEYNOTE LECTURE] During ITP2011 we described a family of fluorescent pI markers (440 < λexcitation < 540 nm) whose members have a common 1,3-bis(sulfonamido)-6-alkoxy-8-aminopyrene core structure with four-to-eight protic groups to control the respective pI values. The pKa values of the protic groups are too closely spaced and too numerous (4-to-8) to be handled by the conventional mobility-based pI determination methods developed for diprotic ampholytes. The Off-Gel Electrophoretic system (Agilent) can be used for either in-gel or off-gel IEF separation of ampholytes using an IPG dry strip as the pH gradient controlling element. Since the new pI markers can be easily detected by conventional fluorescence scanners, we tried to use the Off-Gel device in the in-gel IEF mode with nominally identical IPG dry strips from different manufacturers to determine the pI values of the markers. We found the even after individual optimization, different IPG strips yielded different pI values for the same markers indicating that the actual pH gradient in the strips differed from the claimed nominal one. Measurement of the pH of the fractions obtained in the Off-Gel device when operated in the off-gel mode did not deliver the correct pI value of the markers either, because the concentration of the isolated markers was too low to yield the hydronium concentration that would correspond to the correct pI value (i.e., the measured pH values were much closer to pH=7 than predicted by the true pI value). Therefore, we designed a new pI determination method: we determined the focusing conditions (the time integrals of current and separation voltage) and the minimum required carrier ampholyte concentrations above which the pH of the isolated carrier ampholyte fractions remained constant. When the pI markers were co-separated with carrier ampholytes under such conditions, the markers focused into a single compartment where they were easily detected due to their visible fluorescence and where the pH of the fraction (containing the trapped carrier ampholyte fraction and the markers together) could be determined accurately, independently of their concentration, reflecting their true pI values. By using the maximum number of compartments in the Off-Gel device (24 compartments) and stepwisely reducing the nominal width of the pH gradient of the dry strips from ∆pH=7 through ∆pH =3 to ∆pH =1.5, a 0.06 wide pH range could be determined that included the actual pI values of the markers. L-208 New Simulation Tools for Understanding and Predicting Separation in Electrophoresis. Bohuslav Gas1, Jana Svobodova1, Vlastimil Hruska2, Martina Riesova1, Martin Benes1, Iva Zuskova1, 1Charles University, Faculty of Science, Physical Chemistry Department, Prague, CZECH REPUBLIC; 2Agilent Technologies GmbH, Waldbronn, GERMANY Formulation of basic laws able to describe movement of charged particles in liquid solution under in the electric field is not difficult in itself, however, to solve them in a full complexity has not been possible until now. We presented two software tools how to obtain information about behavior of the separation systems without necessity of wet experiments. The numerical simulation software Simul enables visualizing the movement of all constituents in the separation channel: of both background electrolyte constituents and samples. This allows to inspect various phenomena observed in experiments and understand what takes place during the whole electrophoretic run. The second approach is linearization of the electromigration equations which can be then formulated as a matrix the eigenvalues of which are certain mobilities − eigenmobilites. This concept is implemented in PeakMaster series of software, which can predict some experimental characteristics of the separated analytes: the positions of system peaks, the response in direct, indirect, and conductivity detection, the extent of electromigration dispersion. Recently we made a progress in two directions: (i) Including complex forming equilibria in the electromigration system. This allows to study such important class of analytical tasks as are enatioseparations in the systems with chiral selectors. The new software we developed, Simul 5 Complex, offers a deep insight into the systems with complexation agents, and is able to predict and explain, e.g., the significant electromigration dispersion of analyte peaks in systems with chiral selectors. We also implemented the model to the new version of PeakMaster, PeakMaster 5.3 Complex, that can calculate some characteristic parameters of the electrophoretic system with a neutral complex-forming agent and can be advantageously used for optimization of the separation conditions to avoid high electromigration dispersion in complex-forming systems. (ii) Formulation of the simplified nonlinear model of electrophoresis, which is more rigorous and more accurate than the linear model and which is still solvable in the matrix notation. Such model was implemented in the new version of PeakMaster, PeakMaster 5.3. In addition to the previous versions this will allow to obtain practically important information about peak shapes of both the system peaks and peaks of the analytes in zone electrophoresis in much shorter time when compared to numerical simulation. Both Simul and PeakMaster have a status of freeware (www.natur.cuni.cz/gas) and run in fully graphical environment.

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L-209 Modeling and Simulation of Particle Chaining using DC Dielectrophoresis. Prashanta Dutta, Robiul Hossan, Washington State University, Mechanical and Materials Engineering Department, Pullman, WA, USA Dielectrophoresis is used in many engineering applications including manipulation, separation, characterization, and actuation of particles such as biological cells. Dielectrophoresis requires a spatially non-uniform electric field in a fluid medium. The applied electric field polarizes the dielectric particles and causes a net force on particles due to nonuniform electric field on the accumulated charges. This induced body force drives particles towards higher or lower electric field region depending on the properties of the medium and particles. In recent years, dielectrophoretic manipulation has emerged as a popular mechanism to assemble colloidal particles for bottom-up nanofabrication, to gather biological cells in tissue engineering, to attach biomolecules on engineered surface for biosensing, etc. Although dielectrophoresis is a very powerful manipulation technique, there are not many robust numerical models/tools that can reveal the underlying complex transient physics considering the size of the particle. In this talk, we will present a hybrid immersed interface-immersed boundary method to study dielectrophoretic particle assembly where both dielectrophoretic and hydrodynamic forces are calculated with interface-resolved approach instead of point-particle method. Immersed interface method is employed to capture physics of electrostatics in a fluid media with suspending particles, while immersed boundary method is used to study hydrodynamics with rigid or flexible immersed boundaries. The dielectrophoretic force is obtained from Maxwell’s stress tensor by solving electric fields in both fluid and particle domains, and used as a body force term in the momentum conservation equations. We will particularly demonstrate the particle chaining phenomena for similar and dissimilar type particles in a DC electric field. Moreover, we will discuss the key limitations of the point based dielectrophoretic force measurement techniques commonly used in the literature. L-210 Comparative Investigations on Two Isotachophoretic Methods based on Moving Boundary System (MBS) and Reaction Boundary (MRB). Cheng-Xi Cao, Liu-Yin Fan, Shanghai Jiao Tong University, School of Life Science and Biotechology, Shanghai, CHINA In this paper, we performed theroetical and experimental investigations on the classic isotachophoretic (ITP) method and the newly-developed ITP technique. First, the comparative investigations reveal that the classic ITP technique is based on physico- chemical model of moving boundary system, while the newly-devloped ITP procedure relied on the model of moving reaction boundary (MRB). Second, the separation of analytes in the classic ITP is according to the differences of analyte mobilities, while that in the newly-devloped ITP is in line with the differences of chelation constants or solubility products. Third, there are leading and terminating ions in the classic ITP rather than the newly developed ITP method, whereas there are decisive metal ion and chelate or precipitate reagent in the newly-devloped ITP rather than the classic ITP method. Fourth, the boundary motion in the classic ITP is controlled via the leading electrolyte or ion, however the boundary movement in the newly developed ITP is adjusted via decisvie metal ion together with chelat or precipitate reagent. Fifth, in a given classic ITP, the boundary moves monotonically towards the anode but not the cathode, or towards the cathode rather than the anode, but in a newly-developed ITP, the boundary motion has three possibilities, viz., towards the anode, the cathode and stationary relied on the relative flux of decisive metal ion. L-211 Development of Lectin Affinity Separation Technologies and their Applications to Bioscience/Bioindustry. Jun Hirabayashi, National Institute of Advanced Industrial Science and Technology, Research Center for Stem Cell Engineering, Tsukuba, JAPAN [KEYNOTE LECTURE] Lectin microarray is an emergent technology, which enables accelerated research and development of glycomics. We developed the system in 2005 (1) together with a commercialized machine for lectin microarray by means of an evanescent-field-activated fluorescence detection principle; i.e., GlycoStation Reader 1200 and LecChip (both from GP BioSciences, Ltd., Japan). Becasue the method requires no prior liberation of glycans from the core protein and lipid, but is rather directly applied to extensive biological samples in a crude state(e.g., body fluids, cell/tissue extracts, intact cultured cells, bacteria, and fungi), the method/procedure using lectin microarray is now exploiting its application fields; e.g., glyco-biomarker development (2) and stem cell evaluation (3). Although the number of scientific papers describing GlycoStation Reader 1200 has reached only ~40 as of May 1st, 2012, the method should be propagated into much wider fields as a generalized form (4). One of such application fields include bio-pharmaceuticals, especially in quality control of bio-similar products, for which various analytical methods are necessary in a developmental-specific manner. Both merits and demerits of individual methods including lectin microarray will be described. 1. "Evanescent-field fluorescence-assisted lectin microarray: a new strategy for glycan profiling" Kuno A, Uchiyama N, et al. (2005) Nat Methods. 2, 851-6. 2. "Wisteria floribunda agglutinin-positive MUC1 is a sensitive biliary marker for human cholangiocarcinoma" Matsuda A et al. (2010) Hepatology. 52, 174-82. 3. "Glycome diagnosis of human induced pluripotent stem cells using lectin microarray" Tateno H et al. (2011) J Biol Chem. 286, 20345-53. 4. "Lectin-based structural glycomics: a practical approach to complex glycans" Hirabayashi J, et al. (2011) Electrophoresis. 32, 1118-28

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L-212 Assessing the Glycomic Changes of Breast Cancer Tissues by LC-MS. Yehia Mechref, Yunli Hu, Janie DeSantos-Garcia, Shiyue Zhou, Texas Tech University, Department of Chemistry and Biochemistry, Lubbock, TX, USA Glycosylation is the most prevalent posttranslational modification of proteins in mammalian cells. It is involved in modulating the activities and functions of proteins in health and disease. Thus far, many mass spectrometry based methods has been utilized to quantify the changes associated with glycans derived from biological fluid as a result of disease development and progression. Since permethylation of glycan results in an increase in the ionization efficiency, it has been widely used in MS analysis of glycans. This study compares the quantification of permethylated glycans using different stat-of-the-art mass spectrometers, including Velos Orbitrap, Vantage triple-quadrupole, MALDI-TOFTOF and Exactive. The quantification was based on both MS and LC-MS data. Permethylated N-glycans derived from model glycoproteins were initially employed to optimize both the separation and MS conditions. Hydrophobicity induced by the permethylation of the glycan structures allowed their efficient separation using capillary C18 columns. This separation allowed the detection of glycan structures derived from blood serum using LC-MALDI-MS or LC-ESI-MS. The latter allowed the simultaneous acquisition of tandem MS. More permethylated glycans were observed in the MALDI-MS analysis relative to direct infusion ESI-MS. It appears that chromatographic separation substantially reduces competitive ionization, thus enhancing sensitivity. We used tandem mass spectra of permethylated glycans to assign characteristic fragment ions which are specific for particular glycan type. For example, a fragment ion at m/z value of 825 is only observed in the tandem mass spectra of sialylated glycans. This fragment contains terminal sialic acid moiety attached to HexHexNAc. These characteristic fragments were subsequently utilized to quantify permethylated glycans derived from breast cancer cells and tissues using MRM experiment. The utility of this approach to assess the glycomic changes associated with breast cancer brain metastasis will be also presented and discussed. Also, enzymatic release of glycans from the surface of tissues prior to permethylation and LC-MS analysis will be described and presented.

L-213 Novel Non-reductive Labeling Method for N-glycans Sheathless CE-MS Analysis. Cai Tie, Xin-Xiang Zhang, Peking University, College of Chemistry, Beijing, CHINA Glycosylation is one of the most common and crucial modifications in both post- and co- translation. It plays an important role in many biological processes. It has been reported that different glycosylations can affect the conformations and functions of proteins. Due to their hydrophilicity, low responses on both optical and mass detectors, structures and diverse glycosylation sites, glycans analysis is much more challenging than gene-typing or protein sequencing. Although many labeling methods were developed to facilitate glycans analysis, there are few methods designed for CE-MS which is feasible at analysis for highly polar samples. In this article, a novel non-reductive labeling method is reported for glycans analysis by sheath-less CE-MS. Highly toxic reductive reagent is avoided in the process and the labeled samples could be analyzed without further purification. To reduce the chance of desialylation, the labeling reaction is also optimized to be fast and mild. As low as 30 ng/ml of maltopentaose could be detected with CE-MS, one magnitude lower concentration than traditional HPLC-MS. Furthermore, this method was also successfully applied to human IgG N-glycans analysis. With this novel non-reductive labeling method, CE-MS could be a powerful tool for glycans analysis and overcome challenges in glycans analysis. L-214 Capillary Electrophoresis Mass Spectrometry for Characterization of Complex Glycans and Glycoproteins. Roxana G. Jayo1, Jianjun Li2, David D. Y. Chen1, 1University of British Columbia, Chemistry Department, Vancouver, CANADA; 2National Research Council Canada, Ottawa, CANADA Capillary electrophoresis mass spectrometry provides a seamless high speed two-dimensional separation, detection, and characterization method for complex biomolecules. Native O-acetylated N-glycans can be separated by CE-MS, and detailed structure information can be determined in the same run by tandem mass spectrometry. The results revealed isomeric details that cannot be obtained by other technologies. Glyco proteins and glycol peptides can also be separated and characterized by CE-MS. Different degrees of glycosylation of RNase B, as well as other glycolprotein characterizations by CE-MS will be discussed. L-215 A New Type of Silicon NanoFET Detector with Single-nanoparticle Sensitivity. Annelise E. Barron1, Denitsa Milanova2, Peter Griffin3, Matthew B. Kerby1, R. Fabian Pease4, 1Stanford University, Bioengineering Department, Stanford, CA, USA; 2Stanford University, Mechanical Engineering Department, 3Stanford University, Genome Technology Center, Stanford, CA, USA; 4Stanford University, Electrical Engineering Department, Stanford, CA, USA [KEYNOTE LECTURE] The next major cost reduction in bioanalysis will be accomplished with solid-state electronic detection, eliminating the need for UV or fluorescence signals and affiliated lasers, lenses, CCDs and dyes. Jumping onto this exciting bandwagon, we have designed, fabricated, and are testing a novel type of silicon Field Effect Transistor (FET) detection system for microfluidic devices. We believe that we can integrate this detector into a low-cost platform that will be beautifully applicable to the solid-state electronic analysis of electrophoresing biomolecules from low-volume samples. We created a planar sensor—no nanopore is necessary!—and

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fabricated the chips using conventional, scalable CMOS techniques. These sensors can be integrated into bioanalysis devices that, in their entirety, will be the size of a USB memory stick, and which will draw their power from and download their data to a laptop computer. Charged particles moving down a microchannel (hydrodynamically or electrophoretically) pass over a thinly insulated gate region. We took our inspiration from a new class of charged proteins (e.g., BSA with a charge of -18) altered the gate potential by ~ 1 mV, to change the source-drain current by an easily detectable ~1 nA. This sort of electronic detection technology has inherent sensitivity up to one million times greater than electrode sensing or nanopore-current blockade measurements, because it measures perturbations to the flow of electrons through the silicon chip itself. Computational methods will translate high-speed electronic pulses into quantitative signals that encode specific information about the bioanalytes of interest. This technology has myriad exciting applications in the fields of proteomics, biomarker discovery, and diagnostics by providing a label-free method to sensitively identify and quantify biological material (e.g., proteins, DNA, RNA, viruses, cells) at the single molecule/particle level. We are aiming to develop our detector for genetic analyses, and will present experimental results for the analysis of anionic nano- and microspheres; and if all goes well, for DNA molecules too. L-216 Mass Spectrometry and Surface-Enhanced Raman Spectrometry Detection in Capillaries and Microchannels. Frantisek Foret, Institute of Analytical Chemistry ASCR v.v.i., Brno, CZECH REPUBLIC Mass spectrometry coupled to high resolution separations is generally viewed as one of the most important tools in modern bioanalysis allowing detection and identification of analytes in very complex mixtures. Electrospray interfaces are now available in several design variations for common separation columns and new designs are under development for coupling of low flow separations such as capillary LC or CE including their microfluidic versions. While extremely useful the mass spectrometer is also a bulky instrument with limited potential for miniaturization. In many cases a simpler instrument providing rapid identification of the separated compounds based on an optical fingerprint would be desirable. When considering sensitivity and analyte identification surface-enhanced Raman scattering (SERS) brings an interesting potential for applications in analytical chemistry, biology, pharmacy and/or biomedicine. Detection of the SERS signal at wavelengths characteristic for functional groups or in the fingerprint region provides a unique combination of selectivity and sensitivity difficult to achieve by other techniques. The sensitivity of the method is based on the enhancement factor, which can reach orders of magnitude. Under such circumstances, SERS becomes a potential alternative to fluorescence detection with the benefit of the qualitative information without the need for chemical labeling of the analyzed substances. This presentation will show some experimental designs for both ESI/MS and SERS interfaces for capillary separations and compare their practical use in bioanalysis. L-217 Strategies for Improving Detectability in Microscale Electrophoresis. Saeko Kinami1, Hiroshi Koino1, Takayuki Kawai1, Kenji Sueyoshi1, Fumihiko Kitagawa2, Koji Otsuka1, 1Kyoto University, Department of Material Chemistry, Graduate School of Engineering, Kyoto, JAPAN; 2Hirosaki University, Department of Frontier Materials Chemistry, Graduate School of Science and Technology, Aomori, JAPAN For high-sensitive and high peak-capacity analysis of biomolecules, a novel two-dimensional microchip electrophoresis (2D-MCE) system employed an on-line sample concentration technique, large-volume sample stacking with an electroosmotic flow pump (LVSEP) [1, 2], was developed. The first-dimension channel was entirely filled with a sample solution, while the second-dimension channel with a sieving matrix. Samples injected into the first-dimension channel were concentrated and separated by LVSEP and microchip zone electrophoresis (MCZE), then transferred into the second-dimension channel to be separated by microchip gel electrophoresis (MCGE). In this system, more than 370-fold enhancement of the sensitivity for a fluorescent dye or fluorescein by LVSEP-MCZE–MCGE was achieved compared to conventional pinched-injection MCE. DNA fragments, YOYO-1 labeled ΦX 174 RF DNA Hae III digest, were analyzed by this system, where 72–1353 bp fragments were concentrated and separated with 50–100 fold sensitivity enhancement. In LVSEP applied capillary electrophoresis (CE) of oligosaccharides, ca. 780-fold sensitivity enhancement was attained without any loss of resolution compared to conventional CE [3]. To achieve further sensitivity improvement, a combination of LVSEP with a field-amplified sample injection (FASI) technique was examined, where an APTS-derivatized maltoheptaose (G7) was analyzed. As a typical result, APTS-G7 was well concentrated and separated from the free APTS with a 10,000-fold sensitivity increase compared to conventional CE. This development was supported in part by SENTAN, JST. [1] He, Y.; Lee, H. K. Anal. Chem. 1999, 71, 995-1001. [2] Kawai, T.; Sueyoshi, K.; Kitagawa, F.; Otsuka, K. Anal. Chem. 2010, 82, 6504-6511. [3] Kawai, T.; Watanabe, M.; Sueyoshi, K.; Kitagawa, F.; Otsuka, K. J. Chromatogr. A 2012, 1232, 52-58.

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L-218 The Role of Order in DNA Separations. Kevin Dorfman, Daniel Olson, Scott King, University of Minnesota, Chemical Engineering and Materials Science, Minneapolis, MN, USA [KEYNOTE LECTURE] One of the most attractive features of microfluidic separation devices is the ability to precisely tune the geometry of the device for a given separation. In the context of long DNA, microfabricated devices with pore spaces much larger than is possible in gels have led to order of magnitude reductions in the separation time. These devices use post arrays or colloidal crystals as the sieving matrix. We have recently been interested in using the precise control over the pore size to investigate a more fundamental aspect of the separation process, namely the order of the sieving medium. Gels are inherently disordered media, and one of the motivations for using microfabricated systems is the potential to reduce the band broadening by avoiding fluctuations in the pore space. However, numerical simulations (in particular, for post arrays) indicate that disordered systems may be superior to their ordered counterparts. We will report the results of systematic experiments on the band broadening of DNA in both post arrays and colloidal crystals as a function of their order. In the former case, we have created a disordered array by allowing the posts to perform a random walk. The disorder in this system is by design and easily characterized after fabrication by electron microscopy. In the latter case, we implemented a crystal growth protocol that disrupts the long range order at a known location in the channel. We have characterized the local crystal structure of these separation devices using a combination of laser light scattering and Bragg diffraction measurements. For the moderate electric fields used in post arrays (10 V/cm), we find that the ordered and disordered arrays give similar results for the electrophoretic mobility and plate height, in stark contrast to predictions from Brownian dynamics simulations. At higher electric fields, the plate height in the disordered array increases dramatically due to multi-post interactions, whereas the plate height is constant in an ordered array. In the colloidal crystal, we find that the resolution always increases more quickly when the crystal has long range order. L-219 Capillary Electrophoresis Studies of Molecular Beacon-like DNA Hairpins. Nancy Stellwagen, Chun Yaw (Joel) Chang, University of Iowa, Department of Biochemistry, Iowa City, IA, USA Capillary electrophoresis can be used to determine the free solution electrophoretic mobilities and thermal stabilities of DNA hairpins. In many cases, the observed melting temperatures of hairpins with complementary sequences differ by ~5ºC. However, two complementary DNAs that are predicted to form molecular beacon-like hairpins with 5 base pairs in the stem and 16 nucleotides in the loop exhibit melting temperatures that differ by nearly 20ºC. The greater thermal stability of one of the hairpins (called hairpin 2) is due to tandem GA residues on opposite sides of the loop. These residues appear to form pairs of non-Watson-Crick GA base pairs across the loop, effectively increasing the length of the stem and increasing hairpin stability. If the corresponding TC residues in the loop of the complementary hairpin (called hairpin 1) are replaced by tandem GA residues, the melting temperatures of the modified hairpin 1 are close to the values observed for hairpin 2. Eliminating the tandem GA residues in the loop of hairpin 2 makes the stability of the modified hairpin 2 essentially equal to that of hairpin 1. If the loops in the two hairpins are replaced by a loop containing 16 thymine residues, the thermal stability of the T-loop hairpin is equal to that of hairpin 1. Hence, the difference in the thermal stabilities of the two molecular beacon-like hairpins can be attributed to differences in base stacking of the residues in the loop. L-220 A Two-dimensional Microfluidic Platform for DNA Profiling by Fragment Length and Sequence. Linda B. McGown, Xueru Zhang, Steven Cramer, Rensselaer Polytechnic Institute, Chemistry and Chemical Biology, Troy, NY, USA Separation of DNA has been central to advances in our understanding of human biology and health. Genome sequencing, genotyping, phenotyping as well as metagenomic analysis of pathogenic organisms and microbial communities have all relied on methods for DNA separation, most commonly based on DNA fragment length. Same-length fragments must then be sequenced or separated by sequence. We are working to develop a microfluidic chip for simple, rapid, low-cost separation of DNA in two dimensions: length and sequence. This will be achieved by first separating DNA by length using conventional sieving polymers and then separating the same-length DNA fragments in the second dimension of sequence using a new gel medium that is formed by self-association of guanosine compounds. This guanosine gel, or “G-gel”, shows promise for separation of DNA on the basis of sequence without the need for mobility shifts induced by three-dimensional structure or resistance to thermal/chemical denaturation that are the basis for existing techniques. We present here the first steps toward realization of an integrated platform for real-time DNA analysis that would offer important advantages for wide-ranging applications, for example, routine genomic analysis at point-of-care and in challenging settings such as remote areas or disaster sites, and for profiling pathogenic organisms and health-related microbial communities and monitoring their responses to environmental stressors.

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L-221 Investigations of MS Compatible Capillary Coatings for Intact Protein Analysis by CE-MS. Isabelle Kohler1, Julie Schappler1, Marc Augsburger2, Serge Rudaz1, 1University of Geneva, School of Pharmaceutical Sciences, Geneva, SWITZERLAND; 2University Center of Legal Medicine, Lausanne, SWITZERLAND Capillary electrophoresis (CE) with UV detection is currently recognized as being a high effective separation technique for proteins analysis. Post-translational modifications such as glycosylation, phosphorylation and deamidation can be highlighted thanks to the high selectivity and resolution of CE-UV. However, protein adsorption onto the negatively charged capillary surface is an inherent drawback and numerous approaches can be envisaged to minimize it, e.g. extreme buffers pH, high ionic strength, ionic liquids, or the use of strong rinses between analyses. The most common approach is to coat the capillary wall with adsorbed polymers to prevent protein attachment. Due to the lack of sensitivity and selectivity of UV detection, CE can be successfully hyphenated with mass spectrometry (MS). However, a lot of common capillary coatings cannot be used in CE-MS configuration due to MS incompatibility (bleeding of coating material, ionization suppression). This issue was illustrated with carbohydrate-deficient transferrin (CDT) analysis. Transferrin (Tf) is the most important iron transporting protein containing two metal binding sites and two N-linked carbohydrate chains with a total of up to eight negatively charged sialic acid terminals. The major glycoform of Tf contains four sialic acid residues (tetrasialo-Tf) but minor isoforms with two to eight sialic acid residues can be identified in normal human serum. CDT is the most specific marker for chronic alcohol intake and encompasses isoforms with zero (asialo-Tf) and two sialic acid residues (disialo-Tf). Asialo-Tf is highly specific to alcoholic patients since it is only observed in pathological situation. For routine analyses, a commercial CE-UV method providing a bilayer dynamic capillary coating is used for Tf-isoforms analysis. This coating is not volatile and is therefore not compatible with MS detection. However, valuable information could be obtained with CE-MS configuration to readily assess the presence of specific asialo-Tf glycoform. In this study, various MS compatible non-covalent capillary coatings were investigated for CDT analysis by CE hyphenated with time-of-flight mass spectrometry (TOF/MS). Physically adsorbed coatings as monolayer or successive multiple ionic polymer layer coatings (SMIL), e.g. polybrene (PB), poly(vinylsulfonate) (PVS), dextran sulphate (DS), poly(ethyleneimine) (PEI) were studied, as well as commercial SMIL coatings. Coating procedures (concentration of polymers, coating time, coating regeneration), and analytical conditions were optimized to enhance CE efficiencies and run-to-run stability. L-222 Experimental and Theoretical Study of DNA Origami Dielectrophoresis in a Microfluidic System. Lin Gan1, Baoquan Ding2, Hao Yan2, Alexandra Ros1, 1Arizona State University, Department of Chemistry and Biochemistry, Tempe, AZ, USA; 2Arizona State University, Biodesign Institute, Tempe, AZ, USA In this study we focus on a fundamental study of dielectrophoresis (DEP) of structure-defined DNA nanoparticles in an insulator-based dielectrophoretic microfluidic system. DNA was chosen as model biomolecule due to it’s well known structure and the possibility to form molecular assemblies with defined shapes. Such DNA origamis can be created based on the principle of complementary base pairing. The resulting shapes of DNA origamis provide possibilities to further study the DEP mechanism for biomolecules. In particular, we explore the polarizability dependent dielectrophoretic behavior of 6-helix bundle and triangle DNA origamis with large structural difference and demonstrate distinct insulator-based dielectrophoresis (iDEP) behavior. In this study we employ poly(dimethylsiloxane) microchannels integrating an array of insulating posts to create electric field gradients upon application of external potentials. The DNA molecules are intercalated with a fluorescent dye and DEP manipulation of DNA origamis is recorded via fluorescence video microscopy. According to their particular structures, the polarizabilities for 6-helix bundle origami and triangle origami are estimated to be 7.756 * 10-31 Fm2 and 1.282 * 10-30 Fm2 , respectively. Hence, it is expected that the two origamis perform differently in iDEP experiments. Experimentally, for 6-helix bundle origami, a frequency dependence (60 to 1000 Hz) of the trapping of particles around the insulating posts was monitored at applied electric field of 500 V/cm. DEP trapping behavior for triangle origami was not observed under the same conditions. Increasing the frequency up to 1500 Hz while also augmenting the applied electric field to 1500 V/cm at a lower the buffer conductivity reveals a distinct transition to iDEP trapping for triangle origami. The frequency dependent trapping behavior is also studied by numerical simulation employing a confusion-diffusion model in time dependence manner. These simulations revealed that the specific frequency dependent trapping areas resulted from of the electrophoretic migration within the DEP trapping regions. In conclusion, we demonstrated the DEP manipulation for DNA origami with the sizes down to 130 nm. The two investigated DNA origamis showed distinct difference in trapping strength. and frequency dependence in the trapping behavior. Our on going work is dedicated to the experimental determination of the DNA origami polarizabilities to exploit the their trapping behavior quantitatively and apply the gained knowledge for the optimization of separation techniques based on DEP.

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L-223 Electrokinetic Motion of Particles in Ratchet Microchannels. Saurin Patel, Xiangchun Xuan, Clemson University, Mechanical Engineering, Clemson, SC, USA Ratchet microchannels have been demonstrated to implement the electrokinetic focusing, trapping, and separation of various particles. These manipulations are all based on the particle dielectrophoresis that is induced by the electric field gradients formed around the ratchets. To date, however, no work has been done on the effects of the structure of ratchets on the transport and manipulation of particles in ratchet microchannels. We use a combined experimental and numerical method to study the electrokinetic motion of particles of various sizes in both a symmetric and an asymmetric ratchet microchannel under a series of electrical conditions (including electric field magnitude, AC to DC field ratio, and field frequency). It is found that the asymmetric ratchet can provide either a better or a worse focusing and trapping effectiveness than the symmetric ratchet, which is dependent on the direction of the flow. The structure of the ratchet can also affect the transport speed of particles, which is a function of both the electric field and the particle size. The obtained knowledge from this work should aid in the design and optimization of ratchet microchannels for electrical handling of particles and cells in microfluidic devices. L-224 Sensitive CE-LIF Determination of Ubiquitin in Biological Fluids using an On-line Derivatization Method. Kiarach Mesbah1, Farid Oukacine1, Markus Otto2, Myriam Taverna1, 1Universite Paris-Sud, CNRS, LPNSS Faculte de Pharmacie, Chatenay-Malabry, FRANCE; 2University of Ulm, Department of Neurology, Ulm, GERMANY Alzheimer’s disease (AD) affects more than 35 million people in the world and represents one of the most expensive diseases for developed countries with more than 600 billion USD. To date, ELISA measurement of β-amyloid (1–42), total tau and phospho-tau-181 in cerebrospinal fluid (CSF) is the most advanced and accepted biochemical method to diagnose probable AD with high specificity and sensitivity (1). However, to increase the diagnosis performances both on the side of precocity and discrimination power, new biomarkers such as ubiquitin are to be considered (2). In fact, ubiquitin levels in brain homogenates of AD patients have been shown to be significantly higher than those of both non-neurological aged and neurological control groups (3). One recent publication also reported the increase of ubiquitin CSF level in AD patients compared to healthy controls for a north Indian population (4). The aim of our work was therefore to develop a sensitive and reproducible method to quantify ubiquitin in biological samples and particularly in CSF. We selected an on-line derivatization strategy in order to label ubiquitin on-line and during the separation step. We first optimized basic parameters to achieve not only a satisfactory resolution between ubiquitin, other proteins and fluorescent dye by-products, but also efficient on-line derivatization of ubiquitin in fused silica capillaries. Thus, we compared different separation buffers and fluorescent agent solutions. In addition, the rinsing procedure was adjusted to suppress the non-specific adsorption of ubiquitin to the capillary surface. We then optimized the labelling process by studying several critical parameters: the molar ratio between protein and fluorescent dye, the injected volume of sample and dye solution and the applied voltage. In order to improve the sensitivity, we finally modified the composition of the sample and the labelling solution to enhance the derivatization reaction and to allow a stacking effect. The sensitivity decreased to the nanomolar level and a LOD less than 100 nM was reached. The linearity of the CE assay was demonstrated in the range of physiological concentrations expected in CSF, as well as the reproducibility of the method. The CE-LIF method was successfully applied to human CSF to detect ubiquitin. References: (1) J. Marksteiner, Drugs Today (2007), 43:423-431. (2) K. Blennow, NeuroRx (2004), 1:213-225. (3) T. Kudo et al., Brain research (1994), 639:1-7. (4) R. Kandimalla et al., Neuroscience Letters (2011), 487:134-138. L-225 Development of an In-situ Immobilized Enzyme Reactor for Peptide Mapping by Capillary Electrophoresis. Golfam Ghafourifar, Antoine Fleitz, Karen Waldron, University of Montreal, Department of Chemistry, Montreal, CANADA Protein digestions in solution phase or in-gel suffer from long incubation times, single use of the enzyme and enzyme autolysis that leads to interfering background peptides during separation and identification. By using immobilized enzymes, auto-proteolysis can be limited so that a high enzyme concentration can be used to achieve faster digestion of substrate. Also, since immobilized enzymes can be reused, this offsets the extra expense of using them in larger quantities. Automation in fluidic and microfluidic systems is made possible by using immobilized enzyme microreactors (IMERs). We are using chymotrypsin, an enzyme that cleaves peptide bonds at the C-terminal side of aromatic residues, which we insolubilize by cross-linking it with glutaraldehyde (GA) 2.5% under mild, room temperature conditions for 2h. Our goal is to develop a simple and rapid enzyme immobilization procedure in-situ that is compatible with micro column separations for peptide mapping. Firstly, we studied the necessity of denaturation by 8M urea for large proteins digested by GA-chymotrypsin prepared in batch form. Secondly, the reusability of the enzyme preparation, which results in soft, gel-like particles, was also studied. The various conditions needed to cross-link enzyme directly inside the microreactor will be discussed. The IMER is made of a fused silica capillary whose internal walls are derivatized with 3-Aminopropyltriethoxysilane at 37°C for 4h in a series of steps to stabilize the crosslinked enzyme on the wall. Digestion is achieved by passing protein through the IMER, with the peptide map generated by CE separation of

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the digest. This is important because we are simultaneously developing a CE-based protein fractionation strategy to couple to the IMER for mapping by CE-MS. Our efforts to optimize the GA-mediated cross-linking of enzyme inside the IMER for digestion of native as well as denatured protein will be presented. At low substrate concentrations (<1 μM), peptides are not be easily detected by CE-UV or even MALDI/MS so we used laser induced fluorescence detection (CE-LIF) to map peptides from fluorescently labeled protein substrate to facilitate characterization of the IMER. The long term goal is to have sequential LIF and MS detection for efficient quantitative and qualitative characterization of the IMER. L-226 A Low-cost, Centrifugally-driven Polyester-toner (PeT) Microchip with Passive Valve Flow Control for Protein Quantitation. Yiwen Ouyang, J Li, James P. Landers, University of Virginia Health Sciences Center, Department of Chemistry and Mechanical Engineering, Charlottesville, VA, USA [KEYNOTE LECTURE] Interest in Lab-on-a-Chip (LOC) technology has been escalated by application to real-world problems. The advantages of LOC technology include low-volume consumption of reagents, miniaturization, improved portability, and potential for automation. As microfluidic devices become more application-driven, the focus shifts from functionality to flow control and manufacturability in a cost-effective manner from inexpensive materials. In this work, using polyester (overhead transparencies) as the substrate and printer toner, we show that CD-like PeT microchips can be fabricated rapidly in a cost-effective manner with fluidic transport controlled by centrifugal forces. The chip is designed for automated, parallel serial dilution of sample for colometric-based quantitation of the protein concentration in samples exemplified by artificial human plasma. The five-layer PeT microchip was fabricated by laser ablation of microfeautures in a polyester sheet and toner bonding with a standard office laminator. With low-tech fabrication and materials, this type of device is inexpensive (1 USD per chip) and amenable for use as a single-use, disposable chip (). Various centrifuge fluidic functions have been introduced on this CD-like PeT microchip: (1) hydrophobic valving by simply depositing printer toner on the hydrophilic surface of the microchannel; (2) digital metering by an ‘overflow’ method and (3) chaotic mixing. Control of the fluidic delivering was achieved by simply manipulating the rotation speed of the CD-like polyester-toner microchip. Colorimetric image analysis was used to quantitatively validate the metering and dilution performance of the CD-like microchip. We show that, by changing the geometry of the metering chambers, volume ranges from ~200 nL to 2000 nL could be metered. We show serial dilution of sample through up to eight different concentrations in 2 min requiring less than 30 µL sample and reagent in total, with the empirical dilution ratio correlating well with the calculated ones (CV <5%). Due to the intrinsic nature of the parallel metering microfluidic network, error propagation from manual pipetting is circumvented. As a proof of principle, we explored a quantitative protein assay based on a tetrabromophenol blue indicator and the CD chip was challenged to determine the protein level in artificial blood plasma at clinical relevant concentration. To our best knowledge, it is the first demonstration of using a PeT material to construct a microchip with complex 3-D structure for centrifugal system in less than 1 hour. This has the potential to reduce workload, the handling of biohazardous material while providing a simple and cost-effective strategy for a quantitative assays. L-227 Metabolite Profiling in Urine of Cancer Patients by LC-TOF-MS and LC-MS/MS. Michal J. Markuszewski1, Wiktoria Struck1, Małgorzata Waszczuk-Jankowska1, Arlette Yumba Mpanga1, Piotr Koslinski2, Danuta Siluk1, Marcin Markuszewski3, Roman Kaliszan1, 1Medical University of Gdansk, Department of Biopharmaceutics and Pharmacodynamics, Gdansk, POLAND; 2Nicolaus Copernicus University in Torun, Department of Toxicology Ludwik Rydygier Collegium Medicum in Bydgoszcz, Torun, POLAND; 3Medical University of Gdansk, Department of Urology, Gdansk, POLAND During RNA turnover, nucleosides are excreted intact into the urine and their levels are higher in cancer patients in comparison to the healthy ones. There is a need to find the appropriate biomarkers that could diagnose cancer in its early stage. The aim of our study was to verify if nucleoside metabolic patterns may be treated as biomarkers of urogenital cancer diseases. Quantitative analysis of metabonomics techniques, combined with advanced statistical approaches, could enable the development of a metabolic profile that characterizes early changes of specific organ functions. Urinary nucleosides profiles, as other metabolites profiles, are closely related to pathophysiological status of the organism and can be effectively studied by metabolomic approach. The study includes modified nucleoside, pterines and pteridines profiles from urogenital tract cancer patients and healthy controls, acquired by CE, HPLC and UHPLC techniques. Also, urine samples were qualitatively and quantitatively analyzed using LC-TOF-MS and LC-MS/MS, respectively. Urine samples (n>300) were collected from healthy (with no diagnosed cancer disease) controls and cancer patients. The U-Mann Whitney test was used to distinguish differences in the concentration of metabolites in urine. Comparison of mean values from the healthy and cancer groups, revealed statistically significant differences (p < 0.01) for most of the metabolites studied. Observed elevated levels of nucleosides mean values in urine in the case of cancer patients are between 1.5 and 2.0. Based on logistic regression appropriate model was build which was characterized by sensitivity and specificity above 70 and 90%, respectively. The metabolomic approach developed here confirms significant relations between the changes of urinary metabolic patterns and the presence of cancer diseases.

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L-228 The Basis of Protein, Bacteria and Cell Aptamers Sieving Based on Capillary Electrophoresis. Feng Qu, Xinying Zhao, Xiaomei Zhou, Zhaoyang Meng, Beilei Lou, Beijing Institute of Technology, School of Life Science, Beijing, CHINA Capillary electrophoresis (CE) is one of the most powerful methods for aptamers sieving (known as CE-SELEX), which has the advantages of fast, high resolution, low sample consumption and smart separation modes of capillary zone electrophoresis (CZE), affinity capillary electrophoresis (ACE) and capillary isoelectric focus (CIEF). Moreover, the binding of target and synthetic single stranded DNA (ss-DNA) occurs in free solution, which eliminates the biases caused by stationary support and linker. Several protein aptamers have been successfully obtained based on CE, in which the selection efficiency is greatly improved. Before SELEX, the evaluation of random ss-DNA pool is very important for subsequent aptamers selection which makes great influence to selection efficiency. The quality of ss-DNA pool obtained from different company was compared, and the effects of dissolve buffer, freeze-thaw cycles, storage temperature, and degeneration times of ss-DNA pool were demonstrated. Furthermore, the interaction assessment of target and ss-DNA pool is also a key point before selection, which will significantly improve the selection effectiveness and reduce the blindness of sieving. Moreover, the complete separation of target-ss-DNA complex with ss-DNA pool determined the easily collection of desired target-ss-DNA complex, which benefit the subsequent iterative selection. We have been made efforts based on CE on the evaluation of ss-DNA pool, the interaction assessment of several protein targets (BSA, Thr, Trf, Mb, Hb, Cyt c ) and bacteria (E. coli, Lactobacillus acidophilus, Samonella typhimurium, Staphylococcus aureus) as well as cells (human glioma cells-U251, human T lymphocytic leukemia cell line-Jurkat, human cervical cancer cells–HeLa, human prostate cancer cell-PC3) with ss-DNA pool, and more separation methods for effective target-ss-DNA complex collection. Fortunately, CE plays an irreplaceable and perfect role on above tasks. ACKNOWLEDGEMENT The National Natural Science Foundation of China (No. 21175011) and the National Basic Research Program of China (973 Program, No. 2012CB910603). L-229 Recent Studies on Separation Mechanism of Enantiomers in Capillary Electrophoresis. Bezhan Chankvetadze, Tbilisi State University, Institute of Physical and Analytical Chemistry, Tbilisi, GEORGIA [KEYNOTE LECTURE] Capillary electrophoresis (CE) represents one of the major techniques not only for analytical scale enantioseparations but is also a powerful tool for a better understanding of the fine mechanisms of enantioselective intermolecular recognition. The major advantages of CE from the viewpoint of enantioselective molecular recognition studies are the following: 1. CE allows very fast screening of selector-selectand pairs. 2. The high peak efficiency in CE permits observing enantioselective features in selector-selectand interactions which are invisible by other techniques. 3. A small thermodynamic selectivity of recognition can be transformed into a high separation factor in CE. 4. CE is very flexible technique for adjustment of enantioseparation. The major disadvantage of CE for studies of non-covalent intermolecular interactions is that this technique does not provide any direct information regarding the structure of intermolecular diastereomeric associates. The experiments based on the nuclear Overhauser effect (NOE) in nuclear magnetic resonance (NMR) spectroscopy complement CE from this viewpoint very well. In addition, NMR-spectroscopy is very useful technique for determination of stoichiometry and enantioselective binding constants of selector-selectand associates. However, NMR spectroscopy fails when mixed complexes are formed between a selector and selectand. Mass spectrometry (MS) may appear useful in this case. This presentation summarizes our recent studies on the combined application of CE, NMR and MS methodologies to mechanistic studies of enantioselective selector-selectand interaction in the liquid phase. The methodology is illustrated with the examples including interaction of chiral drugs such as ketoconazole and terconazole, ephedrine, norephedrine and ketoprofen with various cyclodextrins. L-230 Nano-LC and CEC for Chiral Separations using Modified Polysaccharides Coated on Core-shell Silica Particles. Salvatore Fanali1, Zeineb Aturki1, Giovanni D'Orazio1, Anna Rocco1, Bezhan Chankvetadze2, 1Institute of Chemical Methodologies, Italian National Council of Research, Monterotondo, ITALY; 2Tbilisi State University, Tbilisi, GEORGIA The separation of chiral compounds is a challenging topic widely studied in various application fields including pharmaceutical industry, bio-medicine, agro-chemistry, environment, nutrition and food. In most cases, the importance of this research is that many compounds of this type (enantiomers) are, directly or indirectly, related to human health. The use of miniaturized technique for the enantiomers separation and analysis has been widely demonstrated employing a large number of chiral selectors such as cyclodextrins, metal ions-amino acids/peptides complexes, glycopeptide antibiotics, chiral crown-ethers, polysaccharide derivatives etc. Among them electromigration techniques, mainly capillary zone electrophoresis (CZE), has been applied for this purpose. Polysaccharide-coated silica-based stationary phases, have been successfully used in high-performance liquid chromatography for separation of enantiomers of a large number of chiral compounds. Recently, such CSPs have also been studied in capillary-/nano-Liquid chromatography and capillary electrochromatography (CEC). In this study two types of silica particles, one fully porous and the other superficial porous (core-shell) were modified with a polysaccharide-type chiral selector and evaluated for the

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separation of enantiomers in nano-LC and CEC. The major goal of the study was to critically evaluate the contribution of the “flow through particles” to enhancing peak efficiency in CEC compared to nano-LC. The results obtained for fused-core silica particles compared with silica particles of comparable size but having through pores questions the previous assumption that “flow through particles” is the major contributor to enhancing peak efficiencies observed in CEC. In addition, based on the results of this study it is suggested that contrary to previous reports on core-shell particles behaving poorly in narrow bore columns, these materials are useful for CEC, at least in capillary columns of 100 µm I.D. L-231 Comparative Enantioseparation of Chiral Drug Compounds with Different Cyclodextrins in Aqueous and Non-aqueous CE and Study of Related Selector-selectand Interactions by NMR Spectroscopy. Anne-Catherine Servais1, Marianne Fillet1, Bezhan Chankvetadze2, Jacques Crommen1, 1University of Liege, Laboratory of Pharmaceutical Analytical Chemistry, Department of Pharmaceutical Sciences, Liege, BELGIUM; 2Tbilisi State University, Tbilisi, GEORGIA The chiral β-blocker drugs propranolol and talinolol have been enantioseparated using two single component anionic β-cyclodextrin (CD) derivatives, namely heptakis (2,3-di-O-methyl-6-sulfo)-β-cyclodextrin (HDMS-β-CD) and heptakis (2,3-di-O-acetyl-6-sulfo)-β-cyclodextrin (HDAS-β-CD) in aqueous and non-aqueous capillary electrophoresis (CE). The enantiomer affinity patterns of talinolol towards these two CDs were found to be opposite to each other in both aqueous and non-aqueous CE (NACE) while in the case of propranolol opposite affinity patterns towards the two CDs were only observed in NACE. However, the enantiomer affinity pattern for a given CD derivative did not change when the aqueous background electrolyte (BGE) was replaced with a non-aqueous methanolic BGE, except for propranolol in the presence of HDMS-β-CD. The possible molecular mechanisms leading to these changes in the enantiomer affinity pattern were studied in both aqueous and non-aqueous BGEs using rotating frame nuclear Overhauser effect (1D ROESY) NMR spectroscopy. In the aqueous electrolyte, the formation of inclusion complexes between talinolol enantiomers and HDAS-β-CD as well as between propranolol enantiomers and HDMS-β-CD was confirmed while the formation of external complexes of talinolol with HDMS-β-CD and of propranolol with HDAS-β-CD could be suggested. In the non-aqueous electrolyte, all analyte-CD complexes were found to be of external type, except those between propranolol enantiomers and HDAS-β-CD. In spite of the fact that only external complexes were formed in many cases, excellent separation of propranolol and talinolol enantiomers could be observed with these two CDs in both aqueous and non-aqueous CE. L-232 Applications of Micro Free Flow Electrophoresis. Michael Bowser, Meng Jing, Nicholas Frost, University of Minnesota, Department of Chemistry, Minneapolis, MN, USA [KEYNOTE LECTURE] In free flow electrophoresis (FFE) a thin stream of sample is continuously introduced into a planar flow chamber. An electric field is applied perpendicularly to the flow through the separation chamber. Analytes are deflected laterally in the electric field according to their electrophoretic mobility giving rise to individual stream paths. FFE has recently been miniaturized into a microfluidic format (micro FFE), requiring less sample and reagents, a simplified flow profile and better heat dissipation. The continuous nature of micro FFE separations suggests a number of novel analytical applications. For example, we have demonstrated how introducing a buffer gradient into the micro FFE device can be used to efficiently optimize a range of separation conditions or estimate dissociation constants in as little as five minutes. We have also explored introducing a gradient at the sample channel to titrate a fluorescently labeled aptamer with increasing concentrations of its protein target. Due to the continuous nature of gradient micro FFE, complete coverage of the binding curve is possible in as little as five minutes. Micro FFE is an ideal second stage for two dimensional separations. Continuous micro FFE separation allows complete sampling of peaks as they migrate off the first dimension column with minimal loss of efficiency. Coupling to micro FFE results in a 10-20 fold improvement in peak capacity without sacrificing separation time. Micro FFE also offers the potential for impressive microscale purification. We will demonstrate isolation of high affinity aptamers using micro FFE. L-233 Three Dimensional Selection of Leptin Aptamers using Capillary Electrophoresis and Implications for Clone Validation. Jon Ashley, Sam Li, National University of Singapore, Department of Chemistry, Singapore, SINGAPORE CE-SELEX has been used as a fast and efficient way to select aptamers against protein targets and offers the advantage of separating bound DNA from unbound DNA in a free solution 3- dimensional environment. CE-SELEX was used to select aptamers against human leptin protein. Two methods used to validate the aptamers binding affinity against the target were performed and gave differing results. Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) yielded KD values in the high nanomolar range whereas the fluorescence intensity method gave KD values in the low micromolar range. These results suggest that aptamer validation must be carried out in a similar environment to that of the selection partitioning step and the environment in which the aptamer is intended to be used.. We also note that affinity binding by fluorescence

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intensity using microplate readers may be limited to targets which have relatively low Koff rates and that selection of aptamers in free solution can result in sequences which bind around the whole target. L-234 Herbal Fingerprints: Extraction of Information, Focussing on Similarity Analyses. Yvan Vander Heyden, Goedele Alaerts, Melanie Dumarey, Mohammad Goodarzi, Christophe Tistaert, Bieke Dejaegher, Vrije Universiteit Brussel - VUB-FABI, Brussels, BELGIUM Worldwide, herbs are used for preventive and therapeutic goals. Therefore, identification and quality control of these products of natural origin is required. Determination of some of the active compounds does not always allow assessing their total intrinsic quality. Since 1991 the World Health Organization accepts fingerprint chromatography as identification and quality evaluation technique for medicinal herbs. In fingerprint development, the goal is to create general conditions to maximize the peak capacity within an acceptable analysis time. A fingerprint can be developed for a number of reasons: identification, classification or calibration purposes. Identification is to confirm that a sample is originating from the herb expected and to exclude that it is another, i.e. to attain a better quality control of the herbs. Classification can be performed to classify samples according to, for instance, their origin. This can be either a geographic origin or to distinguish between natural and synthetic compounds, e.g. vanillin from herbal, synthetic or microbiologic origin. Such evaluation is most often done by a principal component analysis, occasionally by a cluster analysis. However, it might also be by building a classification model. A multivariate calibration can be performed when the herb or its extract also can be characterized by an activity, e.g. an antioxidant or a cytotoxic activity. The activity then can be modelled as a function of the complete chromatogram. The most commonly used modelling techniques are stepwise multivariate regression, principal component regression and partial least squares. The goal of the modelling can be either to built models that are able to predict the activity for future samples based on the chromatogram (e.g. the antioxidant activity from green tea) or to identify the main compounds/peaks responsible for a given activity. In the presentation the different applications will be considered, but the identification by means similarity analyses will be focused on. L-235 Dielectrophoretic Manipulation of Particles: Mathematical Modeling and Experimental Results. Blanca Lapizco-Encinas, Rochester Institute of Technology, Rochester, NY, USA [KEYNOTE LECTURE] Miniaturization is becoming increasingly important in for many analytical applications; working on the microscale offers significant advantages over traditional bench scale systems, such as: shorter response time, lower requirement of samples and reagents, higher resolution and sensitivity. Important research efforts are devoted toward the development of miniaturized devices for the analysis and manipulation of particles, especially for biological applications. Electric fields allow to drive fluid flows and particles without any moving parts, therefore studying electrokinetics is critical to further the advance of microfluidics. Electrokinetic transport mechanisms, such as electrophoresis and dielectrophoresis, are among the most employed techniques in miniaturized systems. Electrokinetics probes dielectric properties of target particles without causing denaturation or destruction, which makes these techniques suitable for many biotechnological applications in the manipulation of bioparticles, in both nano and microscales, such as macromolecules and microorganisms. Dielectrophoresis (DEP) is the motion of particles due to polarization effects in nonuniform electric fields; this electrokinetic transport mechanism has a great potential for the manipulation of a wide array of bioparticles. DEP offers great flexibility, since it can be carried out employing DC and AC electric fields, and neutral and charge particle can be manipulated. The majority of the studies on DEP have employed arrays of microelectrodes; however, employing microelectrodes has some drawbacks such as higher cost and loss of functionality due to fouling. Insulator-based dielectrophoresis (iDEP) is an attractive alternative, since it employs arrays of insulating structures, instead of electrodes, to create nonuniform electric fields; resulting in inexpensive and robust devices. This talk is focused on the use of AC-iDEP and DC-iDEP for the manipulation, concentration and separation of different types of microparticles. Microdevices containing channels with cylindrical insulating posts were employed to trap and sort particles. Results obtained from mathematical modeling of the device performance will be presented in order to explain the fundamentals behind iDEP. Experimental results demonstrate how different particles were immobilized at the dielectrophoretic traps created along the array of insulating structures when applying AC or DC electric potentials. Videos showing highly controlled particle manipulation with iDEP will be presented and discussed, including applications for cell assessment and microdevice performance characterization. The results demonstrate the versatility and great potential of iDEP as a microscale technique for effective and fast particle manipulation.

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L-236 Constriction-based Dielectrophoresis in Nanofluidic Devices for Protein Pre-concentration. Nathan Swami1, Mikiyas Tsegaye1, Kuo-Tang Liao1, Chia-Fu Chou2, 1University of Virginia, Electrical and Computer Engineering Department, Charlottesville, VA, USA; 2Academia Sinica, Taipei, TAIWAN The challenge of sensing rare numbers of biomarkers against a background of high concentration of other matrix proteins within physiologically relevant media requires methodologies for selective pre-concentration of the biomarker in proximity of the sensor. Antibody affinity based chemical depletion methods are unable to achieve the necessary degree of pre-concentration, since the biomarkers are present at billion-fold lower levels than the background proteins in blood. Hence, there is great interest in applying electrokinetic methods, especially within nanofluidic devices, where million-fold levels of pre-concentration have been reported using ion exclusion-enrichment effects due to electrical double layer overlap at the micro-to-nanofluidic interface. However, ion exclusion-enrichment methods require several tens of minutes to achieve this pre-concentration and they cannot effectively separate target biomarker proteins against a background of similarly charged and sized proteins in samples of physiological fluids. Dielectrophoresis enables highly selective trapping of bio-particles based on the characteristic frequency response of the dielectric permittivity of the bio-particle versus that of the medium, and it has been extensively applied towards sorting of somewhat similar sized biological cells with differing dielectric frequency response. However, its application to trapping ss-DNA and proteins, requires methods to enhance the local field to offset the steep fall in dielectrophoretic trapping forces with particle size. Furthermore, within physiological media of high conductivity, dielectrophoretic trapping is in competition with electrothermal flow due to Joule heating. Herein, we report on the application of dielectrophoresis using ~200 Vpp/cm fields at sub-micron constriction gaps within nanofluidic device channels to enhance the local electric field to enable million-fold levels of biomarker pre-concentration within physiological media of high conductivity. To enhance the degree of pre-concentration of proteins over that achieved by positive dielectrophoresis at ~100 kHz and negative dielectrophoresis at 1 MHz, we report on the application of successively smaller constriction gaps and addition of a critical DC offset to the AC field to exponentially enhance the extent of the protein depletion zone across the device. Using a potential energy diagram across the device, we explain how the application of sub-100 nm constrictions to enable a significant energy barrier towards protein transport, in conjunction to a critical level of DC field to cause energy wells due to significant tilting of the profile, results in an exponential degree of protein pre-concentration. We envision that these protein pre-concentration methodologies may be applied towards biomarker discovery, protein crystallization, and rare target sensing for early disease diagnostics. L-237 Reservoir-based Dielectrophoresis (rDEP) for Microfluidic Particle Separation. Xiangchun Xuan, Clemson University, Department of Mechanical Engineering, Clemson, SC, USA Dielectrophoresis has been widely used to manipulate a variety of particles (e.g., beads, cells, viruses, DNA, and protein molecules). Traditional electrode-based dielectrophoresis (eDEP) uses patterned in-channel microelectrodes to create gradients of high-frequency AC electric fields. This method requires complicated device fabrication, and is prone to device fouling due to electrode degradation. Insulator-based dielectrophoresis (iDEP) is another increasingly used technique for particle manipulation, which has been demonstrated by exploring the use of in-channel micro-insulators and channel curvatures, respectively. However, this method either suffers from Joule heating effects and particle clogging issues due to the involvement of micro-obstacles or requires a long curving microchannel that increases the fabrication and operation difficulty. In this talk I will introduce a new dielectrophoretic method that has been recently developed and demonstrated in my group. This method, termed reservoir-based dielectrophoresis (rDEP), exploits the inherent electric gradients formed at the reservoir-microchannel junction to generate negative dielectrophoresis for continuous particle manipulation. I will present our recent results on particle focusing, trapping and separation (by size, charge and viability) by the use of rDEP. L-238 High-sensitive Analysis of DNA Fragments by EKS-CGE and Possible Damage of the Fragments in Water by Applying High Potential Gradients. Takeshi Hirokawa1, Satomi Mori1, Xiaoxue Ye1, Mihoro Yamada1, Jyun Inoue2, Zhongqi Xu3, 1Hiroshima University, Graduate School of Engineering, Higashi-hiroshima, JAPAN; 2AIST, Tokyo, JAPAN; 3Donghua University, Shanghai, PR CHINA [KEYNOTE LECTURE] Analytical sensitivity of capillary zone electrophoresis can be very high when it is accompanied by appropriate on-line preconcentration methods. The electrokinetic supercharging preconcentration (EKS) can be one of the effective methods, especially when the sample is very dilute and the ionic strength is very small (system-induced termination [1]). EKS can be applied also to DNA fragment analysis as EKS-CGE. Sensitivity of EKS-CGE can be improved by using a BGE playing the role of the leading electrolyte for the analytes (0.2 % HPMC-1xTBE-6 % mannitol [2]) and by applying the electrode configuration with long distance between the capillary-end and electrode. Since the used BGE has enough sieving and preconcentration ability in spite of the low viscosity, it is essential to obtain high sensitivity in DNA analysis: The LOD achieved by using UV detection is less than a few ppb level for the DNA fragments in a 50 bp step ladder sample when the sample volume was 900 uL and the distance was 40 mm. When introducing a dilute sample (e.g. 50 ppb) by EKS-CGE under strong potential gradient, however, unknown peaks were frequently encountered. After careful exploration including the

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analysis of the step ladder sample that was intentionally applied by 200 V/cm before EKS, we came to the conclusion that the peaks might be formed by electrical decomposition of the original fragments under a high potential gradient during injection. A possible mechanism of the decomposition will be discussed by using a computer simulation of the EKS process. References: 1. Zhongqi Xu, K. KawahIto, X. Ye, A.R. Timerbaev, T. Hirokawa, Electrophoresis, 32, 1195-1200 (2011). 2. F.T. Han, B.H. Huynh, Y.F. Ma, B.C. Lin, Anal. Chem. 71 2385-2389 (1999). L-239 Monovalent Cations Control DNA Bending. Earle Stellwagen, Qian Dong, Nancy Stellwagen, University of Iowa, Department of Biochemistry, Iowa City, IA, USA We have used capillary electrophoresis to investigate the effect of monovalent cations on the bending of 26bp DNA oligomers containing A-tracts. All measurements were done at a constant ionic strength of 0.40 M, blending a given tetraalkylammonium ion with Li+, Na+, K+, NH4+ or Tris+. Contrary to expectation, the tetraalkylammonium ions influence the bent=straight equilibrium. Tetrabutylammonium (TBA+), tetrapropylammonium (TPA+) and tetramethylammonium (TMA+) ions preferentially stabilize the straight conformation, while tetraethylammonium (TEA+) ions stabilize the bent conformation. The hydrogen bonding cations Li+, NH4+ or Tris+ stabilize the bent conformation more efficiently than the non-hydrogen bonding cations, Na+ and K+. Surprisingly, high concentrations of all these cations stabilize the straight conformation. The cation concentration needed to stabilize the bent conformation is dependent upon the length of the A-tract, the number of A-tracts in the oligomer and the identity of the bases flanking the A-tracts. We attribute these observations to the properties of monovalent cation mixtures in the counterion cloud surrounding the oligonucleotides. L-240 Entropophoresis: A Novel Nanofluidic Transport Method for DNA Analysis. Samuel M. Stavis2, Jon Geist2, Michael Gaitan2, Laurie E. Locascio1, Elizabeth A. Strychalski1, 1Material Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, MD, USA; 2Physical Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, MD, USA Nanofluidic devices are ideally suited to manipulate biomolecules, such as DNA, because the structural dimensions can be engineered to match those of the analyte and harness nanoscale physicochemical phenomena. Considered in this way, the functionality of a nanofluidic device is determined fundamentally by its structure. More complex nanofluidic geometries generally enable more sophisticated control over nanoscale analytes and phenomena. Despite having delivered impressive advances for manipulating and measuring DNA molecules, nanofluidic technology has been limited by the simple structural geometries that are practical to fabricate with conventional lithographic processes and the application of external electric potential or hydrostatic pressure differentials for DNA transport. To demonstrate a new approach to nanofluidics that transcends these common limitations, we used grayscale photolithography to fabricate the first nanofluidic structures with complex three-dimensional surfaces formed by numerous nanoscale depths. As a prototype device, we created a nanofluidic staircase, which is a conceptually simple but structurally complex geometry, to explore the possibilities of this device for DNA analysis. The staircase imposed a complex free energy landscape on confined DNA molecules, inducing spontaneous transport “down” the staircase. Because DNA self-transport was directed by a gradient in conformational entropy, we termed this new nanofluidic transport method “entropophoresis.” DNA molecules descended the nanofluidic staircase without the application of external forces, eliminating associated technological limitations, such as continuous power input, monitoring, feedback, lag, transients, heating, and engineered attachment to particles or surfaces. We determined that the rate of entropophoresis was influenced by DNA topology, length, and degree of labeling with fluorescent dye molecules. Entropophoretic transport down the nanofluidic staircase enabled unprecedented confinement resolution for quantitative measurements of DNA size through the transition from strong to moderate slitlike confinement, which is a topic of enduring interest and continuing controversy in soft matter physics. The nanofluidic staircase and entropophoresis demonstrate a passive nanofluidic approach to the spontaneous transport, separation, concentration, and organization of DNA molecules and other nanoscale objects in solution. This confluence of capabilities promises to enhance the basic scientific relevance and practical utility of nanofluidic technology, presenting new possibilities in polymer physics, biophysics, separation science, and lab on a chip technology. L-241 CESI-MS, a Promising Tool for the Analysis of Peptides and their Post-translationally Modified Forms in Proteomic Studies. Herbert Lindner, Innsbruck Medical University, Division of Clinical Biochemistry Biocenter, Innsbruck, AUSTRIA [KEYNOTE LECTURE] HPLC in combination with high-resolution mass spectrometry is the commonly used technique for the analysis of complex protein mixtures in proteomics. The use of CZE as a complementary separation method to reversed-phase HPLC has not become fully accepted as an alternative up to now. Here we represent a highly sensitive sheathless CE-MS-interface (CESI-MS) consisting of a separation capillary with a porous tip as an alternative to nano-HPLC-MS. Neutral and uncoated capillaries were used for CE separations upstream from mass spectrometry characterization. Various peptide mixtures were analyzed with CE- and LC-ESI-MS and the results

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were compared with respect to number of identified peptides, proteins and protein sequence coverage. A protein standard with a dynamic concentration range containing 48 proteins divided into six abundance classes was also used to evaluate the capabilities of the CE- and LC-ESI-MS system. The protein amount loaded was ranging from 40 fmol to 0.4 amol. It was found that protein identification with CESI-MS is spanning a range of four abundance classes. Furthermore, a main focus was to investigate the application of this CESI-MS platform for the characterization of various posttranslationally modified peptides including mono- and multiphosphorylated, acetylated, mono- and multimethylated, nitrosylated and sulphated forms. L-242 Enhanced Coverage of the Anionic Urine Metabolome by CE-TOF-MS. Govert Somsen, Miranda Kok, Gerhardus de Jong, Utrecht University, Biomolecular Analysis, Utrecht, THE NETHERLANDS Coupled capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS) can provide unique coverage of the highly polar metabolome. Large part of CE-MS efforts in metabolomics so far have been focused on the analysis of cationogenic metabolites using positive electrospray ionization (ESI). This might be related to the fact that current negative-mode CE-TOF-MS provides relatively low responses for anionic metabolites. Still, a significant number of essential urine metabolites is acidic and can only be detected using negative ESI. In this lecture an improved CE-TOF-MS method for the analysis of anionic metabolites in urine will be presented. It will be shown that addition of volatile amines to the background electrolyte and sheath liquid can yield enhanced metabolite ionization efficiencies. When compared with conventional CE-MS methods employing ammonium acetate, sensitivity gain factors of up to seven were achieved, while the number of metabolites detected in urine was more than doubled. The new CE-MS method was used to study microbial-host co-metabolism by analyzing urine samples from antibiotic-treated rats. Based on the obtained urinary metabolic profiles, groups of rats receiving different treatments could be adequately distinguished. Seventeen discriminatory metabolites were found, of which nine were putatively identified. Comparison with an NMR study of the same urine samples showed that CE-MS revealed at least ten unique discriminatory compounds, exemplifying the added value of CE-MS in non-targeted profiling of anionic metabolites. L-243 CE-ESI-MS Interfaces: Effect on Protein Ionization Profiles. Grégoire Bonvin, Serge Rudaz, Julie Schappler, University of Geneva, School of Pharmaceutical Sciences, Geneva, SWITZERLAND The on-line combination of capillary electrophoresis (CE) with mass spectrometry (MS) is an attractive option for the analysis of intact proteins that are multi-charged with electrospray ionization (ESI). Nowadays several ESI interfaces exist to realize this coupling and they can be divided into two major classes according to their operating flow rate. The first group works at the µL/min flow rate (electrospray regime) and is generally pneumatically assisted by addition of a nebulizing gas and a sheath liquid. In this particular configuration, the sheath liquid is a key parameter of the ionization and any change in composition and flow rate leads to a significant alteration of the molecule ionization. The second approach operates in the nL/min flow rate range (nanospray regime) without any pneumatic assistance. Here the composition of the background electrolyte (BGE) mainly drives the ionization. In this study, each type of CE-MS interfaces (i.e., sheath liquid and sheathless) were investigated regarding the ionization profiles of insulin selected as model protein and analyzed by CE with different conditions (e.g., pH, buffer salts, addition of organic solvent). According to their respective spray regime, both interfaces led to distinct droplets formation, which influenced the protein ionization. As expected with the sheath liquid interface, ESI largely depended on the sheath liquid composition but interestingly the BGE composition also affected the protein ionization profile. The sheathless interface provided significantly different ionization profile and higher charge states were detected, as well as lower limits of detection compared to the sheath liquid interface. Performance, in terms of repeatability and sensitivity, was eventually assessed for both interfaces. L-244 Easy Peak Tracking in CE-UV-ESI-MS by Incorporating the Drift in Current in the Mobility Calculations. Nickolaj J. Petersen, Steen Honoré Hansen, University of Copenhagen, Copenhagen, DENMARK In CE-MS it is difficult to maintain thermostatic control along the whole separation capillary. In addition to the non-uniform temperature along the capillary, Joule heating will cause the temperature to increase during the separation. As a result the analytes may accelerate and pass detectors along the capillary with different speed. This is especially problematic for identification of small impurities in complex samples and in trace analysis, where it is desired to correlate peaks in the UV trace with their corresponding mass spectra. Recently we introduced a new way of transforming the CE separation data into the mobility scale by taking into account the temperature dependent change in viscosity of the separation buffer (1). The method is based on the fact that the relative change in conductivity during the separation reflects a similar relative change in the mobility of all analytes. The mobilities derived using our method are called “temperature-correlated mobilities”. The method allows CE-UV data performed under full thermostatic control to be directly comparable with CE-UV-ESI-MS data independent of capillary dimensions, applied field strengths, temperature control and voltage ramping. Representing data in the temperature-correlated mobility scale further allows an easy way of aligning the electropherograms for changes in the Zeta potential and even suctions effects caused by the ESI interface.

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Some practical examples using this technique will be illustrated. References 1) N. J. Petersen, S. H. Hansen: "Improving the reproducibility in capillary electrophoresis by incorporating current drift in mobility and peak area calculations" Electrophoresis, 2012, 33 (6), p. 1021-1031 L-245 Magnetophoretic Separation and Detection in Microchannels. Je-Kyun Park, Korea Advanced Institute of Science and Technology (KAIST), Department of Bio and Brain Engineering, Daejeon, REPUBLIC OF KOREA [KEYNOTE LECTURE] Magnetophoresis is a phenomenon explaining particle migration driven by magnetic force exerted on a particle. The intensity and direction of magnetic force depend on the intrinsic magnetic properties of the materials and surroundings of them, including diamagnetism and paramagnetism. Previously, we have developed a new magnetophoretic separation scheme, isomagnetophoresis, which can be used to discriminate subtle differences in magnetic susceptibility by using a magnetic susceptibility gradient in a microfluidic channel. The magnetic force exerted on a microbead is proportional to the magnetic susceptibility difference between the microbead and its surrounding solution, and the microbead moves toward or away from the high magnetic field in proportion to the magnetic susceptibility difference and stays at this isomagnetophoretic position. In isomagnetophoretic immunoassays, the magnetic nanoparticles are used as labels on microbeads in sandwich-type immunoassay, detecting the amount of bound analytes by isomagnetophoretic focusing the solid-support microbeads under the magnetic susceptibility gradient and magnetic field in a microchannel. One advantage of the method is that the dynamic range of the isomagnetophoretic immunoassay system can be adjusted by altering the magnetic susceptibility gradient. In addition, this isomagnetophoretic immunoassay system can be used to analyze the selected concentration of target analytes in detail by tuning the dynamic ranges. The proposed immunoassay can be useful to accurately quantify the concentrations of biomarkers over the whole range of analyte concentrations, based on the current status and needs of the patient. L-246 Microchip Capillary Electrophoresis for Extraterrestrial In situ Chemical Analyses Part I: Expanding the Range of Targets and Dealing with Complex Samples. Maria F. Mora, Amanda M. Stockton, Morgan L. Cable, Peter A. Willis, California Institute of Technology, Jet Propulsion Laboratory, Pasadena, CA, USA Despite multiple orbiter and landed missions to extraterrestrial bodies in the solar system, we still know relatively little about the detailed chemical composition and quantity of organics present on them. Microchip capillary electrophoresis (µCE) with laser-induced fluorescence (LIF) detection could be employed on future missions to greatly expand our knowledge in this regard. µCE-LIF provides pptr sensitivity of organics while maintaining small instrument volume, low mass, and low power consumption, making it ideal for the payloads of in situ robotic explorers. The most interesting chemical analyses on planetary samples are undeniably those that involve the search for life. The typical approach in this search focuses on finding organics representative of life as we know it on Earth. However, by broadening the search to include multiple compound classes, we could account for the biomolecular diversity that may exist on other worlds where life may have evolved under very different chemical and thermodynamic conditions. To date, organics targeted by µCE instrumentation for planetary exploration (e.g. the Mars Organic Analyzer, MOA) have included amines, amino acids, aldehydes, carboxylic acids, and polycyclic aromatic hydrocarbons. In this presentation we will describe our work in the expansion of our chemical repertoire to include thiols, dipeptides, long-chain amines, and fatty acids. Thiols are ubiquitous in terrestrial biology and play a role in the alkaline hydrothermal model of the origin of life. As such, their detection on extraterrestrial bodies could provide evidence for life as well as information about other abiological processes. We have developed a protocol to analyze thiols by µCE-LIF employing a fluorescent probe that is compatible with MOA-like technology thus broadening the number of species that could be analyzed in situ. We have also developed methods for the analysis of long-chain amines at low temperature using ethanol-based non-aqueous µCE. This method enables analyses at significantly reduced temperatures such the ones required for analysis on Titan (having a surface of temperature 90 K). We demonstrated analyses down to -20 °C. This method was employed to analyze laboratory-generated Titan analogues (tholins) demonstrating the capabilities of this technique for in situ analysis. We will also summarize past work about how analyses are performed on complex samples relevant to astrobiology and how the analyses of different molecular classes could serve as indicators of a host of different types of biological, prebiological or abiological processes, hence illuminating our understanding of the processes taking place on alien worlds. L-247 Microchip Capillary Electrophoresis for Extraterrestrial In situ Chemical Analyses Part II: Making it Work on Another Planet. Peter Willis, Amanda Stockton, Fernanda Mora, Morgan Cable, California Institute of Technology, Jet Propulsion Laboratory, Pasadena, CA, USA The search for signs of life on extraterrestrial targets is among NASA’s top priorities in solar system exploration. Quantitative compositional analysis of organic material could enable us to find those signs, as abiotic processes yield a statistical distribution of racemic organics, while biotic processes homochirally enrich those organic molecules useful to life. Microchip capillary electrophoresis (μCE) with laser-induced fluorescence (LIF) detection is capable of measuring these distributions via highly sensitive analyses (pptr) of amines, amino acids,

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aldehydes, ketones, carboxylic acids, thiols, and polycyclic aromatic hydrocarbons in complex samples ranging from Titan aerosol analogues to the Murchison meteorite. However, although μCE-LIF instrument prototypes have been developed and tested in the field (e.g. the Mars Organic Analyzers of UC Berkeley), these instruments have lacked the automation and miniaturized packaging required for spaceflight operation. In this presentation we will summarize our efforts to mature this technology so it is ready for eventual implementation on another planet. These developments begin with “flight-ready” microfluidic devices based upon pneumatically actuated monolithic membrane valves fabricated in glass/polymer hybrid stacks that can tolerate the extreme temperature cycling of spaceflight. We integrated this technology with microchip electrophoresis into a single stack device capable of completely automated sample processing (labeling, dilution, standard spiking) and electrophoretic analysis of amino acids. To expand the system’s capabilities to include analysis of a wider range of chemical targets on a single chip, we subsequently developed a fluidic processor capable of automated, rapid, contamination-free fluidic analyses utilizing a general purpose 32-valve microfluidic sample processor which can be reprogrammed for a practically limitless number of mixing operations. Furthermore, preliminary efforts have extended the operation of the microchip analysis system to -20°C for non-aqueous CE analysis of Titan aerosol analogue samples. Additionally, whereas our previous work has exclusively involved the analysis of liquid samples, we also have conducted proof of concept demonstrations for storage, manipulation and extraction of solids (both reagents and unknown samples) on chip. Finally, with our SBIR partners at HJ Sci & Tech, we are currently packaging all the above technology into a battery-powered, automated, reprogrammable, portable astrobiology instrument (the Chemical Laptop). The next stage in our technology development will involve the use of this system to perform a series of automated, end-to-end μCE-LIF analyses of multiple chemical functionalities present in a single sample, which is the ultimate goal of an in situ astrobiology instrument deployed on an extraterrestrial target. L-248 Miniaturizing Western Blot through Protein Capture in Hydrogel. Anais Ali-Cherif, Marine Verhusel, Laurent Malaquin, Jean-Louis Viovy, Stephanie Descroix, Institut Curie, Paris, FRANCE Bioanalytical methods based on immune reactions are widely used especially in the field of biomedical diagnosis. Among them, immunoblotting is currently performed to detect specific analytes in complex sample such as plasma or serum. Conventionally, proteins are separated by electrophoresis on a polyacrylamide gel, and thereafter are blotted on a membrane before being targeted with a specific labeled antibody. This method is highly sensitive and efficient nevertheless it is time-consuming and the initial volume of sample required is important. And as the blotting complex depends on various parameters it leads to low reproducibility of this bioanalytical method. There have been few attempts to combine microfluidics and conventional immunological methods to develop original immunoblotting approaches such as this open micropillar platform (1). And the group of A. Herr has provided important contributions in this field in particular with strategy based on polyacrylamide photografting and immobilized antibody to capture analyte of interest (2,3). We report here an original method based on a native separation of a mixture of protein by zone electrophoresis in presence of a photoactivable prepolymer. After a given separation time, the microchip is illuminated leading to the photopolymerization of the whole electrolyte. The proteins of interest are thus trapped within the photopolymerized hydrogel and specific biomolecules can be flown through the matrix to bind specifically the protein of interest. In this work, polyethyleneglycol-diacrylate (PEG-DA) was selected as photoactivable prepolymer. The pore size of the photopolymerized matrix has been evaluated as a function of PEG-DA concentration to get a better characterisation of proteins trapping. We have demonstrated a good efficiency of protein trapping in this hydrogel using fluorescently labelled proteins. Finally, the specific binding step has been studied in the photopolymerized matrix containing separated proteins using antibody and aptamer. Fluorescent aptamers were successfully used to replace antibody, they were electrokinetically driven trough the hydrogel and allow for the trapped proteins specific detection. This novel immunoblott approach should thus allow the complete integration of the western blot process within the microchip with high efficiency, good reproducibility and short analysis time. References: 1. Li H, Dauriac V, Thibert V, Senechal H, Peltre H, Zhang X, Descroix S (2010) Lab Chip, 10, 2597. 2. Q Tia, M He, D Kim, AE Herr (2011) Anal. Chem., 3581-8. 3. M He, AE Herr. Nature Prot, (2010) 5, 1844 L-249 Comparative Proteomic Profiling of Human Breast Cell Lines Exposed to the Phytoestrogen Genistein. Tim Veenstra, Frederick National Laboratory for Cancer Research, Laboratory of Proteomics and Analytical Technologies, Frederick, MD, USA [KEYNOTE LECTURE] Diet is implicated in approximately 50% of all newly diagnosed breast cancers. Genistein is an isoflavone which is abundant in soy products and epidemiological studies suggest it may exert protective effects against breast cancer. However, the exact mechanism by which genistein may exert its antitumorigenic effects is not clearly understood and there is some controversy with respect to the relationship between cancer incidence and doses of genistein. Herein we present a method that relies on label-free comparative shotgun proteomics to investigate the changes in relative protein concentrations in non-cancerous breast epithelial MCF10A cells, Hras transfected MCF10A-Hras cells, and breast cancer estrogen receptor-positive T47D cells following treatment with genistein at four different doses (0, 3, 10, and 30 µM) for 18 h. These cells were lyzed, trypsin digested and

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subjected to two dimensional LC separation followed by LTQ-FT MS detection. A total of 8914, 7992, and 8165 proteins were identified in MCF10A, T47D, and MCF10A-Hras, respectively. Changes at protein level were determined by spectral counting showing significant semi-synchronic changes in relative abundance for 544 proteins in all of three types of cells. A concurrent cell cycle analysis with the same dosing schedule showed that the genistein treatment induced G1-phase arrest in MCF10A-Hras and G2/M-phase arrest in T47D cells, but it did not alter cell cycle distribution in MCF10A cells. In the proteomic data, the arrest in G1-phase of MCF10A-Hras cells coincided with down-regulation of the proliferation marker Ki67, epidermal growth factor receptor and various components of the mitogen-activated protein kinase family of enzymes. These data highlight the differential response between breast cancer and non-malignant epithelial cells to genistein and the usefulness of proteomic approaches to study the effects of bioactive food components in nutrition and cancer prevention. L-250 Concentration of Low-abundance Proteins from Serum using Microchip Isotachophoresis. Cornelius Ivory, Danny Bottenus, Washington State University, Voiland School of Chemical Engineering and Bioengineering, Pullman, WA, USA Isotachophoresis (ITP) is used to isolate very low-abundance basic proteins from the high-abundance proteins in serum and then concentrate them about 20,000-fold. This is done using a PMMA microchip which contains a reducing union that decreases the channel's cross-sectional area by a factor of 100. Proteins migrating in ITP "peak" mode will concentrate 200-fold in the approach channel, and then will concentrate another 100-fold as they pass through the reducing union. This paper reports both the experimental application of ITP under basic, acidic and neutral conditions as well as the 3D numerical simulation of ITP in this type of chip. In particular, several challenges had to be met in each different ITP system including formation of carbonate ion from dissolved carbon dioxide at alkaline pH and the adsorption or precipitation of serum proteins at low pH. L-251 Profiling of Comprehensive Proteomics in Inflammatory Diseases by Immunoaffinity Capillary Electrophoresis. Norberto Guzman, Princeton Biochemicals Inc., Bioanalysis, Biomarkers, Princeton, NJ, USA Proteomic technologies represent useful strategies towards high-throughput, simultaneously analysis of hundreds or even thousands of proteins and peptides leading to the discovery of biomarkers for early diagnosis and prognosis of inflammatory diseases. Furthermore, panels of biomarkers are crucial for monitoring disease recurrence and therapeutic treatment efficacy to improve long-term survival of patients suffering of inflammatory diseases. In most instances chronic inflammatory diseases are very complex and it might require a panel of multiple biomarkers in order to achieve sufficient clinical efficacy. Recent evidence suggests that each patient affected by an inflammatory disease may have a unique subset of molecular patho-genetic/proteomic derangements, and the biomarkers may circulate in biological fluids in a number of distinct molecular forms. Common co- and post-translational modifications of proteins include phosphorylation, glycosylation, citrullination, nitration, ubiquitination, acetylation, prenylation, hydroxylation, chlorination, methylation, and others. In fact, it is known that modifications of tyrosine in certain proteins can elicit significant changes in protein structure and function, which, in some cases, may contribute to biological aging and age-related pathologies, such as atherosclerosis, neurodegeneration, and cataracts. Protein-bound tyrosine has been found to be modified to 3-nitrotyrosine, 3,4-dihydroxyphenylalanine (DOPA), 3,3’-dityrosine or 3-chlorotyrosine. When using Avastin (bevacizumab) as a protein drug in certain cancer treatments, there is some evidence that modification of tyrosine groups, such as nitrotyrosine or aminotyrosine, may contribute or be responsible for side effects of the drug. The need for a panel of biomarkers reflects the multi-dimensional nature of chronic inflammatory diseases, which includes cardiovascular diseases, cancer, Crohn’s disease, systemic lupus, trauma, rheumatoid arthritis, ulcerative colitis, psoriasis, diabetes, and others, all of which require distinct treatments and medical interventions. The labor-intensive task of isolating and characterizing individual modified proteins must continue, especially given the expanding list of unknown co- and post-translational modifications. Immunoaffinity capillary electrophoresis (IACE) is a technology that is becoming a powerful tool as a multi-dimensional platform for the rapid and effective isolation and characterization of a panel of biomarkers. The constant evolution of technologies makes multi-dimensional IACE strategy a crucial player in clinic and basic comprehensive proteomics. In this presentation, I will review important aspects of the technology of IACE and its use in the search for a panel of novel biomarkers of inflammatory diseases, and I will discuss future directions for this rapidly evolving field of proteomics than can aid in diagnosing disease, estimating prognosis, and monitoring treatment. Protein modifications can change a protein’s physical or chemical properties, conformation, activity, cellular localization, stability, and interactions. References: 1. Guzman NA, Phillips TM. Electrophoresis 32 (13), 1565-1578 (2011). 2. Fujigaki H, Seishima M, Saito K. Anal. Bioanal. Chem. 2012, in press. 3. Fu Q, Zhu J, Van Eyk JE. Clin. Chem. 56 (2), 314-318 (2010). 4. Woods JA, Wilund KR, Martin SA, Kistler BM. Aging Dis. 3 (1), 130-140 (2012).

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L-252 Gold Nanoparticle (AuNP)-based Immuno Probes for Targeting Proteomics. Shu-Hui Chen, National Cheng Kung University, Taiwan, RO CHINA Immunoprecipitation combined with mass spectrometry (MS) is a promising technique for targeting proteomics in characterizing sub-micrograms of target protein and interacting proteins in living cells. This method, however, is limited by interference arising from nonspecific binding. We report a novel gold nanoparticle (AuNP)-based immuno probe approach for immunoprecipitation. By crosslinking the antibody Fc domain to protein G covalently modified on AuNPs, the probe was fabricated and characterized to have 60 protein G and 30 immunoglobins per AuNP. We used human immunoglobin against the target and mouse immunoglobin with the same isotype (IgG) to fabricate the target and pre-clear probe, respectively, and termed it as the dual probe approach. Our results showed that the pre-clear probe (AuNP-IgG) and the target probe (AuNP-anti-ERα) share a similar panel of nonspecific binders but dramatic different specificity towards the target. Thus, using the dual probe method, we showed major nonspecific binders in the cell lysate could be largely removed without sacrificing the target protein. Compared to the conventional agarose gel, AuNP-based probe exhibited less non-specific interference and higher recovery yield for ERα. Moreover, AuNP-based probe is more inert than the agarose gel under harsh conditions and does not induce dissociation of the cross-linked IgG that could interfere with target identification. Using AuNP-based dual probes, ERα was shown to be purified from MCF-7 cells with minimum nonspecific binding. Moreover, the identity and phosphorylation sites on the C-terminus of the purified ERα could be positively confirmed by MS using only 1mg of cellular protein. L-253 Improved Analytical Methods for the Determination of Carbohydrate Deficient Transferrin (CDT), the Most Reliable Biochemical Marker of Chronic Abuse of Alcohol. Jennifer Pascali, Daniela Sorio, Federica Bortolotti, Franco Tagliaro, University of Verona, Public Health and Community Medicine, Verona, ITALY [KEYNOTE LECTURE] Transferrin is the major iron transporting serum protein bearing two branched oligosaccharide chains, which include four monosaccarides, acetylglucosamine, galactose, mannose and sialic acid. According to the number of sialic residues present in the transferrin molecule, different glycoforms have been identified, which in the serum of a normal subject are present in the following percentages: tetrasialo-Tf 75%, pentasialo-Tf 15%, trisialo-Tf 5%, disialo-Tf 2%, hexasialo-Tf 2%, a-, mono-, hepta-, octa- sialo-Tf < 1%. Carbohydrate Deficient Transferrin (CDT) refers to the sum of the disialo-, monosialo-, and asialo- Tf, and represents the most important biomarker of chronic alcohol abuse. Research has indicated that subjects with a pattern of alcohol intake > 60-80 grams per day for at least 7 consecutive days have an increased CDT concentration. The diagnostic sensitivity and specificity of CDT are about 60-80% and 100%, respectively. On this basis, CDT is used as a marker of chronic alcohol abuse in both clinical and forensic settings. At present, the analytical methods for CDT determination include immunoassays and methods based on capillary electrophoresis (CE) or HPLC. CE methods show high selectivity associated to high productivity, particularly when applied in capillary array instruments [1,2]. Aim of the present work was to investigate new analytical approaches for improving the performances of the CDT determinations, in terms of specificity and sensitivity, with a special focus on CE based methods. In order to achieve unequivocal recognition of the CDT peaks, an automated and reproducible in-capillary immunosubtraction was developed by injecting a plug of serum sample followed by a plug of anti-Tf antiserum. After the application of the electric field (direct polarity), the anionic charge of the Tf molecule made it move backward inside the capillary, being it drawn toward the detector by a strong electroosmotic flow. Contrariwise, the plug of antiserum moved forward under the electric field. Thus the voltage application provided the mixing of the sample with the anti-Tf antiserum with in-capillary formation of immunocomplexes, which, displaying a different mobility, were removed from the ‘transferrin region’ of the electropherogram. This analytical approach was applied successfully to recognize CDT peaks in the presence of potential interferences. Further analytical strategies were investigated in order to increase the analytical sensitivity of CE including the use of fluorescence detection, pre-precipitation of potentially interfering proteins, and use of high sensitivity detection cells. [1] Delanghe JR, De Buyzere ML. Clin Chim Acta. 2009, 406:1-7. [2] Bortolotti F, De Paoli G, Tagliaro F. J Chromatogr B 2006 841:96-109. L-254 A Microchip-Capillary Hybrid Device for 2D Protein Separations. Joann J. Lu, Shili Wang, Guanbin Li, Wei Wang, Shaorong Liu, University of Oklahoma, Chemistry and Biochemistry, Norman, OK, USA One of the major problems that the proteomic research community faces is lack of analytical tools to separate and measure the cellular proteins with adequate resolution, sensitivity, dynamic range and throughput. Deciphering the human proteome is a lot harder than mapping the human genome, as much of a challenge as that was, since the complexity of alternative splicing and post-translational modifications generates great complexity, over a widely variable dynamic range. The payoff, though, will be profound, in terms of mechanistic understanding of physiology and disease. 2-D (IEF and SDS-PAGE) gel electrophoresis has, arguablely, the highest resolution power for protein analysis. It was the first that was able to achieve proteomic analysis. 2-D gel has been used for more than thirty years and is still the major workhorse for separations of proteins in complex biological samples. However, it is far from dealing with the real complexity of the proteomes. Traditional 2-D gel

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electrophoresis with coomassie or silver staining is fundamentally a semi-quantitative analysis technique. Its sensitivity is inadequate for detecting low abundance proteins and its linear dynamic range is insufficient for handling the extremely divergent protein concentrations in cells. The technique usually requires tens of micrograms of sample for each assay, which inhibits the use of 2-D gel electrophoresis when the sample is scarce. In addition, because the IEF and SDS-PAGE are two isolated separation steps, the operation is labor intensive and its automation is challenging, and the throughput is low. In this project, we make our initial attempt to develop a microfabricated hybrid chip-capillary device to address the above issues. The hybrid device consists of three butted chip pieces. While two end chips are fixed, the middle chip can be shifted back and forth between two positions. At one position, CIEF is performed. As the middle chip is shifted to the other position, the CIEF separated proteins are fractionated and each fraction is injected between two capillaries for capillary gel electrophoresis. In this presentation, we will illustrate the working principle and demonstrate the feasibility of this approach for automated 2D protein separations. L-255 A Decade-long Advances in Electrodeless Dielectrophoresis (eDEP) and its Future Bioanalytical and Biomedical Applications. Chia-Fu Chou, Academia Sinica, Institute of Physics, Taipei, TAIWAN Ever since the introduction of electrodeless dielectrophoresis (eDEP) for DNA manipulation in 2002, eDEP has been extended to the manipulation of cells, RNA, virus, and microparticles, by exploiting the dielectric response of the particles and its interaction with enhanced non-uniform AC electric fields at dielectric constrictions. In parallel to the developments of using insulator-based dielectrophoresis, a similar concept of eDEP but running mostly with DC field, various creative designs have been proposed and demonstrated for broader applications of this technique. In particular, eDEP and its integration of biosensors demonstrated simultaneous sample enrichment and sensing are feasible which opens up the possibility of using eDEP for bioanalytical applications. Thanks to the advancement of nanofabrication and the nanofluidic chip bonding techniques, most recently proteins are shown to be highly enriched in seconds with eDEP using nanoscale structures, where the field focusing factor may reach 10^5-fold, with the molecular damming effect. In this review talk, we will highlight some of these developments of eDEP in the last decade and discuss its future perspectives for bioanalytical and biomedical applications. L-256 Microfluidic Devices for Comparative Profiling of Serum N-Glycans Indranil Mitra, William Alley, John Goetz, Jackie Vasseur, Milos Novotny, Stephen Jacobson, Indiana University, Department of Chemistry, Jacobson Lab, Bloomington, IN, USA Ovarian cancer is the fifth leading cause of cancer-related mortalities for women in the United States and often asymptomatic during the early stages. Aberrant glycosylation has been linked to several human diseases and may be highly informative for clinical diagnosis and prognosis. We are developing methods that use microchip electrophoresis to determine quantitative differences in the serum N-glycan profiles of disease-free individuals and patients with cancer. N-Glycans are enzymatically cleaved from serum glycoproteins, labeled with a charged, fluorescent tag, and analyzed on microfluidic devices that have a 22-cm long separation channel, are operated at 1250 V/cm, and are coupled to fluorescence detection. These devices generate analysis times under 100 s, separation efficiencies up to 700,000 plates, and migration time reproducibility better than 0.5% RSD after peak alignment. Low and high abundance glycans and their structural isomers are easily resolved and are used in the statistical analysis of the samples. Principal component analysis and analysis of variance tests reveal differences among the N-glycan profiles of disease-free individuals, patients with ovarian cancer, and patients after late-stage treatment. We are also comparing results for the same samples analyzed by microchip electrophoresis and MALDI-MS to better understand alterations in the glycomic profile linked to disease progression. L-301 Immobilized Enzyme Reactor (IMER) Enhanced Proteomics. Fred Regnier, JinHee Kim, Purdue University, Department of Chemistry, West Lafayette, IN, USA [PLENARY LECTURE] The significance of how proteins are prepared by mass spectral analysis has generally been under appreciated in proteomics. Whereas MS analysis can be achieved in milliseconds, preliminary fractionation along with reduction, alkylation, and trypsin digest it can take hours and be variable. The manner in which these steps are executed can even impact the nature and quality of mass spectra. Two of the slowest steps are i) reduction and alkylation (R&A) and ii) trypsin digestion. This paper will show that a continuous flow immobilized enzyme reactor (IMER) can reduce sample preparation to less than 5 min and allow R&A to be circumvented. It will also be shown that IMER based trypsin digestion is generally not identical to that in solution and that in many cases different proteins are identified with R&A than by direct proteolysis with the IMER. With identification and quantification of Lewis x antigen bearing glycoproteins affinity selected from human plasma approximately 30% more proteins were identified by combining results from both methods. The great advantage of the IMER was that it enabled automation along with allowing rapid method validation and a greater breadth of protein identification. Proteomics based on MS identification of peptides is clearly dependent on the mode of sample preparation. It is concluded that perhaps 1/3 of all biomarker candidates may have been missed in current studies by using a single sample preparation method.

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L-302 New Advancements in Chromatographic Techniques for the Enrichment and Separation of Biomolecules. Guenther Bonn, University of Innsbruck, Institute of Analytical Chemistry and Radiochemistry, Innsbruck, AUSTRIA [PLENARY LECTURE] The move towards miniaturization in bioanalysis has prompted the development of advanced enrichment and separation technologies. Considerable progress has been made in the development of stationary phases for sample preparation and separation. Novel enrichment and desalting methods based on modern solid-phase extraction (SPE) technologies will be presented for reducing the complexity of biological samples while µ-HPLC is used for separation, preconcentration and fractionation. Several chemistries (e.g. fullerene-silica) are available for a wide range of applications which can be tailored to a specific application allowing endless possibilities in terms of selectivity tuning. SPE has been performed with various nanomaterials packed, embedded or coated on the walls of the pipette tip. The micropipette tip format permits the handling of submicrolitre amounts of samples. In another approach precipitation of phosphorylated proteins and peptides is performed by trivalent lanthanide ions for further investigations by LC-MS. Major focus is placed on the development of novel innovative analytical techniques using high performance single- and multi-dimensional enrichment and separation methods including capillary electrophoresis CE coupled to mass spectrometry (MS). Separation of complex mixtures is a crucial point during analysis including identification, structural elucidation and quantification. Novel polymeric capillary monoliths especially tailored for miniaturized liquid chromatography (μ–LC) offer a highly efficient unique separation tool to overcome all these problems in bioanalysis. Separation efficiencies of these capillaries are outstanding, as they have no inter-particular void volume and retaining frits, which results in a reduced resistance to mass transfer as a result of convective flow. Literature: Rania Bakry , Matthias Rainer , Christian W. Huck & Günther K. Bonn. New stationary phases for enrichment and separation in the ‘omics’ era. Bioanalysis, (2009), 1(1), 151-169. Stefan Bachmann, Rainer Vallant, Rania Bakry, Christian W. Huck, Danilo Corradini & Günther K Bonn. CE coupled to MALDI with novel covalently coated capillaries. Electrophoresis (2010), 31(4), 618-29. L-303 Direct-EI LC-MS Interface: Can Liquid Chromatography and Electron Ionization Mass Spectrometry Work Together Again? Achille Cappiello, University of Urbino, Urbino, ITALY [KEYNOTE LECTURE] Electrospray Ionization (ESI) has become a de facto standard methodology for interfacing HPLC with MS. However, many shortcomings of ESI are overlooked or neglected especially when complex matrices are involved. Ion suppression limits reproducibility and accuracy. Cappiello et al. have been pioneering the efficient direct coupling of capillary HPLC with EI-MS and have recently demonstrated feasibility of working in the nanoflow-HPLC coupling with GC-MS type detectors. The technique has proven its effectiveness for a wide range of compounds. In this presentation, the author wants to give an overview about the principle methodology, figures of merit and up-to-date validation studies executed. Special focus will be on new high-temperature applications. The interfacing mechanism is based on direct introduction of a liquid phase into the EI source followed by complete conversion to the gas phase prior to a conventional, electron-assisted ionization (1). The mass spectrometer is an Agilent 5975B Inert MSD, a single quadrupole instrument coupled to an Agilent 1100 Series nano-HPLC system. The transition mechanism is based on the formation of an aerosol in high-vacuum conditions, followed by vaporization of the solute upon contact on the hot surface. Nano liquid chromatography was performed in reversed and normal phase conditions using a mixture of water, acetonitrile or hexane and isopropanol respectively. 75 µm i.d. columns, packed with C18 particles were used for all RP separations. The new, EI-based interface shows no matrix effect under many experimental conditions and allows an alternative LC-MS detection for innovative fields of application. Acknowledgments: The study has been supported by Agilent Technologies. References: 1) A. Cappiello, G. Famiglini, E. Pierini, P. Palma, and H. Trufelli Advanced Liquid Chromatography-Mass Spectrometry Interface Based on Electron Ionization Anal Chem. 79, (2007), 5364-5372 L-304 Surface-Enhanced Raman Scattering Detection in Microfluidic Devices. Dosil Pereira de Jesus1, Grazielle Oliveira Setti2, Angelo Gobbi3, Ronei Jesus Poppi1, Ednan Joanni2, 1State University of Campinas, Institute of Chemistry, Analytical Chemistry Department, Campinas, BRAZIL; 2Center for Information Technology Renato Archer, Campinas, BRAZIL; 3Laboratory of Microfabrication, Brazilian Synchrotron Light Laboratory, Campinas, BRAZIL [KEYNOTE LECTURE] Usually, analytical microfluidic devices require high sensitivity detection systems. Consequently laser induced fluorescence (LIF) detection has been the first choice for many applications. Nevertheless surface-enhanced Raman scattering (SERS) detection has been emerging as an alternative to LIF since it can also provide low limits of detection, suitable for microfluidic systems. We synthetized oxide nanowires such as indium tin oxide (ITO) into microchannels by using sputtering, thermal synthesis or pulsed laser deposition (PLD). Although the depositions made by the sputtering methods have advantages in terms of reproducibility and uniformity over large areas, there are just a few reports on the use of this technique for nanostructural growth. We growth ITO nanowires by RF sputtering using an In catalyst layer and alternated depositions from ITO and In targets. The

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SERS effect was obtained by depositing a thin gold layer on the nanostructured substrate. The SERS spectra were measured with a Raman Station 400F (Perkin Elmer) equipped with a 750 nm excitation laser. High peak intensities could be attained at 1176 cm-1 for diluted (µM range) aqueous solutions of crystal violet. L-305 Determination of Polyamines in Arabidopsis by Capillary Electrophoresis using Salicylaldehyde-5-sulfonate as a Derivatizing Reagent. Takashi Kaneta1, Genki Inoue1, Toshio Takayanagi2, Junichi Kakehi1, Hiroyasu Motose1, Taku Takahashi1, 1Okayama University, Department of Chemistry, Okayama, JAPAN; 2Tokushima University, Tokushima, JAPAN Polyamines are important biological molecules which play roles of growth factors involved in cell division and biosynthesis of proteins. Therefore, a simple method for the determination of polyamines is needed not only for clinical test and food analysis but also clarifying biological roles of polyamines. High performance liquid chromatography (HPLC) is conventionally used for the separation and determination of polyamines. Conversely, capillary electrophoresis (CE) is preferred to HPLC for the determination of biomolecules since it needs a small amount of sample for the separation and determination. In this study, we report on a novel method for the determination of polyamines by CE using salicylaldehyde-5-sulfonate (SAS) as a derivatizing reagent. An aldehyde group of SAS forms Schiff base with primary amino groups of aliphatic polyamines, resulting in anionic species with absorption band at ultraviolet region. The derivatization method is simple, i. e. the derivatives are formed in a water-ethanol solution only by mixing a sample and the derivatizing reagent under a condition of neutral pH. In addition, the negative charges induced by SAS lead to high resolution with a short analysis time. The method permits the separation of five polyamines which play important roles in plants. Further improvement in sensitivity was needed for the determination of the polyamines in real plant samples. Therefore, the CE method was coupled with C18 solid phase extraction using ion-pairing formation with sodium dodecyl benzene sulfonate. Accumulation of polyamines by solid phase extraction enhanced sensitivity 10-times. The calibration curves of cadaverine (CAD), putrescine (PUT), and spermidine (SPD) were linear in the concentration range from 1 to 20 uM. The limits of detection for CAD, PUT, and SPD were 0.25, 0.37, and 0.44 uM, respectively. The sensitivity of this method was comparable to that of a conventional HPLC method. The proposed method was applicable to the determination of SPD in an Arabidopsis plant sample. Acknowledgements We thank Professor Masaru Niitsu (Joysai University) for his generous sample of synthesized thermospermine. L-306 Formation and Properties of Monoalkyl Carbonates: CE-C4D Beyond Analytical Chemistry. Claudimir do Lago, Universidade de Sao Paulo, Instituto de Quimica, Department Quimica Fundamental, Sao Paulo, BRAZIL A monoalkyl carbonates (MAC) is the anionic form of a hemi-ester of carbonic acid. They were first identified as an intermediary of the hydrolysis of diethyl carbonate during the 1920’s. The hemi-ester or alkyl carbonic acid was first synthesized at the beginning of the 1970’s. Since then, the most studied property is the hydrolysis, which releases carbon dioxide and the corresponding alcohol. During the last years we could demonstrate that MACs can be easily formed in aqueous medium and detected by capillary electrophoresis with capacitively coupled contactless conductivity detector (C4D). Ionic mobilities, diffusion coefficients, and hydrodynamic radii were obtained for 21 adducts, including aliphatic alcohols, polyols, and sugars. Although not precisely assessed, a first estimate for the pKa of monomethyl carbonic acids suggests that this acid is similar to carbonic acid. After migration is started into the capillary, the MACs equilibrium is shifted toward bicarbonate. This hydrolysis process can be monitored by using two C4D along the capillary, which allowed us to calculate the kinetic constants of hydrolysis and formation as well as the equilibrium constant. The highest equilibrium constant was 4.2 for monomethyl carbonate. For increasing chain length of the alcohols, the equilibrium constant tends to the unit, which suggests that fatty alcohols can also form MACs. The presence of monoethyl carbonate (MEC) in alcoholic drinks and beer was also confirmed by this technique. Despite the low concentration of MEC in a distilled beverage, MEC is readily formed when it is mixed with a carbonated soft drink. The low pH of the resulting mixture accounts for the fast formation of MEC. This MAC is naturally found in beers in concentration of ca. 1 mmol/L. The occurrence of this MAC in a fermentation product and the formation of adduct for fatty alcohols and sugars suggests that this class of substances may be present in biological medium. L-307 Capillary Zone Electrophoresis in Narrow Capillaries with Auxiliary Hydrodynamic Pumping. Thanh Duc Mai, Peter C. Hauser, University of Basel, Department of Chemistry, Basel, SWITZERLAND Contactless conductivity detection can be used with capillaries of internal diameters as little as 10 µm with negligible loss of sensitivity. For such narrow capillaries it was found possible to introduce hydrodynamic pumping as dispersion due to the laminar nature of the bulk flow is minor. This then opens up a new parameter for flexible and rapidly implemented modification of the residence time of analytes, which can be used to advantage either for the optimization of separation efficiency, or otherwise for optimization of analysis time. Furthermore, it is possible to compensate the electroosmotic flow, enabling the determination of anions without using a surface modifier. Also feasible are different new schemes for the concurrent determination of cations and anions based on the manipulation of sample plugs by supplementary pumping. This is aided by the fact that

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the conductivity measurement allows the facile detection of non-UV-active inorganic and organic ions of both charges. L-308 Building Nanochannels in Thermoplastics: An Approach for Single-Molecule Electrochromatography. Steven Soper1, Jiahao Wu2, Nyote Oliver2, Park Sunggook2, Dorel Moldovan2, Brian Novak2, Heungjoo Shin3, Yoonkyoung Cho3, Dimitris Nikitopoulos2, DongKyu Park3, Franklin Uba1, 1University of North Carolina, Biomedical Engineering and Chemistry, Chapel Hill, NC, USA; 2Louisana State University, Baton Rouge, LA, USA; 3UNIST, Ulsan, SOUTH KOREA [KEYNOTE LECTURE] We are developing techniques and methodologies for the fabrication of electrophoretic or chromatographic columns on the nanometer-scale that can be used for sorting single-molecules (DNAs, RNAs, peptides and proteins). Selecting columns with nanometer dimensions is predicated on two operational parameters associated with processing single molecules: (1) Infinite wall effects. In the case of chromatographically separating single molecules, columns with nanometer-scale dimensions allow frequent solute/wall interactions to provide high resolving power; columns with µm dimensions reduce potential solute/wall interaction frequency and result in poor resolution. (2) Detection scaling effects for single molecules. In both the electrophoretic and chromatographic cases, appending labels onto the target will heavily influence the migration/elution process. As such, we must depend on label-less detection techniques to prevent the separation process from being dominated by the label. We are thus using electrical conductivity readout using two pairs of nano-electrodes poised on both the input and output ends of the column. In this way, the migration/elution time can be discerned by reading the time required for single molecules to move through a fixed-length column. Electropherograms/chromatograms are constructed from the migration/elution times for individual molecules. To prepare the columns, NanoImprint Lithography (NIL) is used, which employs the molding of columns with diameters <50 nm and lengths >50 µm using a resin stamp. These columns can be made from a variety of thermoplastics, such as poly(methylmetharcylate), PMMA. The walls of a PMMA nano-column can be functionalized in a variety of ways to make the stationary phase or to modify the electroosmotic flow, such as plasma oxidizing the surface of pristine PMMA or appending monolayers to the plasma functionalized surface. Single molecule separations are currently being undertaken using a variety of model compounds, such as mononucleotides, oligonucleotides, peptides and proteins. Preliminary results from Molecular Dynamic (MD) simulations have supported the viability of sorting mononucleotides using PMMA-based columns. For single-molecule electrical detection, calculations using single molecules have shown that the gap size as well as the bias voltage between the electrodes has a profound impact on the signal-to-noise ratio for transducing single molecules. In this presentation, simulation and experimental results of sorting single molecules in nanometer polymer columns will be demonstrated. L-309 Biomimetic Approaches for Organ on Chips: Neuron and Kidney Chip for Tissue Engineering and Drug Screening. Kyung-Jin Jang1, Kahp-Yang Suh2, 1Wyss Institute at Harvard, Boston, MA, USA; 2Seoul National University, Seoul, KOREA [KEYNOTE LECTURE] In vivo, cells respond to surrounding mechanical/chemical stimulations and complex three-dimensional anisotropic environments. To understand these mechanisms, cells need to be maintained in an in vivo-like environment that reproduces key structural, functional, and mechanical properties of the target organ. In this talk, I will describe two biomimetic micro and nano systems that reconstitute critical functional aspects of the renal and nervous system. To emulate the kidney tubular environment, microfluidic-based techniques were used to recapitulate the complexity of the renal microenvironment in vitro, including fluid shear stress and transepithelial chemical gradient. The device included the integreation of a polydimethyl siloxane (PDMS) microfluidic channel and a porous membrane. Culture of renal tubule cells in the microfluidic device under fluid shear stress resulted in enhanced cell polarization, cytoskeletal reorganization, and cilia formation. In addition, to study the complex morphogenetic event of neurite outgrowth, we used UV-assisted capillary force lithography with polyurethane acrylate to construct different topographic patterns. These nanotopographical patterns can provide an insight into how neuronal growth cones can sense geometric ECM cues, which possibly leads to new applications in tissue regeneration after nerve injury as well as the treatment of neuropathological conditions. We also have on-going work on human kidney proximal tubule-on-a-chip for more predictive in vitro human kidney models for investigating absorption, distribution, metabolism, excretion, and toxicological properties of new chemical entities during the drug development process. This novel system may also provide a useful and cost-effective tool for studying biotransformation profiles, pharmacology, drug transport and toxicity, and hence help to facilitate the drug development process with a more human-relevant model.

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L-310 Dispersed Microgradient-based Separations. Mark Hayes, Arizona State University, Chemistry Department, Tempe, AZ, USA Core to all separations science is resolution: causing important components to move in time or space away from each other. Although a description of core processes controlling resolution across all separations systems does not exist, the most successful techniques are considered to be electrophoretic in nature. Early demonstrations of capillary- and microchip- based electrophoresis have shown absolutely extraordinary resolution using high fields showing deuterated vs. hydrogenated and sub-millisecond separations. These results were obtained in systems operating in a linear mode—spreading and diluting a mixture out along a single axis. Our work focusses on a new approach enabled by the short length-scale of microdevices which exploits local field gradients. The core to this discovery is setting up punctuated microgradients separated by flat field zones. This uniquely minimizes the effects of diffusion by dramatically increasing the local restoring forces. As a result, not only does the resolution increase by a factor tied to the strength of the local gradients, but can be accomplished in parallel while increasing concentration of the targets (focusing). This opens up several new venues for exploitation, including dynamic and programmable collection of specific analytes at higher resolution than is available from any traditional technique in an array format. Speeds of separations are also dramatically increased. We describe the underlying theoretical principles and provide examples of flow/electric field and electrokinetic/dielectrophoretic devices in a microfluidic format. The underlying models extend to any force which can be induced into dispersed microgradients in the presence of a static counter field. L-311 Electrochromatography on Acrylate-based Monolith in Cyclic Olefin Copolymer Microchip: A Robust and Easy-to-use Technology. Yoann Ladner, Gerard Cretier, Karine Faure, Universite de Lyon, Institut des Sciences Analytiques, Lyon, FRANCE For many years, the development of electrochromatography on chip has been slowed down because the synthesis of the stationary phase inside a microfluidic device was a difficult, non reproductive task. Recently, we have demonstrated that it is possible to synthesize an acrylate monolith by photopolymerization in cyclic olefin copolymer chip, with high reproducibility and perfect control of both structure and anchoring, in less than 10 min. This presentation will highlight that photopolymerization conditions are critical issues that shall not be neglected in order to get the most efficient stationary phase. Exciting reversed-phase chromatographic performances on chip are illustrated by 1) high efficiencies: 250000 plates/m on polycyclic aromatic hydrocarbons and 2) fast analysis: baseline separation of 8 neurotransmitters in less than 3.5 min through the combination of electrophoretic and chromatographic processes. This monolith-based electrochromatography device demonstrates many interesting features such as strong resistance to aggressive conditions (organic solvents, extreme pH), application to a wide range of analytes (non-polar or polar, neutral or charged), high separation efficiency and ability for on-line preconcentration. Preconcentration of solutes is demonstrated through two examples: polar biogenic amines derivatized with naphthalene-2,3-dicarboxaldehyde (NDA), and non-polar solutes such as PAH, for which a preconcentration step of 4.5 hours could be applied to reach a LOD of 1 ppb. The robustness of this new electrochromatographic microchip was demonstrated on real samples through the quantitative analysis of histamine and tyramine at pH 13 in French wines. The chip could withstand over 100 injections of unfiltered wine samples. L-312 A Nanoporous Membrane Electrokinetic Molecular Sensing Platform. Senapati Satyajyoti1, Zdenek Slouka2, Sunny Shah1, Li-Jing Cheng1, Hsueh-Chia Chang1, 1University of Notre Dame, Chemical and Biomolecular Engineering Department, Notre Dame, IN, USA; 2Prague Institute of Chemical Technology, Prague, CZECH REPUBLIC We report a new molecular (nucleic acid and protein) microfluidic sensor platform that is economical to mass produce, label-free, multi-target (>5), rapid (<10 min), selective (SNP) and sensitive (pM)----and is hence most suitable for portable point-of-care diagnostic devices. The new membrane sensor platform employs fabrication, sol gel and photocuring chemistry to embed and synthesize the membrane in polymer and glass chips. When a DC field is applied across a nanoporous ion-selective membrane (or a fabricated nano-channel or nanostructure assembly), a molecular-sized charge polarized layer develops on the depletion side after concentration polarization. The presence of target molecules in this polarized layer can hence sensitively change the limiting and overlimiting currents of the membrane. The overlimiting current shift is particularly large (~1 V) because large nucleic acids can change the hydrodynamic slip length and hence suppress the surface electrokinetic vortices responsible for the overlimiting current. Oppositely charged small molecules, on the other hand, can produce a bipolar membrane like a PN semiconductor junction that can produce a large water-splitting ion current signal when a high field develops at the carrier-depleted junction with a reverse bias. Without using electron transfer to detect the molecules, we eliminate the need for SAM in classical electrochemical sensing and prevent any robustness issue due to spurious reactions. This new molecular sensing technology also reduces the transport-limited assay time by several orders of magnitude because the high field in the polarized layer can attract large nucleic acids by dielectrophoresis and the depletion region can concentrate smaller ones in a flowing stream at the membrane sensor location. We also use flow to linearize hairpin structures/collapsed globules and remove non-specifically bound molecules. An optimum shear rate is found empirically as a function

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of the target molecule size and the probe-target association constant-----and is accurately captured by an activated theory for hybridization. Since each membrane is only a micron-wide strip, extension to more than 10 targets can be easily done. We have successfully tested and quantified this platform in double blind tests for RNAs of 4 Dengue virus seroltypes, microRNA of ovarian cancer cells, cytokine and interferon-gamma signal proteins, Enterococcus, E coli, Brucella, Listeria, MRSA, Pseudomonas, Meningitis and four aquatic invasive species: quagga mussel, D bugenesis, E sinensis, L fortunei. RNA detection is particularly exciting because of microRNA as a new cancer biomarker and the fact that many bacteria have tens of thousands of mRNA but only one copy of DNA implies that PCR amplification is not necessary even with only one pathogen per sample. We also describe current extension of this sensor platform to massively parallel target detection and regenerable real-time sensors for immune cell cultures and remote applications. Yossifon, G. and Chang, H.-C., “Selection of Nonequilibrium Overlimiting Currents: Universal Depletion Layer Formation Dynamics and Vortex Instability”, Phys Rev Lett., 101, 254501(2008). Chang, H.-C. and Yossifon, G., "Understanding Electrokineitcs at the Nanoscale--a Perspective", Biomicrofluidics, 3, 012001 (2009). Basuray, S. , Senapati, S., Ajian, A. , Mahon, A. R. and Chang, H.-C., “Shear and AC Field Enhanced Carbon Nanotube Impedance Assay for Rapid, Sensitive and Mismatch-Discriminating DNA Hybridization”, ACS Nano, 3, 1823 (2009). Cheng, I.-F., S. Senapati, X. Cheng, S. Basuray, H-C Chang and H.-C. Chang, “A Rapid Field-Use Assay for Mismatch Number and Location of Hybridized DNAs”, Lab-on-a-Chip,10, 828-831 (2010). Mahon, A. R., Barnes, M. A., Senapati, S., Feder, J., Chang, H.-C. and Lodge, D. M., “Molecular Detection of Invasive Species in Heterogeneous Mixtures using a Carbon Nanotube Platform”, PLOS One, 6,17280 (2011). Senapati, S., Basuray, S., Slouka, Z., Cheng, L.-J. and Chang, H.-C., "A Nanomembrane Based Nucleic Acid Sensing Platform for Portable Diagnostics" , Topics in Current Chemistry, 304, , 153-169(2011). Chang, H.-C., Yossifon, G. and Demekhin, E. A. , “Nanoscale Electrokinetics and Microvortices: How Microhydrodynamics Affects Nanofluidic Ion Flux”, Annual Rev of Fluid Mech, 44, 401-426 (2012) L-313 Analysis of Potentially Immunogenic Carbohydrate Residues in Biologics. Andras Guttman, Zoltan Szabo, Jonathan Bones, Barry L. Karger, Northeastern University, Barnett Institute, Boston, MA, USA [KEYNOTE LECTURE] Recent rapid increase in the number of protein therapeutics on the market requires novel high resolution bioanalytical techniques to detect potentially immunogenic residues, such as galactose-α-1,3-galactose and N-glycolylneuraminic acid (NGNA). A capillary electrophoresis method was developed for the analysis of 8-aminopyrene-1,3,6-trisulfonic-acid (APTS) labeled galactose-α-1,3-galactose containing moieties in conjunction with exoglycosidase array digestion, using commercially available reagents and systems readily applicable for implementation and validation within the biotechnology industry. The excellent reproducibility of the CE method enabled precise GU value determination some key α-1,3-Gal containing structures. The limit of detection and limit of quantitation for α-1,3-Gal containing glycans were 1 and 2 μg of intact IgG, respectively. Detection of N-glycolylneuraminic (NGNA) content in immunoglobulins was accomplished after enzymatic release of the sialic acids and labeling with a non-charged fluorophore 2-aminoacridone (AMAC). Derivatization conditions under both aqueous and non-aqueous conditions were evaluated and resulted in different degree of decomposition due to the loss of the carboxylic group, requiring a high borate concentration buffer for CE based analysis under suppressed EOF conditions. The separation buffer system was optimized in respect to boric acid concentration, analysis temperature and applied electric field strength/column length. NGNA analysis was very reproducible after complete heat mediated decarboxylation with the limit of detection and limit of quantitation of 0.15 pmol/ul and 0.32 pmol/ul, respectively. L-314 Urinary Evaluation of Amino Acids and Peptides of Children Affected by Vesicoureteral Reflux. Aline Vitor1, Edgar Moraes2, Marcelo Tavares3, João Farah1, Marina Tavares1, 1University of Sao Paulo, Institute of Chemistry, Sao Paulo, BRAZIL; 2Federal University of Rio Grande do Norte, Natal, BRAZIL; 3Federal University of Minas Gerais, Belo Horizonte, BRAZIL [KEYNOTE LECTURE] Vesicoureteral reflux (VUR) is the most commonly found urinary tract abnormality associated with urinary tract infection in children. It is characterized by the returning of the urine contained in the bladder into the upper urinary tract. Its importance relies on the fact that recurrent urinary tract infections may cause renal damage in genetically predisposed children and therefore may lead to chronic kidney disease, hypertension episodes and progressive loss of renal function. As a result of the injury of the renal parenchyma, kidney functions are changed and many constituents are eliminated in the urine in large concentrations, instead of being reabsorbed by the renal system. Therefore, inspection of urinary metabolites such as primary amino acids and peptides could indicate potential biomarkers for the disease providing means for the development of a clinical noninvasive diagnosis. In this work, a pilot study was devised comprising 24 children, previously tested positive to VUR by voiding cystourethrography, and 15 healthy children. Separate aliquots of each urine sample were filtered and cleaned-up and/or preconcentrated by different solid-phase extraction procedures before analysis. One aliquot was submitted to analysis by two CE-DAD methods with direct and indirect detection for the quantitative assessment of 20 amino acids. Another aliquot was submitted to CE-MS peptidomics analysis. Raw CE-MS data files were preprocessed by in-house developed scripts where the most intense m/z peaks were

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binned in time. Data was visualized by principal component analysis and further classified by projection to latent structures-discriminant analysis and support vector machine. In both classification procedures, separation of VUR patients and healthy individual classes was achieved indicating that the data carry discriminatory information which will possibly lead to biomarker discovery. Although the existence of specific biomarkers for VUR is still uncertain and a much more thorough investigation needs to be pursued, the results strongly suggest that CE-DAD and CE-MS methods may offer a promising alternative approach to the noninvasive diagnosis of VUR in the clinical practice. Acnowledgements: CNPq, Fapesp, INCTBio L-315 A CE Method for the Separation of Paspalic Acid, Lysergic Acid and Iso-Lysergic Acid. Christian Klampfl, Markus Himmelsbach, Wolfgang Buchberger, Martin Stiftinger, Johannes Kepler University, Linz, AUSTRIA Naturally occuring alkaloids as well as their synthetic and semi-synthetic analogues are key substances in pharmaceutical industries. Pharamceuticals produced from these classes of compounds are used for a series of different medical indications comprising Parkinson´s disease, Hyperprolactinaemia or vascular disorder. D-lysergic acid and its inactive from D-iso lysergic acid which are naturally occurring alkaloids are used within a series of synthetical processes for producing ergoline alkaloids. Paspalic acid is pre-cursor in the synthesis of D-lysergic acid, which mostly is isomerized using potassium hydroxide. As this isomerization process never yields 100% lysergic acid, a subsequent careful clean-up process, as well as analytical procedures allowing to evaluate the clompleteness of this clean-up process are compulsory. In the present paper we described the development of a CE method, allowing the separation and quantification of the relevant species paspalic acid, lysergic acid and iso lysergic acid in such reaction mixtures. Best results were obtained using a carrier electrolyte based on sodium tetra borate (pH 8.3) with 40% methanol and UV detection at 282 nm and 308 nm. Subsequently, to enhance the performance of the developed method, also when more complex matrices are expected, several MS compatible carrier electrolyte for this specific separation problem were developed and subsequently tested. Using high resolution MS (with exact mass determination) and MS-MS as a detector allows the reliable extraction of the analyte signals also from rather problematic matrices, where analysis with the CE-UV method would be impossible due to interferences. Common validation parameters like linearity, reproducibility and repeatability were determined for both the CE-UV and the CE-MS method. L-316 Fractionation of Carbon-based Nanomaterials by Liquid Chromatography and Capillary Electrophoresis. Luis A. Colon, John C. Vinci, Ivonne M. Ferrer, Zuqin Xue, Allen Bourdon, University at Buffalo, Department of Chemistry, Buffalo, NY, USA The study of carbonaceous nanomaterials continues to grow, as these materials are finding a variety of applications in many areas (e.g., drug delivery, chemical sensors, and separation sciences). Our interests are in sensing applications and their use as stationary phases for chromatography. Several approaches are used to synthesize carbon-based nanoparticles; many are very simple in nature. For the production of graphene oxide, for example, oxidation of graphite and subsequent exfoliation produces graphene oxide sheets, which can be used for a given application or treated to create chemically converted graphene if desired. There is a variety of graphite starting materials and synthetic procedures available, yielding, in most cases, a crude bulk of carbonaceous nanoparticles with a wide variety of size and surface functionality. Fractionation of these nanomaterials can be crucial to evaluating the complexity of the materials as they are produced by a given synthetic approach. By means of HPLC fractionation and capillary electrophoresis (CE) we demonstrate that the heterogeneity of these nanomaterials can be simplified, rendering fractions for further studies. Various graphite starting materials and synthetic conditions were studied. HPLC allowed for scalability, producing quantities of material to be studied further and that could be effectively used in desired applications. CE allowed for further fractionation based on the charge-to-size ratio characteristic, providing information on how such a characteristic can affect other material’s properties (e.g., photoluminescence). We found that there are differences in photoluminescence properties among the fractionated material. Details of our investigations will be the focus of this presentation. L-317 Rapid Free Solution Electrophoretic Separation of Long DNA. Angela Jones, James Schneider, Max Fahrenkopf, Carnegie Mellon University, Pittsburgh, PA, USA We have developed a rapid, free solution electrophoretic DNA separation technique for long DNA (greater than 1 kb) that is capable of separating up to 6 kb with 1 kb resolution, with the potential to theoretically separate up to 48 kb with hundred-base resolution in a matter of minutes. In our system, nonionic micelles are transiently attached to the end of the DNA molecules and lower the DNA’s electrophoretic mobility in a length-dependent manner. By tuning the size of the micelle, we can separate DNA in a specific size range. Because the separation takes place in free solution rather than in a gel, the separation time is also extremely low. Traditionally, sieving matrices in the form of gels have been used in capillary electrophoresis to perform length-based separation of DNA molecules. However, long DNA molecules cannot be separated in these traditional gels because the DNA undergoes biased reptation, aligning in the direction of the electric field and migrating with a length-independent mobility. In the 1980s, pulsed-field gel electrophoresis was developed which increased the length of DNA that could be separated to the millions of base pairs, but has the disadvantage of

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requiring several days for separation. Techniques that have been developed in the past decade to separate long DNA include top-down approaches such as nanofabricated pillars, and bottom-up approaches such as self-assembled magnetic particles. The major drawbacks to these methods include the time consuming and expensive nature of nanofabrication processes, and the low resolution capabilities of the matrices. Instead of single base resolution achieved with traditional gels, these artificial matrices can typically only resolve DNA molecules that differ in length by thousands of bases. There are many applications such as genome mapping and DNA fingerprinting which involve separating DNA thousands of bases long, and improving the resolution of these separations is critical. L-318 Enantioselective Retention on Cellular Membrane Affinity Chromatography Columns: Using Chirality to Probe Small Molecule-protein Interactions. Ruin Moaddel, Prateek Bhatia, Chester Frazier, Sarangan Ravichandran, Sylvestre Dossou, Irving Wainer, National Institutes of Health, Baltimore, MD, USA [KEYNOTE LECTURE] Cellular membrane fragments have been immobilized on silica supports and on the surface of activated open tubular glass capillaries to create cellular membrane affinity chromatography (CMAC) columns [1]. CMAC columns have been prepared from cell lines expressing target receptors or transporters or cell line that were stably transfected to express the target protein, CMAC(+) columns. CMAC columns were also constructed using membranes from cell lines in which the expression of the target protein had been reduced using siRNA or from the wild type of the cell line used in the transfections, CMAC(-) columns. Frontal affinity chromatography (FAC) studies using small molecule ligands were carried out on the CMAC(+) and CMAC(-) columns and the relative difference between the two columns used to characterize the binding of the ligands to the immobilized target protein. When chiral ligands were employed, enantioselective differences in retention were used to differentiate between receptor subtypes and to characterize the effects of single nucleotide polymorphisms (SNPs). This approach will be illustrated using a series of CMAC columns developed using cell lines expressing ATP-drug transporters, Pgp, MRP1, MRP2, BCRP and the human organic cation transporter (hOCT1). In the latter studies, the effects of two SNPs on the binding of chiral substrates were used to create a pharmacophore model of the hOCT1. In addition, this presentation will discuss the use of CMAC columns containing beta2-adrenergic receptors to study the interactions of the stereoisomers of fenoterol and fenoterol analogs, beta2-adrenergic receptor agonists being developed for use in the treatment of congestive heart failure and glioblastoma brain cancers. References [1] R. Moaddel, I.W. Wainer, Nature Protocols, 4, 197-205 (2009). [2] Bhatia P, Moaddel R, Wainer IW. Talanta, 81, 1477-1481 (2010) L-319 Enantioseparation of Cathinone Derivatives used as Recreational Drugs by Capillary Electrophoresis. Martin G. Schmid, Karl-Franzens-University, Department of Pharm. Chemistry, Graz, AUSTRIA In the past 3 years cathinone derivatives have entered the global drug market being misused as “recreational drugs”. They cause serious social problems in many countries worldwide. Modification of the basic structure of cathinone leads to a multitude of derivatives and all these “research chemicals” are chiral. As many chiral active pharmaceutical ingredients also the pharmacological effect of the enantiomers of those psychoactive compounds is supposed to differ. In this study, a capillary zone electrophoresis method was developed for chiral separation of at least 22 cathinone derivatives. First, the enantioseparation ability of different types of cyclodextrin (CD) was compared. Then, the effect of CD concentration, degree of substitution, temperature and addition of organic modifiers to the background electrolyte on separation was studied. Furthermore, other chiral selectors such as chiral crown ethers and chiral metal complexes were checked for enantioseparation. Using optimal conditions most of the compounds were baseline resolved. It turned out that all samples purchased via the internet consisted of racemic mixtures. L-320 Rapid CE Separa tions with Ultra Fas t Time-of Fligh t Mas s Spec trometry Detec tion and the ir Ap plica tions in Metabo lomics . Roza Wojcik1, Matthew Giardina1, Norman Dovichi2, 1LECO Corp, St Joseph, MI, USA; 2University of Notre Dame, Notre Dame, IN, USA Since the early 1990s time-of-flight mass spectrometry has been recognized as a detection method of choice for capillary electrophoresis, with its fast acquisition rates providing a good match for narrow CE peaks. However, fast CE-MS separations have been rarely applied in practice. The small injection volumes and high electric fields employed in fast and efficient CE separations pose challenges to CE-MS interface designs, require fast and sensitive MS instrumentation and are difficult to perform with bulky commercial CE instruments. In this work we evaluate rapid CE separations of metabolites with the LECO Citius™ high resolution time-of flight mass spectrometer, capable of acquisition rates of up to 200 spectra per second and < 1ppm mass accuracy. Our experiments employ a nanospray CE-MS interface design based on an electrokinetically pumped sheath flow. CE separations with electric fields up to 1.3kV/cm were achieved owing to a compact CE instrument built in-house. Our proof of principle experiments employ amino acid standards and a urine sample, as an example of a complex biological sample matrix. We demonstrate that CE separations of urine metabolites at electric fields of 1kV/cm can generate peak widths as narrow as 100 milliseconds at full width half height, requiring 200 spectra

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per second acquisition speed for adequate peak sampling. Such separations, which last less than two minutes, allow for detection of hundreds of molecular features, falling within 2ppm tolerance match in metabolomic databases. The 2% RSD migration time repeatabi lity achieved with minimal sample pretreatment and capillary conditioning indicates usefulness of fast CE separations for high throughput analysis. Combining fast acquisition rates with high electric field CE separations can be beneficial for targeted metabolomic applications. Separation of isomers, such as Leucine and Isoleucine typically require employment of high ionic strength separation buffers with organic solvent additives. We demonstrate Leucine and Isoleucine separation at 1kV/cm with 200mM formic acid as a background electrolyte. The Citius™ comprehensive CID capability at fast data acquisition rates allows for additional validation of isomer identity based on diagnostic fragment ions. L-321 Inves tiga tion o f Bio -SPME Technology fo r the Enrichment o f Illic it Pheneth ylamine and Cath inone Compounds from Bio log ica l Samples . David Bell1, Craig Aurand1, Robert Shirey1, Janusz Pawliszyn2, Leonard Sidisky2, 1Sigma-Aldrich/Supelco, Bellefonte, PA, USA; 2University of Waterloo, Waterloo, Ontario, CANADA Introduction: Recent advances in SPME devices, termed Bio-SPME, are physically stable toward polar media. Bio-SPME devices, when coupled with LC/MS, provide analysts with an interesting alternative set of tools for bioanalyses. Some potential advantages of Bio-SPME over traditional sample preparation may include improved selectivity toward target analytes, micro sampling, in-vivo sampling, and high throughput analyses through automation. Methods: Bio-SPME fibers prepared in a 96 tip array were utilized for the enrichment of nine illicit “Bath Salts” from urine and plasma samples. Bio-SPME fibers functionalized with hydrophobic and cation exchange particles were employed. Following enrichment, the bio-SPME fibers were directly desorbed in a 96 well plate for LC/MS TOF analysis. Key parameters that impact analyte enrichment efficiency, such as sample pH, and extraction time were explored along with the impact of biological sample matrix. In addition to extracted analyte, overall sample matrix interferences were monitored and compared with typical dilution/precipitation techniques. Results: Extraction studies demonstrated the capability of analyzing sub 10 ng/mL concentration levels in both plasma and urine samples. Unlike traditional SPE approaches, the unique utility of Bio-SPME enables the direct enrichment of targeted small molecules without the need for sample dilution or precipitation. Plasma samples prepared using the Bio-SPME technique exhibited a 5X reduction in detected endogenous matrix as compared to standard precipitation techniques, while demonstrating increased analyte response. The capability to configure the fiber tips in a 96 tip array allows for handling high numbers of samples comparable to that of 96 well plate formats. L-322 Plasmon-Controlled Fluorescence: Applications to Biophysics and Biotechnology. Joseph Lakowicz, University of Maryland, School of Medicine, Department of Biochemistry and Molecular Biology, Baltimore, MD, USA [KEYNOTE LECTURE] Fluorescence spectroscopy is widely used in chemical and biological research. Until recently essentially all fluorescence experiments were performed in the far-field regime. By far-field we mean at least several wavelengths from the fluorophore. In recent years there has been a growing interest in the interactions of fluorophores with metallic surfaces or particles. Near-field interactions are those occurring within a wavelength distance of an excited fluorophore. The spectral properties of fluorophores can dramatically be altered by near-field interactions with the electron clouds present in metals. These interactions modify the emission in ways not seen in classical fluorescence experiments. Fluorophores in the excited state can create plasmons that radiate into the far-field and that fluorophores in the ground state can interact with and be excited by surface plasmons. These reciprocal interactions suggest that the novel optical absorption and scattering properties of metallic nanostructures can be used to control the decay rates, location, and direction of fluorophore emission. We refer to these phenomena as plasmon-controlled fluorescence (PCF). We will present a review of the recent work on metal-fluorophore interactions and suggest how these effects could result in new classes of experimental procedures, novel probes, bioassays and devices. While there are many complexities to fluorophore-metal interactions there are also many opportunities. In addition to control of the chemical structure of the fluorophore we can now control, to some extent, the processes of excitation and emission with nearby metallic nanostructures resulting in new approaches to high-throughput biology, diagnostics and molecular imaging. L-323 Neurochemical Patterns in CNS Disorders – A Molecular Diagnostic Tool. Jonas Bergquist, Uppsala University, Uppsala, SWEDEN Most diseases manifest themselves by more or less severe changes in human physiology. This forms the basis for clinical chemistry and its value in helping to diagnose disease correctly and in following therapeutic interventions. Presently, many biochemical and cellular parameters are routinely measured in blood, plasma, serum or urine in any major hospital and the results of these measurements support decision making by clinicians. However, the number of biomarkers is still insufficient for diagnosis of e.g. neurodegenerative diseases as well as for many other common diseases and some of the samples (like cerebrospinal fluid, microdialysis of the central nervous system and minute tissue samples) have yet not been explored in great detail. Modern analytical tools including high resolution liquid based separation in combination with high

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resolution mass spectrometry opens up earlier unexplored depths of the molecular complexity in these neurochemical environments. L-324 Monitoring Oxidative Stress using Capillary Electrophoresis with Electrochemical and Fluorescence Detection. Craig Lunte, University of Kansas, Department of Chemistry, R.N. Adams Institute for Bioanalytical Chemistry, Lawrence, KS, USA Capillary electrophoresis (CE) is a powerful separation technique with excellent mass detection limits owing to the small volume of sample typically injected. Unfortunately with common detectors, such as UV absorbance, the limited sample volume and detector pathlength result in compromised concentration detection limits. This has fuelled the implementation of highly sensitive detection schemes such as laser induced fluorescence (LIF) and electrochemical detection (EC). In addition, several on-capillary concentrating technique have been developed to improve concentration detection limts. In this presentation, our work at implementation of CE in combination with all of these approaches as applied to studying oxidative stress will be described. Several target analytes that serve as biomarkers of oxidative stress have been identified and analytical methods for these targets developed. Glutathione is a key marker of the oxidative state of a tissue. It is necessary to measure both the oxidized (disulfide) and reduced (thiol) forms of glutathione. A method that employs on-capillary stacking to achieve acceptable concentration detection limits and a dual-electrode CEEC (capillary electrophoresis with electrochemical detection) method will be described. This method has been applied to monitoring lipid peroxidation occurring as a function of ischemia-reperfusion. 8-Oxo-guanine (8-oxoG) and 8-hydroxy-deoxyguanosine (8-OHdG) are biomarkers for DNA oxidation. These biomarkers can also be detected using CEEC with on-capillary concentration. Another important biomarker of oxidative stress is malondialdhyde (MDA), which is a product of lipid peroxidation. MDA must be derivatized to a fluorescent product prior to detection using CE-LIF. Finally, a CE-LIF method for detecting arginine, citrulline, and ornithine will be described. L-325 Mass Spectrometry Analysis of Extra-virgin Olive Oil. Alfonsina D'Amato1, Clara Esteve2, M. L. Marina2, M. C. Garcia2, Pier Giorgio Righetti1, 1Department of Chemistry, Materials and Chemical Engineering “Giulio Natta”, Politecnico di Milano, Milano, ITALY; 2Department of Analytical Chemistry, Faculty of Chemistry, University of Alcala, Ctra. Madrid-Barcelona, Alcala de Henares, Madrid, SPAIN The characterization of food components and functional molecules, authentication and traceability of foodstuffs, quality control, identification and quantitation of additives, allergens, chemical and microbiological contaminants, have widespread consequences on economy, agriculture, industry, health. Olive oil consumption has been associated with the decrease of the incidence of important illnesses such as cardiovascular diseases and cancer. Despite their highly informative value and role in food stability and allergenicity, olive oil proteins have been scarcely investigated [1]. This fact is probably due to the difficulty of working with a lipid matrix and the dramatically low abundance of the proteins potentially present. The aim of this study was the investigation of extra-virgin olive oil in order to see if enough proteins could be detected so as to classify the various oils according to different cultivars and if high-quality oils could be distinguished from industrial oils available in supermarket. As a pre-requisite to that, we first started investigating the proteome content of the olive fruit, on which not much was known up to the present. To that aim, both the proteinaceous content of the seed as well as of the pulp were analyzed, since both compartments could contribute to the presence, if any, of trace proteins in the final olive oil processed. Indeed our work resulted in a very extensive exploration of both proteomes, amounting to the identification of a grand total of 61 proteins in the seed and as many as 231 species in the pulp [2]. Having established these two proteomes, we started investigating the trace proteins in commercial olive oils. We had to test different extraction protocols in order to obtain reasonable results. The SDS PAGE was performed by using the extracted protein mixture, the gel was segmented in several slices, digested with trypsin and the resulting peptides analyzed by high-resolution mass spectrometry (LTQ XL Orbitrap). We identified a few proteins coming from the viridiplantae protein database. This is not surprising considering that only traces of proteins can be found in olive oils. In conclusion this is the first time an in depth study of the proteome is performed in the olive seed and pulp, as a first step in the study of the olive oil proteome. References [1] Hidalgo FJ, Alaiz M, Zamora R, Anal Chem. (2001) 73: 698-702. [2] Esteve C, D’Amato A, Marina ML, García MC, Citterio A, Righetti PG. J Proteomics (2012), in press. L-326 Genomic Claustrophobia via Nanoconfinement Systems. David Schwartz, University of Wisconsin-Madison, Laboratory for Molecular and Computational Genomics, Department of Chemistry, Laboratory of Genetics, Madison, WI, USA [KEYNOTE LECTURE] My laboratory’s research efforts embrace physical, biological and computational domains that unify within the context of creating working single molecule systems for addressing hard problems in the genomic sciences. We solve problems through the invention and application of multiple novel genome analysis systems (novel, highly integrated tools encompassing series of novel interlocking components) centered on exploiting the unique attributes of single molecules, or complexes. The Optical Mapping System, the first genomics platform to utilize single molecule analytes, exploits the detection range afforded by restriction fragment length polymorphism

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analysis, but with high throughput and single-DNA molecule precision engendered by automated fluorescence microscopy. Optical mapping enables the construction of genome-wide physical maps from ensembles of ordered, single-DNA molecule restriction maps developed from genomic sources, obviating clone libraries, PCR, and hybridization. Comparisons of optical consensus maps against a reference map reveals structural alterations as “differences,” in the form of novel restriction sites (missing or extra cuts; MCs or ECs), or indels (insertions or deletions), which are statistically assessed, in part, based on the number of single-DNA molecule optical maps collectively represented by the consensus map. Since high-resolution restriction maps intrinsically reveal genome structure, elusive differences such as indels are discoverable and physically characterized. In this talk, I will describe the workings of: the Optical Mapping System, the Nanocoding System, which leverages molecular confinement effects, and single molecule sequencing approaches; presented within the context of numerous applications covering normal and cancer genomes. L-327 Nanopore Graphene-based Electronic Devices. Marija Drndic, University of Pennsylvania, David Rittenhouse Laboratory, Philadelphia, PA, USA Graphene is an exceptional material for high-speed electronics, as well as a revolutionary membrane material due to its strength and atomic thickness. Nanopores in suspended graphene membranes are currently regarded as candidates for ultrafast DNA sequencing. When a single DNA molecule passes through a nanopore, it blocks the field-driven ions passing through the pore and is detected by measuring the ion current reduction. Due to the thin nature of graphene membranes and reduced pore resistance, we observe larger current signals than in the case of traditional solid-state nanopores. Use of graphene as a membrane material opens the door to a new class of nanopore devices in which electronic sensing and control are performed directly at the pore. L-328 Southern Nanopore Sequencing: Building a "Nanoporase" for Diffusion Reduction and Kinetic Proofreading. Xinsheng Ling, Brown University, Physics Department, Providence, RI, USA I will discuss our recent work on the concept of DNA sequencing using solid-state nanopores to detect hybridizing oligos. Our experimental results revealed that thermal smearing effects are significant in both oligo position and de-hybridizaton, posing serious questions concerning the feasibility of DNA sequencing using short hybridization probes. Learning from how Nature accomplishes the task of overcoming these errors in the functions of polymerases and ribosomes, we propose an active kinetic proofreading mechanism using solid-state nanopores [1], we call it a "nanoporase". At least theoretically, one can use a nanoporase to achieve an error rate as low as 1 in 100,000,000,000 (in comparison, in DNA replication in eukaryotic cells, typical error rate for DNA polymerase is about 1 in 1,000,000,000). [1] X. S. Ling, USPTO Patent Application (2012). L-329 Positional Sequencing: Single-molecule, Electronic, Solid-State Sequencing of Long DNA Molecules. John Thompson, Nabsys Inc., Biochemistry, Providence, RI, USA Nabsys’ positional sequencing technology is a single-molecule approach that utilizes solid-state nanodetectors. The platform locates, with sub-diffraction-limit resolution, the positions of tags that have bound to very long DNA fragments. The platform does not require amplification of samples. As a result, the method is capable of providing very long reads (in excess of 100 kb) with unbiased, quantitative information. Positional sequencing does not attempt to discriminate individual nucleotide bases passing through an electrical detector. Instead, the Nabsys approach tags specific regions of very long DNA templates that are then translocated through solid-state nanodetectors. Distances between tags are determined electronically. Long-range information is preserved in these single-molecule reads so structural rearrangements and duplications are easily identified and correctly assembled. Results for deletions of various sizes will be presented. L-330 Isolation of Cancer Stem Cells (CSCs) Based on their Dielectrophoretic Signature. Alireza Salmanzadeh1, Lina Romero2, Scott Cramer2, Rafael Davalos1, 1Virginia Tech - Wake Forest University, Biomedical Engineering, Blacksburg, VA, USA; 2University of Colorado, Denver, CO, USA [KEYNOTE LECTURE] Cancer stem cells (CSCs) are tumor cells which have the ability to generate a new tumor that exhibits a similar histopathology as the tumor from which it was dervied. Isolating these cells is the first step towards understanding their role in the pathogenesis and progression of cancer and is critical for the development of improved cancer specific therapies. Unfortunately, existing approaches, such as flow cytometry, are limited. We invented a new technique known as contactless dielectrophoresis (cDEP) to isolate rare cells of interest from background. Advantages of cDEP over other cell sorting methods include minimal sample preparation, rapid isolation, and maintaining cell viability. Furthermore, cDEP capitalizes on the sensitivity of traditional DEP while eliminating its significant challenges such as bubble formation, electrode delamination, expensive fabrication, and electrode-sample contamination. We characterized the dielectrophoretic response of prostate cancer stem cells from a prostate cancer cell line (PC3) utilizing cDEP. We have shown that the required voltage to completely trap CSCs differs from that of non-CSCs. This data was then used to find the optimal parameters sort and culture enriched CSC populations. We observed that the CSC-identified cells, sorted using cDEP produced significantly more spheroids than control. Additionally, the average size of the cDEP enriched CSC

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spheroids was about 4 times larger than control, and about 17 times larger than cDEP enriched non-CSCs spheroids. L-331 Linear Sweep Dielectrophoresis for Cell Surface Characterization. Cullen Buie, Massachusetts Institute of Technology, Mechanical Engineering, Cambridge, MA, USA At low frequency and DC fields it is known that cell polarizability is dominated by surface properties. We have recently begun exploring the use of DC dielectrophoresis with high electric field gradients in order to characterize bacterial cell envelopes. We have shown that cell surface polarizability correlates with several bacterial phenotypes of interest including electrochemical activity, biofilm formation, and antibiotic resistance. We measure cell polarizability using a process known as linear sweep dielectrophoresis wherein the applied electric field (and therefore the dielectrophoretic force) is slowly increased. For a given cell envelope polarizability there exists a unique electric field where the dielectrophoretic force exceeds the linear electrokinetic effects. In this talk we will discuss our most recent experiments and provide theoretical analysis of this phenomenon. Results of this study will enable engineers and microbiologists to obtain subtle phenotypic information on bacterial populations. L-332 Dielectrophoretic Tweezers as a Platform for Molecular Force Spectroscopy in a Highly Parallel Format. Dmitri Vezenov, Peng Cheng, Michael Barrett, Piercen Oliver, Lehigh University, Department of Chemistry, Bethlehem, PA, USA In the push towards personalized medicine, low cost DNA sequencing is essential. To lower the final cost of sequencing, we have proposed a sequencing strategy that uses molecular force spectroscopy (MFS) to detect the conformational changes of DNA in the course of a stepwise ligation. This approach to sequencing requires a controlled ligation of short DNA strands of known composition to the DNA strand in question followed by mechanical stretching of individual molecules to determine the success or failure of the ligation (as in sequencing by synthesis). In typical MFS experiments, hundreds of measurements must be collected on the same system in order to provide sufficient data for averaging and statistics or to uncover multiple unique mechanical pathways or states of the system. This experimental process is often time-consuming and cannot be readily extended to a parallel format using conventional methods (e.g. optical tweezers and atomic force microscopy). Our aim is to build a force-spectroscopy-on-a-chip device that can detect and manipulate many (millions) single molecules in parallel. Our approach to massively parallel MFS technique is based on dielectrophoresis (DEP), in which the force is applied evenly to all molecules in the system via tethered dielectric probes. We implemented dielectrophoretic tweezers using parallel flat electrodes to manipulate an array of polymer force probes. Instead of a common approach where one fabricates complex micro-electrode arrays to generate the field gradients necessary to manipulate particles with DEP, we used a single set of macroscopic electrodes to generate high electric field gradients in the vicinity of the sample surface that has simple microfabricated dielectric structures (microwells) on one of the electrodes. We evaluated the performance characteristics of several possible designs for DEP tweezers using numerical analysis and then used this chip-based force spectroscopy technique to stretch single stranded DNA. The combination of a simple device fabrication, uniform distribution of high axial forces, and simultaneous detection of probe positions makes DEP tweezers ideal for surface analysis of single biomolecules. L-333 Elec trode-les s Die lec trophores is fo r Biomolecu lar and Nanos truc ture Sep a ra tions . Yi-Hsuan Su, Walter Varhue, Nathan Swami, Electrical Engineering, University of Virginia, Charlottesville, VA, USA Diagnosis of diseases and detection of their pathogenesis requires the characterization of the concentration levels of a spectrum of closely related biomarkers, in conjunction to characterizing their interaction with small biological molecules. However, these closely related biomarkers are present in extremely small quantities (~ng-pg/mL) and need to be detected in complex biological fluids, such as blood serum, which contain several other proteins at million to billion-fold higher concentration levels (mg/mL). To address this need for separation and selective enrichment of closely-related biomarkers over other proteins in the bio-fluid media, we explore the application of frequency-selective electrokinetic methods, such as dielectrophoresis, on a microfluidic device platform. Dielectrophoresis (DEP) enables highly selective translation of polarized bio-particles based on the characteristic frequency response of the dielectric permittivity of the bio-particle versus that of the medium, and it has been extensively applied towards sorting of somewhat similar sized biological cells with differing dielectric frequency response. However, its application to low polarizability bio-particles, such as microbial cells (~1-3 µm) and nanostructures (ss-DNA, proteins, and nanoparticles) requires micro- or nano-device geometries to enhance the local field to offset the steep fall in dielectrophoretic trapping forces with particle size [1], [2], [3]. Device designs based on electrode-less DEP cause bio-particles to be trapped in the vicinity of insulating constriction structures rather than at metal electrode-deges, thereby avoiding damage to the trapped bio-particle and disruptions due to electrolysis at ~kHz frequencies. Herein, we demonstrate two examples of separations that can be accomplished only within microfluidic devices using electrode-less DEP. The first utilizes interfacial polarization of the bio-particle to separate viable versus non-viable oocysts of Cryptosporidium parvum, a highly infectious water-borne parasite, after various levels of treatment. The second utilizes double layer induced

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screening of the dielectrophoretic field to separate closely-sized nanostructures with differing charge distributions. We envision the application of these separation methodologies towards biomarker discovery, arresting infectious diseases, and rare target sensing for early disease diagnostics. References: [1] Swami, N.; Chou, C.-F.; Ramamurthy, V.; Chaurey, V. Enhancing DNA hybridization kinetics through constriction-based. Lab on a Chip 2009, 9, 3212–3220. [2] Chaurey, V.; Polanco, C. F.; Chou, C.-F.; Swami, N. S. Floating electrode enhanced constriction dielectrophoresis for biomolecular trapping in physiological media of high conductivity. Biomicrofluidics 2012, 6, 012806:1-10. DOI: 10.1063/1.3676069. [3] K.T. Liao, M. Tsegaye, V. Chaurey, C.F. Chou, N. Swami. “Nano-constriction device for rapid protein pre-concentration in physiological media by electrokinetic force balance”, Electrophoresis (2012), 33, 1958-1966. L-334 Application of Biomimetic Molecularly Imprinted Polymer Thin Films in the Electro- and Liquid Phase-Separation of Biomolecules. Reinhard Boysen, Shuyang Li, Jamil Chowdhury, Lachlan Schwarz, Milton Hearn, Monash University, Centre for Green Chemistry, Clayton, AUSTRALIA [KEYNOTE LECTURE] Molecularly imprinted polymers (MIPs) are potentially useful as recognition elements in chemical and biological sensors. MIPs are porous polymeric materials that possess built-in molecular recognition capabilities. These attributes are achieved by exploiting molecular self-assembly processes mediated by the interaction between a template molecule and chemical monomers. Subsequent polymerisation of the monomers with a suitable cross-linkage reagent generates functionalised cavities that are chemically and spatially defined to accommodate the template molecule. The derived polymeric materials thus contain nano-cavities of pre-determined shape and complimentary binding site features towards the template molecule. It is well documented that MIPs can be made prepared in reusable monolith, membrane, and bead formats. However, much less effort has been expended on the generation of thin film MIPs. Our previous investigations have led to new ways to achieve the cost-effective optimisation of the polymerisation chemistry with thin film systems, guided by advanced molecular modelling and spectroscopic methods to determine the choice of the monomer(s) that best interact(s) with the chosen imprint molecule, based inter alia on spin-coating procedures to allow efficient control over film thickness. Although chemically robust MIP thin films can be produced in this manner, an effective method to prepare double layered and ‘patterned’ MIP thin films, in terms of well defined physical architecture, morphology and regularity, for as use biomimetic nano-arrays in biomolecule analysis and discovery has hitherto represented a significant constraint. Here, we describe the development of procedures enabling the generation of ‘patterned’ MIP thin films compatible with nano-printing processes, soft (stamp) lithography, etching using an electron beam or photolithographic techniques. In addition, methods to optimise target selectivity through the use nano-droplet deposition procedures have been established, resulting in double layered nano-structured functional polymer thin film surfaces, whereby each surfaces contains multiple regions of differing and pre-determined selectivity for the molecular target(s). Examples of the application of these new types of biomimetic molecularly imprinted polymer thin films in the electro- and liquid phase-separation and analysis of low and mid molecular weight biomolecules will be described. L-335 CE-UV Characterization of New Polydopamine- and Polypyrrole-coated Magnetic Nanoparticles for Selective Extraction of Aromatic Contaminants and Pharmaceuticals in Water Treatment. Edward P.C. Lai, Zafar Iqbal, Musharraf Miah, Banu Ormeci, Carleton University, Ottawa, Ontario, CANADA Molecularly imprinted or non-imprinted polymer-coated magnetic nanoparticles are attracting interest in environmental and industrial applications for the extraction or clean-up of different classes of contaminants from various complex matrices. Magnetism can enhance the efficiency and rate of removing these particles from water. New polydopamine- and polypyrrole-coated particles have recently been synthesized in our lab to demonstrate selectivity towards aromatic compounds including pharmaceuticals. They were tested at the analytical scale for both in-vitro and in-situ bindings by capillary electrophoresis (CE) under a wide range of water characteristics. The application of this technology platform will be demonstrated in the presence of both organic and inorganic contaminants. Moreover, the feasibility of a novel magnetic separator design will be highlighted for water treatment at the sub-pilot scale. A comparison to removal efficiencies obtained with centrifugation, coagulation, membrane filtration and sedimentation will be elaborated. The regeneration and reuse potential of these recovered polymer-coated magnetic particles can be rapidly assessed by CE with UV or fluorescence detection. Based on the data and results obtained so far, these particles promise great effectiveness for application in water treatment from the home scale to full industrial scale. L-336 Use of Capillary Electrophoresis to Assess the Purity and Potency of Genuine and Knockoff Dietary Supplements Containing Anatabine. Robert Weinberger1, Curtis Wright IV2, 1CE Technologies Inc., Chappaqua, NY, USA; 2Rock Creek Pharmaceuticals, Gloucester, MA, USA The dietary supplement industry consists of some 55,000 products including vitamins, minerals and broadly defined and poorly regulated products with a variety of uses including weight loss, energy boost and sexual enhancement. There are over 100,000,000 users in the United States that must rely on the integrity of the supplier to ensure the quality and safety of the product. Until recently, the only products recalled from the marketplace were those containing ephedra. Anatabine is a natural product found in common plants such as

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tomatoes, eggplants and peppers. The compound has been shown to have anti-inflammatory properties. Since anatabine is a normal component of the diet, non-toxic in animal testing, and since Rock Creek Pharmaceuticals does not make any claims other than anti-inflammatory support, it fits into the “supplement” category in the US regulatory system. Counterfeiting of pharmaceuticals is a major problem especially in underdeveloped nations. Because dietary supplements are inexpensive, counterfeiting is seldom seen but imitation is more common. Recently, a product appeared on the internet that claimed among it’s ingredients to contain anatabine. This talk will briefly review the dietary supplement industry, the differences between drugs and supplements, some of the properties of anatabine and show how capillary electrophoresis was used to determine the anatabine content of genuine and knock-off products. L-337 Implementation of Carbon-based Nanomaterials in Capillary Electrochromatography as an Immunosupport. German Alejandro Messina, Lorena Lujan Sombra, Walter Pedro Stege, Julio Raba, Roberto Antonio Olsina, Patricia Wanda Stege, National University of San Luis, INQUISAL, Department of Chemistry, CONICET, San Luis, ARGENTINA (withdrawn) L-338 Design and Applications of Sequential Injection – Capillary Electrophoresis Systems. Adam Gaudry1, Daniel Gstoettenmayr1, Philip Zakaria1, Sui Ching Phung1, Rosanne Guijt1, Cari Sanger – van de Griend2, Joselito Quirino1, Mirek Macka1, Greg Dicinoski1, Paul Haddad1, Michael Breadmore1, 1University of Tasmania, Hobart, AUSTRALIA; 23 Kantisto Separation Sciences, Baarn, THE NETHERLANDS [KEYNOTE LECTURE] Sequential injection – capillary electrophoresis (SI-CE) has a number of attractive features including the ability to integrate down-stream sample processing, such as extraction, as well as continuous sample and electrolyte introduction to improve sensitivity and robustness. A number of SI-CE systems have been developed to exploit the advantages of this approach. A new system for the simultaneous separation of both anions and cations from a single injection point allows the rapid separation of 23 inorganic anions and cations within 3 min. This system is also ideal for application in the pharmaceutical industry for counter-ion determination. A novel microfluidic approach for isolation of anion and cation separation chemistry has also been developed. Finally, our recent results on the implementation of SI CE in a commercial CE instrument will be discussed. L-339 On-column Preconcentration using Alkylthiol Self-assembly on Gold-deposited Monodisperse Colloidal Silica Packing for Electrochromatographic Analysis with a Fully Packed Microchip. Jongman Park, Shinseon Kim, Department of Chemistry, Microanalytical System Laboratory, Konkuk University, Seoul, KOREA A fully packed microchip with on-column extraction bed for sample preconcentration was prepared. A linear microchannel (80um, 50um and 12mm in width, depth and length) packed with monodisperse colloidal silica particles (800nm) was formed on a COC microchip with two reservoirs on both ends of the microchannel. Gold-deposited silica packing frit structures were formed at the entrances of the microchannel by electroless plating treatment. The frit structure was used for the extraction bed formation by self-assembly adsorption of dodecanethiol on it. The dodecanethiol layer on the gold coating was utilized for the solid phase extraction of organic analytes. Rhodamine B, Sulforhodamine B, and fluorescein were used as probe molecules for the characterization of the microchip. Aqueous borate buffer solutions containing the analytes in nM concentration level were flown through the extraction bed electroosmotically for a given time, and then the analytes were eluted with acetontrile/borate buffer solutions. Approximately thousand times of preconcentration of the analytes followed by electrochromatographic separation was possible. Detailed analytical characteristics will be discussed in terms of the extraction time, concentration, the composition of the solvents. L-340 Single Capillary Multiplexed Electrophoresis for High Sensitivity Analysis of Cell Signaling Components. Craig Aspinwall1, Elyssia Gallagher1, James Hackett1, Troy Comi1, Kevin Braun2, 1University of Arizona, Department of Chemistry and Biochemistry, Tucson, AZ, USA; 2Beloit College, Beloit, WI, USA Cellular signaling involves a diverse array of biological analytes, which are present at low concentrations can change rapidly. The majority of analytes involved in signaling pathways lack suitable optical or electrochemical transducer moieties further complicating detection. We have integrated capillary zone electrophoresis with an online-injection interface controlled by a Hadamard Transformation (HT) sequence to yield a high-speed, single capillary digital multiplexing approach. The resultant HT-CZE approach is readily coupled to a wide range of detectors, including laser-induced fluorescence, contactless conductivity and UV-absorption detection. High speed HT-CZE yields sensitivity enhancements comparable to signal averaging yet increasing the analysis time by as little as two fold to detect a diverse catalog of biological analytes, including neurotransmitters, biological phosphates and etc. Here, we will outline the key instrumental and analysis advances that facilitate HT-CZE and present novel applications that are enabled using this approach.

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L-341 Application of Battery-operated Continuous Flow Electro Membrane Extraction on Biological Anions with Ion Chromatography. Basheer Chanbasha1, Tsze Yin Tan2, Nguyen Thi Le Hang2, Hian Kee Lee2, 1King Fahd University of Petroleum and Minerals, Department of Chemistry, Dhahran, SAUDI ARABIA; 2National University of Singapore, Singapore, SINGAPORE A simple and sensitive single step continuous flow electro membrane extraction (EME) procedure was demonstrated for the first time for biological organic anions with determination by ion chromatography (IC). Nitrite, adipate, oxalate, iodide, fumarate, thiocyanate and perchlorate were extracted from aqueous donor solutions, across a supported liquid membrane (SLM) consisting of methanol impregnated in the walls of a porous polypropylene membrane envelope and into an alkaline aqueous acceptor solution in the lumen of the propylene envelope by the application of potential of 12V applied across the SLM. The acceptor solution was analyzed by IC. Parameters affecting the extraction performance such as type of SLM, extraction time, pH of the donor and acceptor, flow rate of sample solution and extraction voltage were studied. The most favourable EME conditions were methanol as the SLM, extraction time of 5 min, pH of acceptor and sample solutions of 12 and 4, respectively, sample flow rate of 10ml/min and a voltage of 12V. Portable 12V batteries were used in the study. Under these optimized conditions, all anions had enrichment factors ranging from 2.5 to 10.6 with relative standard deviations (RSD) (n=3) of between 5.5 and 14.9%. Good linearity ranging from 0.1 to 10 µg/mL with correlation coefficient (r) of between 0.9981 and 0.9996 were obtained. The limits of detection of the battery-operated EME-IC method were from 0.01 – 0.14 µg/mL. The developed methodology was applied to urine samples to evaluate the feasibility of the method for real applications. L-342 The Silk Road, Marco Polo, a Bible and Its Proteome: A Detective Story. Pier Giorgio Righetti1, Lucia Toniolo1, Alfonsina D'Amato1, Riccardo Saccenti2, Davide Gullotta1, 1Politecnico di Milano, Proteomics, Milano, ITALY; 2Fondazione per le Scienze Religiose, Bologna, ITALY [PLENARY LECTURE] Around the end of XIII century (at the time of young Marco Polo’s first trip to China at the court of Khubilai Khan in Khan Baliq) a pocket Bible was delivered by a Franciscan friar to the Mogul Emperor, in the framework of the evangelization program of the Far East. It was a long journey, some three and half years and 6000 miles on the Silk Road, whose paths were not “silky” at all but extremely rough. Four centuries later, in 1685, this Bible was rediscovered by the Jesuit Philippe Couplet in the house of a rich Chinese in Nanchin (modern Nan-ching or Nánjīng) and donated to Cosimo III, Grand Duke of Tuscany. Cosimo, being not such a religious fellow (and probably unable to read Latin), donated it immediately to the Biblioteca Laurenziana, a renaissance building designed by Michelangelo in the cloisters of the most famous church of San Lorenzo. This Bible was recently “unearthed” in the Biblioteca Medicea Laurenziana in Florence (stored in the pluteo III, capsula 1), wrapped up in a precious yellow silk cloth, in a rather ruined state. After two years of restoration, the Bible will return to China in 2012 for a celebration of its >700 years of life and of its remarkable return trip on the Silk Road. What all of this has to do with a meeting in Separation Science is not clear to us, but come to the presentation and we will try to figure it out.

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POSTER PRESENTATIONS P-101 The Use of a Multi-channel Capillary for CE Separation in Combination with Contactless Conductivity and UV Detection. Petr Tuma1, Frantisek Opekar2, Eva Samcova1, 1Charles University in Prague, Third Faculty of Medicine, Institution of Biochemistry, Cell and Molecular Biology, Prague, CZECH REPUBLIC; 2Charles University in Prague, Faculty of Science, Prague, CZECH REPUBLIC A fused-silica capillary with outer diameter 360 micrometres and a set of seven inner channels with inner diameter 28 micrometres (multi-channel capillary) was tested in CE. The multi-channel capillary is characterized by a high ratio of inner capillary surface to capillary volume, which is important for the Joule heat dissipation. CE separation of epinephrine, norepinephrine and dopamine was performed in the multi-capillary by use of 100 mM acetic acid and 20 mM citric acid/NaOH as background electrolytes. Both contactless conductivity and UV detection exhibit higher sensitivity for analytes in combination with multi-capillary in comparison with a single-channel capillary at same inner diameter. Relative differences of analyte movement in individual channels of multi-capillary don’t differ more than 0.4%, which enable to achieve separation efficiency about 300000 theoretical plates per meter. Advantages of multi-channel capillary are demonstrated at CE separation of neurotransmitters, neutral sugars, small inorganic cations and low molecular organic acids. CE separations were performed in numerous background electrolytes, by use of hydrodynamic and electrokinetic injection and in combination with contactless conductivity and UV detection. P-102 The Use of Large Volume Sample Stacking for the Determination of Sub-micromolar Levels of Neurotransmitters in Microdialyzate of Periaqueductal Gray Matter by CE with Contactless Conductivity Detection. Eva Samcova, Petr Tuma, Klara Malkova, Vaclav Pavlicek, Charles University in Prague, Third Faculty of Medicine, Institution of Biochemistry, Cell and Molecular Biology, Prague, CZECH REPUBLIC The periaqueductal gray matter (PAG) is a region of the central brain, which functions as one of the main control centres of spinal nociception and is thus intensively studied. The pain response in PAG is treated by neurotransmitters as gamma-aminobutyric acid, glycine and glutamate. The in-vivo pharmacological studies of paracetamol administration were performed at rats and the PAG was sampled by microdialysis. The CE method was developed for the direct determination of neurotransmitters in very small amount of microdialyzate (cca 2.5 microlitres). 4 M acetic acid serves as background electrolyte and contactless conductivity detection is used for direct determination of non-absorbing amino acids. The high sensitivity of CE method is achieved by large volume injection of microdialyzate sample into the capillary. The microdialyzate samples are treated only by dilution by acetonitrile. The diluted sample is injected into the separation capillary in amount equals to half of the total capillary length and after switching separation voltage, acetonitrile is pushed away from the capillary. The developed stacking method is characterized by LODs for amino acids around 10-7 mol/L, which is sufficient for the direct quantification of gamma-aminobutyric acid, glycine and glutamate in the PAG microdialyzate. The effect of the paracetamol administration on neurotransmitters levels is demonstrated. P-103 Simultaneous Analysis of Acetylcholinesterase Inhibitors with Antipsychotic Drugs by Capillary Electrophoresis with on Column Field-amplified Sample Injection and its Application in the Clinic. Yi-Rou Wang, Su-Hwei Chen, Kaohsiung Medical University, School of Pharmacy, Kaohsiung City, TAIWAN A simple and sensitive zone capillary electrophoresis (CZE) with UV detection (214 nm) was developed and validated for the simultaneous determination of the acetylcholinesterase inhibitors (AChE I), donepezil and rivastigmine, with antipsychotic drugs in plasma. A sample pretreatment by liquid-liquid extraction and subsequent quantification by CZE with field-amplified sample injection (FASI) was used. The optimum separation for these analytes was achieved in < 20 min at 25°C. Using phenformin as an internal standard (IS), the linear ranges of the proposed method for the simultaneous determination of donepezil, rivastigmine, aripiprazole, quetiapine, risperidone, clozapine, ziprasidone and trazodone were over the range 4.0-80.0 ng/mL; olanzapine and IS concentration were over the range 1.0-20.0 ng/mL and a fixed concentration of 40.0 ng/mL, respectively. The method was applied for concentrations monitoring of AChEIs and antipsychotic drugs in ten Alzheimer’s disease patients with behavioral and psychological symptoms of dementia (BPSD) after oral administration of the commercial products. P-104 Simultaneous Analysis of Epirubicin and Doxorubicin by Capillary Electrophoresis with on Column Field-amplified Sample Injection and their Applications. Ping-Chih Lin, Su-Hwei Chen, Kaohsiung Medical University, School of Pharmacy, Kaohsiung City, TAIWAN Brief description: Anthracyclines are chemotherapeutic drugs which are used in the treatment of lung cancer and ovarian cancer. We developed a sensitive high-performance CZE combining on-column field-amplified sample injection (FASI) method to determinate epirubicin and doxorubicin by capillary electrophoresis (CE) with UV detection at 230 nm. A sample pretreatment by means of liquid–liquid extraction (LLE) (ethyl acetate) was used. The optimal separation of epirubicin and doxorubicin was performed using a BGE containing phosphate buffer (150 mM, pH = 5), 30% MeOH, 10% ACN, 0.03 % PVP (M.W. = 55000) and 3 % α-CD as a dynamic

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coating to reduce interaction of analytes with the capillary wall. Before sample loading, a water plug (0.3 psi, 6 s) was injected to permit FASI for stacking. The samples were injected electrokinetically (8 kV, 30 s) to introduce sample cations and the applied voltage was 15 kV with on-column detection at 230 nm. Several parameters affecting the separation and sensitivity of the anthracyclines were studied, including reconstitution solvent, organic modifier, pH and concentration of phosphate buffer, kinds of polymers and cyclodextrin modified. The linear ranges of the method for test drug in plasma using olanzapine as an internal standard, were over the range 3.0–50.0 ng/mL. Six cancer patients were received transcatheter arterial chemoembolism (TACE) therapy and blood samples were drawn over period times to detect epirubicin. Besides, this method was also applied to detect the doxorubicin in mice plasma. P-105 Two Detector Method for Determination of Accurate Effective Mobilities in Interacting BGE. Ludmila Muellerova, Pavel Dubsky, Jana Svobodova, Bohuslav Gas, Charles University in Prague, Faculty of Science, Department of Physical and Macromolecular Chemistry, Prague, CZECH REPUBLIC Chiral separation in capillary electrophoresis (CE) is based on interaction of enantiomers with a chiral selector contained in a background electrolyte (BGE). This interaction can be characterized by complexation constant and complex mobility. Both these parameters can be determined by CE itself, yet their determination requires accurate measurement of the electroosmotic flow (EOF). The neutral marker method is the simplest and the most popular way of the EOF mobility determination. However, when a charged interacting agent is present in the BGE it can interact not only with analytes as desired, but also impart a nonzero effective mobility to the EOF marker. In this work, we used a two detector pressure mobilisation method (2d method) that enabled us to measure very low mobilities which originally neutral markers gained because of their interaction with a charged selector. The 2d method is based on the same principle as method developed by Williams and Vigh [1]: one marker zone is situated in a BGE containing the charged selector, while second marker zone is surrounded with BGE without the selector, and therefore, its complexation is prevented. The two detector setup is utilized for the method and the distance between the detectors is elongated by a capillary loop. Determination of the initial distance between the two marker zones is based on the known distance from the capillary inlet to the first detector. After a brief voltage application, the final distance between the marker zones is determined based on the known distance from the first to the second detector. Difference between the initial and the final distance determines the effective mobility. By the 2d method, we determined effective mobilities of four compounds often used as EOF markers (namely: dimethyl sulfoxide, mesityl oxide, nitromethane and thiourea) in BGE containing highly sulfated β-cyclodextrin. Based on the results, suitability of these compounds as EOF markers for systems with this selector was discussed. This work was financially supported by the Grant Agency of the Charles University grant number 669412 and the Grant Agency of the Czech Republic, grant numbers 203/08/1428 and P206/12/P630. [1] Williams B. A., Vigh G.: Anal. Chem. 69, 4445 (1997) P-106 A Novel Strategy for Phosphopeptide and Phosphoprotein Enrichment using Lanthanide Phosphate Co-precipitation. Matthias Rainer, Yuksel Guzel, Munazza Raza, Guenther Bonn, University of Innsbruck, Institute of Analytical Chemistry and Radiochemistry, Innsbruck, AUSTRIA The presented work describes a novel method for the isolation of phosphorylated proteins and peptides using precipitation with an excess of lanthanide phosphate. The original idea was that phosphoproteins and –peptides could be precipitated from complex samples just like lanthanide phosphate by the addition of trivalent lanthanide ions. The rare earth element phosphates which also include lanthanides exhibit very low solubility products. Lanthanides are known to be hard “acceptors” with a strong preference for oxygen-containing anions such as phosphates to which they form very tight ionic bonds. This is achieved by the addition of potassium dihydrogen phosphate (KH2PO4) which causes precipitation of both, lanthanide phosphate and lanthanide-phosphopeptide/protein complexes. The co-precipitation can be explained by the ability of trivalent lanthanide cations to form up to nine-coordinate complexes. Thus, coordination occurs between the metal ion and the phosphorylated amino acid residues. The produced pellet is very stable, allows performing stringent washing steps and can easily be separated by centrifugation without loss of enriched phosphopeptides or phosphoproteins. The performance of the presented method was verified by a top-down and bottom-up approach using standard proteins (α-casein, β-casein and ovalbumin), milk and egg-white samples. The high selectivity of this method can be explained by the tremendous increase in free coordination sites of metal ions to attract phosphopeptides. The technology presented here represents an excellent and highly selective tool for phosphopeptide/protein recovery, it is easily applicable and shows several advantages in comparison to standard approaches such as TiO2 or IMAC. Literature: Güzel Y, Rainer M, Mirza MR, Bonn GK (2012) Highly efficient precipitation of phosphoproteins using trivalent europium-, terbium- and erbium ions. Anal Bioanal Chem 403:1323–1331 Mirza MR, Rainer M, Güzel Y, Bonn GK (2012) A novel strategy for phosphopeptide enrichment using lanthanide phosphate co-precipitation. Anal Bioanal Chem DOI: 10.1007/s00216-012-6215-0 (in press)

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P-107 Selective Extraction of Cocaine and its Metabolite Benzoylecgonine from Aqueous Samples by Use of Newly Synthetized Molecularly Imprinted Polymers (MIP’s). Renata Bujak1, Renata Gadzala-Kopciuch2, Joanna Raczak-Gutknecht3, Alicja Nowaczyk4, Boguslaw Buszewski2, Michal J. Markuszewski3, 1Department of Toxicology, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, Bydgoszcz, POLAND; 2Department of Environmental Chemistry & Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, Torun, POLAND; 3Department of Biopharmacy and Pharmacodynamics, Medical University of Gdansk, Gdansk, POLAND; 4Department of Organic Chemistry, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, Bydgoszcz, POLAND Analysis of cocaine and its metabolites in wastewater has been intensively reported recently [1]. The new, selective sorbents are needed to extraction step, especially when the sample is complex and impurities can interfere with quantification. Molecularly imprinted polymers (MIPs) are highly crosslinked synthetic polymers that are characterized by molecular recognition properties towards the template molecules or even group of similar compounds, with specificity and binding selectivity [2]. At the beginning of this investigation, the components of polymers such as: template, functional monomer, cross-linker and porogen have been chosen by computational approach. MIPs synthesis, with atropine and scopolamine, as the templates has been based on non-covalent strategy. The synthesis of Non-Imprinted Polymers (NIPs) has been performed simultaneously. The selectivity of new synthesized polymers has been estimated in binding and adsorption study. The concentrations of cocaine and its metabolite benzoylecgonine have been measured in liquid phase by HPLC method. The application of new synthesized MIPs as a sorbents in solid phase extraction (SPE) will be performed in further studies. The application of these synthetic polymers as sorbents allows not only pre-concentration and cleaning of the sample but first of all, selective extraction of cocaine and its metabolites from wastewater, when supposed concentrations of analytes are on the low levels. [1] S. Castiglioni, E. Zuccato , E. Crisci , Ch. Chiabrando ,R. Fanelli ,R. Bagnati, Identification and measurement of illicit drugs and their metabolites in urban wastewater by liquid chromatography-tandem mass spectrometry. Analytical Chemistry 78 (2006), 8421-8429 [2] M. Lasakova, P. Jandera, Molecularly imprinted polymers and their application in solid phase extraction. Journal of Separation Siences 32 (2009), 799 – 812 P-108 Obtaining Molecular Polymers Printing by Sol-Gel Process for the Determination of Cannabinoids in Treatment Plants by GC/MS Ion Trap. Elizabete Campos de Lima1, Ana Luiza Soares1, Luciana Fernandes1, Kathia Honorio2, Paula Homem de Mello1, 1UFABC-CCNH, SAo Paulo, BRAZIL; 2USP-EACH, SAo Paulo, BRAZIL ∆9-THC is the most widely used and produced illicit drug, consumed by between 125 and 203 million of worldwide people in 2009. The stability of the prevalence rates of drug consumption in the last decade become these substances part of the group of emerging environmental contaminants of interest to new studies. The analysis of incoming sewage in a sewage treatment plant can provide real-time data of drug consumption, considering that the excretion of these substances as active metabolites occur in the urine and feces. Molecularly imprinted polymers (MIPs) have been used for preconcentration and extraction of several analytes from the samples, having greater selectivity than the conventional sorbents used in solid phase extraction. In this study are shown the initial results obtained using sol-gel process phases for obtaining MISPE using tetraethyl orthosilicate (TEOS) Δ9-THC and hydrochloric acid. Parallel to the empirical study we are also developing a study by computational methods (ab initio and semi-empiric Density Functional Theory (DFT) and the continuous method IEF-PCM solvation (Integral EquationFormulationofPolarizableContinuumModel) that will be used to rationalize the design of MIPs, given analysis of interactions between the template molecule and functional monomers potential employees. The synthesis conditions are optimized by experimental design strategy. Polymers obtained will be analyzed by GC / MS ion trap (Varian model 3800) under the following conditions: gas He2 1mLmin-1, injector 260 ° C, temperature of the interface 280 ° C, temperature trap 190 ° C, electron ionization (70eV) parent ions (m/z) 299 Δ9-THC, 231 cannabidiol (CBD) and 295 cannabinol. . Initial results show to be promising for the selective extraction of Δ9-THC from samples of treated water. But the methodology still needs to be validated and applied to real samples. The authors are grateful FAPESP for financial support (Process 2010/20762-4) and CNPq. P-109 Enrichment by Fraction Collection Developed for CE/MALDI-MS to Analyze Proteins by Top Down Strategy. Michael Biacchi, Ricky Bhajun, Yannis-Nicolas Francois, Emmanuelle Leize-Wagner, Laboratoire de Dynamique et Structure Moléculaire par Spectrométrie de Masse (LDSM2), CNRS – UMR7177, University of Strasbourg, Strasbourg, FRANCE Protein identification and characterization seem to be one of the most important challenges posed to analytical sciences these last years. Capillary electrophoresis (CE) coupled with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is a technique highly suitable for the separation and detection of intact proteins. Since 1995, several approaches for coupling CE to MALDI-MS have been described [1-2]. However, due to its miniaturized format, CE suffers from a lower loading capacity which is a major drawback for trace analysis of proteins. In this study, new instrumental developments allowed to improve CE-MALDI/MS coupling for the analysis of intact proteins. CE fractions were collected directly on a MALDI target, using a

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sheath-flow interface. A homemade delivery matrix system was developed in the laboratory to allow dry droplet deposit with matrix addition just after sample deposition. Moreover, a modification of the CE system allows obtaining a CE-UV-MALDI/MS coupling. In a first step, a reproducible separation of protein mixture has been realized in CE/UV. Different background electrolytes and capillary coatings have been tested to allow an optimized separation compatible with CE/MALDI-MS. 1% formic acid aqueous solution was selected and protein adsorption during CE separations was prevented by coating the capillaries with Polybrene. In a second step, we have evaluated the robustness of the homemade CE-MALDI interface. For this reason, all proteins were analyzed by MALDI-TOF-MS directly after fraction collection. In a third step, fraction enrichment has been realized by the repetition on the same MALDI target of several CE separations performed on the same proteins mixture. The ultimate aim of this project is to obtain a suitable CE/MALDI-MS methodology for the analysis of complex proteins mixture by a Top Down approach. P-110 Pulsed Versus Continuous Lasers in CE/LIF Studies: Role of Photodegradation. Audrey Boutonnet1, Arnaud Morin1, Patricia Vicendo2, Florence Benoit-Marquier2, Jacques Fabre1, Véréna Poinsot2, Francois Couderc2, 1Picometrics, Toulouse, FRANCE; 2IMRCP Université de Toulouse, Toulouse, FRANCE LIF as detection mean is now well known and have a lot of applications. In most of applications, 488nm continuous lasers are used. Recently LEDs were introduced and achieved same sensitivities. For over 10 years pulsed lasers are used to provide to analysts a wide range of detection wavelengths. It is specially the case of NdYag 266nm pulsed laser which is used for the native detection of peptides or proteins. However this technique leads to the photodegradation of these compounds. Using continuous lasers with a relatively low power (around 10 mW) we do not observe the photodegradation of peptides and proteins. There is a linear correlation between their concentration and their detected fluorescence. In contrast the use of a powerful pulsed laser (10µJ in 0.5ns) leads to the loss of this linear correlation between the concentration of compounds and their fluorescence. We observed appearance of a plateau at high concentration which may be attributed to the formation of photoproducts or a saturation of the detection system. Using pulsed lasers, which light power is very high (10µJ in 0.5ns) this linearity is only on two orders of magnitude, and does not allow to obtain a complete linearity between fluorescence and concentration. Never the dependence of the fluorescence signal following the concentration was described using these pulsed lasers. In fact, in this case, the obtained detection curve seems to correspond to the combination of two equations one corresponding to low concentrations (nM) with a high slope and the second may be attributed to high concentrations (µM) with a small slope. In this work we relate such dependence, for all the used pulsed laser (355nm, 266nm, 532nm) compared to continuous lasers or LEDs (355nm, 280nm, 532nm) using the same fluorescent molecule (umbelliferone, tryptophane, texas red). Studying umbelliferone, we show that photodegradation during the migration in the detection cell results in a large loss of the fluorescent species and the formation of others fluorescent molecules excitable at the same the wavelength as the starting molecule. A calculation model based on the work of Heywood and Farnsworth is proposed considering that the photodegradation yield of the fluorescent molecule is concentration dependent, because of monomolecular and bimolecular reactions. A very nice correlation between the theoretical model and the experimental results is proposed. Some attempts to limit this photodegradation by the pulses of the lasers are proposed. P-111 Optimizing Conditions to Generate Frits in Capillary Microcolumns. Sonia Keunchkarian, Cecilia B. Castells, Leonardo G. Gagliardi, División Química Analítica, Universidad Nacional de La Plata, La Plata, Buenos Aires, ARGENTINA; División Química Analítica, UNLP y Centro de Investigación y Desarrollo en Tecnología de Pinturas (CIDEPINT-CIC-CONICET), La Plata, Buenos Aires, ARGENTINA Since its first years Capillary Electrodriven Chromatography (CEC) has demonstrated to be a promising separation technique. The flat flow speed profiles and the virtually unlimited possibility to extend the column lengths leads to efficiencies in the order of 100 000 theoretical plates or higher, this is, 10-fold or more of the typical efficiencies obtained in HPLC. However, difficulties for the reproducible production of capillary columns constituted an important drawback that make HPLC techniques to lead the market of standard methods for the analytical separation of liquids. One of the most important source of non-reproducibility is the generation of the porous devices (frits) to retain the particle beds. Due to the “micro” scale, porous devices must be generated in-situ and the control of the experimental conditions at these scales is naturally difficult. Porous devices based on silica particles have some advantages since it is the same material of the tube wall. They have excellent properties from both, the mechanical and chemical point of view. Procedures described on literature for sinterizing silica particles involve radial heating of the capillary tube with electric resistances. This leads to radial inhomogeneities across the frit, localized zones of strong electric fields heating and forming bubbles, differences in permeability, peak dispersion and loss of efficiency. Additionally, with these method it is difficult to control the frit length. In this work we introduce a method to obtain silica frits by frontal exposition of the extreme of capillary tubes filled with silica particles to a controlled source of heat (muffle oven). Experimental details and conditions, such as temperature, exposition time, introduction of particles, etc., have been optimized. SEM imaging, chromatographic and electrophoretic characterizations are also used to study the frits generated in the different conditions.

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P-112 Chemometric-assisted Microextraction Based on an Ionic Liquid as Ion-pairing Agent for the Determination of Chromium Species in Environmental Samples. Paula Berton1,2, Luciana Vera-Candioti2,3, Hector Goicoechea2,3, Rodolfo Wuilloud1,2, 1Laboratory of Analytical Chemistry for Research and Development (QUIANID), Instituto de Ciencias Básicas, Universidad Nacional de Cuyo, Mendoza, ARGENTINA; 2Consejo Nacional de Investigaciones Científicas y Tecnicas, Mendoza, ARGENTINA; 3Laboratory of Analytical Development and Chemometrics (LADAQ), Faculty of Biochemistry and Biological Sciences, National University of the Litoral, Santa Fe, ARGENTINA Ionic liquids (ILs), defined as liquid salts at room temperature, have been proposed, among other applications, for separation processes due to their specific and unique properties. The hydrophobic long-chain quaternary phosphonium IL trihexyl(tetradecyl)phosphonium chloride (CYPHOS® IL 101) has been employed as extraction solvent in Liquid-Liquid Microextraction (LLME) for metal preconcentration. Recently, the use of CYPHOS® IL 101 has been investigated as a potential novel ion-pair reagent dissolved in conventional organic solvents for metal extraction from aqueous phases. In most of these methods, metal anionic chloro- or iodo-complexes are formed before ion-pair formation with CYPHOS® IL 101. In the present work, an IL is proposed for the first time as direct ion-pair reagent for Cr speciation analysis, without the need of an extra ligand reagent. The speciation analysis was developed with initial ion-pair formation between Cr(VI) and CYPHOS® IL 101 followed by a simple and rapid microextraction procedure named ultrasound-assisted emulsification-microextraction (USAEME) for Cr species separation and preconcentration. Determination of Cr was performed by direct injection of the organic phase into an electrothermal atomic absorption spectrometer (ETAAS). A Plackett–Burman screening design and a central composite design were used to optimize the operational conditions for the procedure. The multiple response criterion was successfully used to optimize the extraction of Cr(VI). The combination of microextraction and chemometrics significantly simplify sample processing, and also addresses problems related to improvement in detectability and method validation. Under optimum conditions, the analyte extraction efficiency was higher than 99% and yielded a preconcentration factor of 250 with only 10 mL of sample. The limit of detection obtained was 2.4 ng L−1 and the relative standard deviation was 4.8%, calculated at peak areas. A correlation coefficient of 0.9983 was achieved. The two oxidation states of chromium (Cr) most commonly present in aqueous solution, i.e. Cr(III) and Cr(VI), differ drastically in its physiological and toxicological effects, its chemical transformations as well as its distribution and transport in the environment. Therefore, considerable emphasis has been given to the development of analytical methodologies for Cr species separation and determination. The proposed method was successfully applied to the determination of Cr species at trace levels in natural and drinking water samples. P-201 Chiral Analysis of Aspartate and Glutamate in Biological Samples by Capillary Electrophoresis with Laser-induced Fluorescence (LIF) Detection and Application in Investigating the Relationship with Alzheimer’s Disease. Yi-Rou Wang1, Ya-Hui Hsieh2, Su-Hwei Chen1, 1Kaohsiung Medical University, School of Pharmacy, College of Pharmacy, Kaohsiung, TAIWAN; 2Kaohsiung Medical University, Kaohsiung, TAIWAN A simple CD-mediated CZE method equipped with laser-induced fluorescence (LIF) detector was developed for chiral analysis of excitatory amino acids (EAAs), aspartate and glutamate, and to determine the EAAs concentrations in Alzheimer’s disease patients’ plasma. Before analysis, plasma samples were pretreated with centrifugal filter devices for removing proteins with high molecular weight (molecular weight cut off 3000) and then derivatized with 10 mM 6-carboxyfluorescein N-hydroxysuccinimide ester/ DMSO. The ratio of filtrate to the derivatizing reagent is 5 (v/v= 5：1). Mixed samples were sonicated for 2 h at 25℃ for chemical derivatization. After the derivatization reaction, reacted samples were diluted 100-fold with water and then hydrodynamically injected into CE instrument (0.5 psi for 5 s). The separation buffer was consisting of borate buffer 50 mM (pH 9.0) with 6 mM γ-CD and 0.1% PVP. The separation voltage was set at 20 kV. This method was applied in determining the EAAs concentrations of 26 patients with Alzheimer’s disease. We compared the EAAs concentrations and CDR-SB or MMSE and then discussed the relationship between EAAs concentrations and Alzheimer’s disease. From the results, there’s a moderately negative correlation between L-Asp concentrations with CDR-SB values. P-202 Chiral Capillary Electrophoresis in the Investigation of a New Circulene-like Species. Lukas Severa1, Milan Oncak2, Dusan Koval1, Radek Pohl1, David Saman1, Ivana Cisarova3, Paul E. Reyes-Gutierrez1, Petra Sazelova1, Vaclav Kasicka1, Filip Teply1, Petr Slavicek2, 1Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic v. v. i., Prague, CZECH REPUBLIC; 2Institute of Chemical Technology, Department of Physical Chemistry, Prague, CZECH REPUBLIC; 3Charles University, Department of Inorganic Chemistry, Prague, CZECH REPUBLIC Helquats, a new class of synthetic helical N-heteroaromatic cationic compounds were recently introduced as congeners of helicenes and diquat or paraquat-like structures [1]. A distinctive feature of helquats is helical chirality, thanks to which helquats exist as two enantiomers. Their interconversion, i.e. transition of the left- into the right-handed helix, occurs upon thermal initiation via a transient saddle-shaped configuration. Such intermediate is typically unstable. However, a saddle-shaped intermediate long-lived enough to be isolated was

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found so far for two helquat structures. This intermediate denoted as saddlequat of a [6]helquat derivative, again inherently chiral, was profoundly characterized [2]. Further, another example of saddlequat stemming from [7]helquat family was isolated. In addition, this saddlequat was found to undergo subsequent photoinitiated cycloaddition, which leads to completely new circulene-like structure, named circulenoid. Chiral capillary electrophoresis with sulfated cyclodextrins-based stereoselectors (see also [3,4]) turned out as important part of methodological toolbox used for description of helquats and related structures. In conjunction with spectral methods (NMR, X-ray crystallography, CD) and ab-initio calculations, a newly formed circulenoid was characterized in detail. In addition, a reaction pathway of its photoinitiated formation was described as well as a thermal conversion of the circulenoid to the corresponding helical congener. Financial support from the Czech Science Foundation ( P207/10/2391, 203/08/1428, P206/12/0435) and ASCR (RVO 61388963) is gratefully acknowledged. References: 1. Adriaenssens L. et al. (2009) Chem. Eur. J. 15:1072-1076. 2. Adriaenssens L., Severa L. et al. (2011) Chem. Sci., 2:2314-2320, DOI: 10.1039/C1SC00468A. 3. Koval D. et al. (2011) Electrophoresis, 32:2683–2692, DOI: 10.1002/elps.201100173. 4. Severa L. et al. (2010) New J. Chem., 34:1063-1067, DOI: 10.1039/c0nj00085j P-203 Comparative Enantioseparation of Talinolol in Aqueous and Non-aqueous Capillary Electrophoresis and Study of Related Selector-selectand Interactions by Nuclear Magnetic Resonance Spectroscopy. Lali Chankvetadze1, Anne-Catherine Servais2, Marianne Fillet2, Antonio Salgado3, Jacques Crommen2, Bezhan Chankvetadze1, 1Tbilisi State University, Institute of Physical and Analytical Chemistry, School of Exact and Natural Sciences, Tbilisi, GEORGIA; 2University of Liege, Laboratory of Analytical Pharmaceutical Chemistry, Department of Pharmaceutical Sciences, CIRM, Liege, BELGIUM; 3Centro Nacional de Investigaciones Oncologicas (CNIO), Department of Medicinal Chemistry, Madrid, SPAIN The enantiomers of the chiral β-blocker drug talinolol were separated with two single component sulfated β-cyclodextrin (CD) derivatives, namely heptakis(2,3-di-O-methyl -6-sulfo)-β-CD) (HDMS-β-CD) and heptakis (2,3-di-O-acetyl-6-sulfo)-β-CD) (HDAS-β-CD), in aqueous and non-aqueous capillary electrophoresis (CE). The enantiomer affinity pattern of talinolol towards these two CDs was opposite in both aqueous and non-aqueous CE. However, the enantiomer affinity pattern for a given CD derivative did not change when aqueous buffer was replaced with non-aqueous background electrolyte. The structures of the analyte-selector complexes in both, aqueous and non-aqueous electrolytes were studied using rotating frame nuclear Overhauser effect (ROESY) NMR spectroscopy. Inclusion complex formation between the enantiomers of talinolol and HDAS-β-CD was confirmed in aqueous buffer, while the complex between the enantiomers of talinolol and HDMS-β-CD was of the external type. The complex of the talinolol enantiomers with HDAS-β-CD in non-aqueous electrolyte was also of the external type. In spite of external complex formation excellent separation of the enantiomers was observed in non-aqueous CE. P-204 Use of Chiral Amino Acid Ester-Based Ionic Liquids as Chiral Selectors in Capillary Electrophoresis. Ioannis Stavrou, Constantina Kapnissi-Christodoulou, University of Cyprus, Nicosia, CYPRUS In this study, the utility of using a chiral ionic liquid (CIL) as the sole chiral selector in CE was investigated for the first time. In particular, five amino acid ester-based CILs were synthesized and used as additives in the background electrolyte (BGE) in order to evaluate their chiral recognition ability. The performance of each CIL as the sole chiral selector was evaluated by using 1,1´-Binaphthyl-2,2´-diyl hydrogen phosphate (BNP) as the analyte. Different parameters were examined, such as the alkyl group bulkiness and the configuration of the cation of the CIL, the type of the anion, the concentration of the CIL and the pH of the BGE, in order to optimize the separation of the enantiomers and to demonstrate the effect that each parameter has on the chiral recognition ability of the CIL. Baseline separation of BNP was achieved within thirteen minutes by using a BGE of 100 mM Tris/10 mM sodium tetraborate decahydrate (pH=8) and a chiral selector of 60 mM L-alanine tert butyl ester lactate. The run-to-run and batch-to-batch reproducibilities were also evaluated by computing the RSD values of the electroosmotic flow and the two enantiomer peaks. In both cases, very good reproducibilities were observed, since all RSD values were below 1%. P-205 L-his tid ine Functiona lized Po lymer Nanopartic les fo r Amino Acid Enantio s epara tion b y Ligand Exch ange CE. Cemil Aydogan, Veyis Karakoç, Adil Denizli, Hacettepe University Department of Chemistiry Biochemistry Division, Ankara, TURKEY A novel method of chiral ligand exchange capilary electrophoresis (LE-CE) has been explored for enantioseparation of aliphatic and aromatic amino acids with polymeric nanoparticles with L-histidine as a chiral ligand and copper (II) as a central ion. Polymer nanoparticles (NPs) was prepared by polymerization of N-methacryloyl-L-histidine methyl ester (MAH) and ethylene dimethacrylate (EDMA). NPs were characterized by elemental analysis, fourier transforminfrared spectroscopy (FTIR), atomic force microscopy (AFM). Average particle size and size distribution were also performed. Elemental analysis for MAH was found as 0.2 mmol/g polymer. CE system that contain nanoparticles in running buffer can be thought of as pseudocapillary electrochromatography. Factors affecting enantioseparation of the amino acids were studied. Successful

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enantioseparations of 6 pairs of amino acid namely, D,L-malonic acid, D,L-aspartic acid, D,L-glutamic acid, D,L-histidine, D,L-pheylalanine and D,L-tyrosine have been achieved with a buffer of 4 mM CuSO4, 40 mM (NH4)2SO4 and 0.5 mg/mL (20 mM) NPs at pH 6.0. The optimum pH was selected as 6.0 for the studied amino acids. It was observed that D-amino acids migrated more faster than L-amino acids. This novel method was also successfully achieved using D,L-malic acid from apple juice for real aaplication. P-301 Capillary Electrochromatographic Separation of Sulfonamide Antibiotics using Hydrophilic Monolithic Column. Cemil Aydogan1, Fatma Mutlu2, Adil Denizli1, 1Hacettepe University, Department of Chemistry, Biochemistry Division, Ankara, TURKEY; 2Abant Izzet Baysal University, Vocational School of Gerede, Bolu, TURKEY Hydrophilic monolithic columns, which were prepared by single step in situ polymerization of 2-hydroxyethyl methacrylate (HEMA), ethylene dimethacrylate (EDMA) and methacrylic acid (MAA) in a binary porogenic solvent consisting of toluene and 1-dodecanol, were developed as separation columns of sulfonamides by CEC. The columns with respect to monomer and porogen content were optimized. The resulting monolith was evaluated as hydrophilic interaction chromatography of sulfonamide antibiotics. Effect of salt concentration, ACN content and pH on the separation were also investigated using the prepared monolith. Furthermore, optimization of the mobile phase composition successfully determined the sulfonamide antibiotics in samples. [1] Aydoğan, C., Tuncel, A., Denizli, A., J. Sep.Sci. 2012, 35, 1010-1016. [2] Lin, C.L., Cheng, Y.J., Huang, H.Y., Lee, S., Electrophoresis 2009, 30, 3828–3837 P-302 Using Off-line Orthogonal Selectivity CE-UV – LC-MS/MS to Confirm Cocaine Presence in Banknotes. Melina Heller, Luciano Vitali, Gustavo Micke, UFSC Federal University of Santa Catarina, Department of Chemistry, Santa Catarina, BRAZIL This paper describes the development of a method to separate cocaine from banknotes using capillary electrophoresis (CE) with UV detection and confirming the presence of this drug in the sample using off-line hyphenation with LC-MS/MS [1]. The off-line hyphenation was performed using a fraction of the background electrolyte (BGE), which was inserted on a capillary containing the peak of the cocaine separated by electromigration in the CE-UV method and was collected and injected into the LC-MS/MS equipment [2]. The optimized BGE used in the CE-UV method was composed of 7.0 mmol L-1 2-hydroxyisobutyric acid and 20 mmol L-1 tris(hydroxymethyl)aminomethane at pH 8.4. The separation was conducted in a fused-silica capillary (48.5 cm total length and 40 cm effective length, 75 µm I.D.). A good sensitivity and efficiency were obtained by CE-UV method by extracting the cocaine from banknotes in acetonitrile and hydrodynamic injection at 50 mbar for 30 seconds. A few figures of merit of the proposed method include: good linearity (R2 > 0.99); good intra-day precision and recovery values. The LC-MS/MS system used was: Synergi Polar-RP C18 column (150 mm, 2.0 mm, 4 µm particle size) with its temperature set to 40°C; isocratic mode using a mobile phase 50% of solvent A (H2O + 0.1% formic acid) and 50% of solvent B (95:5 methanol/H2O); and a flow rate 350 µL/min. The cocaine was monitored using a scan type multiple reaction monitoring, and the quantitative ion used was 304/182, in positive mode. The results show that the combination of all the information obtained by CE-UV–LC-MS/MS off-line hyphenation contributed greatly to confirm the cocaine presence in banknotes, increasing the reliability of the result. Keywords: off-line orthogonal selectivity; CE-UV; LC-MS/MS; cocaine; banknotes; confirmation analysis. P-303 Optimization of Microemulsion Electrokinetic Chromatography for the Separation of Charged Pharmaceutical Enantiomers. Erin Ennis, Joe Foley, Drexel University, Chemistry Department, Philadelphia, PA, USA Electrokinetic chromatography (EKC) is a technique used primarily to separate neutral compounds via a pseudostationary phase (PSP) into which the compounds can differentially partition. Charged compounds, including charged enantiomers, can also be separated by EKC via such differential partitioning, although these separations have received less attention. The separation of charged enantiomers via microemulsion EKC is examined at various conditions in order to optimize microemulsion formulation and experimental methods. Experimental emphasis will be on optimization of resolution, migration time, and enantioselectivity by factorial design for pharmaceutical compounds of varying hydrophobicity. Chromatographic figures of merit are compared for all parameters and applications are explored. P-304 Determination of Active Ingredients in Advil® Cold & Sinus Liqui-Gels® Utilizing Dual-Opposite Injection Capillary Zone Electrophoresis. Donna Blackney Beckett, Joe Foley, Drexel University, Department of Chemistry, Philadelphia, PA, USA The ability of dual-opposite injection capillary zone electrophoresis (DOI-CZE) to simultaneously separate oppositely charged active ingredients in an over-the-counter pharmaceutical drug is demonstrated. Suppression of electroosmotic flow (EOF) allows sample injection from each end of the capillary. Therefore, a sample can be subjected to appropriate amounts of pressure and time of injection from opposite ends of the capillary to allow equal concentrations of each active ingredient to be introduced into the capillary. Once voltage is applied, the

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active ingredients will migrate from their respective injection site at opposite ends of the capillary toward the detector. Representative electropherograms from a separation of Advil® Cold & Sinus Liqui-Gels®, where each gel contains almost seven times larger amount of ibuprofen, a nonsteroidal anti-inflammatory drug, compared to pseudoephedrine are shown. P-305 Simultaneous Analysis of Cocaine and Adulterants in Street Samples Seized by Police in Sao Paulo State by HPLC-DAD and Ultra-fast HPLC-DAD. Elizabete Campos de Lima, Viviane Bianchi, Universidade Federal do ABC, Sao Paulo, BRAZIL The determination and quantification of cocaine and its impurities is of great interest to forensic toxicology. The knowledge of the various contaminants that make up the samples seized by the police and even predictive detect possible interactions between the active and adulterants enabling improvements in emergency care and ambulatory patient and helping the police intelligence service in tracing the route of entry the same country, and contribute to the understanding the problem in the country and help in public policies to control use of these drugs. In this context, the objective of this study was to develop a simple and fast methodology for determination of cocaine and adulterants in seized street samples using high performance liquid chromatography (HPLC) with DAD detection. An HPLC-DAD method was optimized using a Shimadzu 20 Series HPLC equipped with DAD detector, online degassing system, column oven, autosampler and software acquisition and data analysis LC Solution version 1.22 SP1. The analytical conditions were: C18 column (2.1mmx250mm, 5μm from Supelco), mobile phase 35:65 (v / v) methanol: sodium phosphate buffer pH 3.2, flow rate of 1.0 mLmin-1 injection 10 μL, T = 25 °C and λ = 232 nm. The methodology was validated using ICH Q2R1 protocol, the method showed good linearity for the studied compounds (R ²> 0.99), and coefficients of variation intra-assay and inter-assay ranged from 0.18% to 7.66%. A pool of samples of cocaine powder and crack rocks analyzed in powdered form. The results show that the pool of samples cocaine and crack contained respectively: 13.33% and 1.43 of caffeine, 11.63 and 1.27% of lidocaine, 6.37 and 28.17% of cocaine. The methodology is now being optimized for ultrafast-chromatography using a C18 column (3.0mmx50mmx1.8μm, from Agilent). This is a work of an undergraduate research and has the financial support of FAPESP (Process 2010/20762-4). P-306 Determination of Citrus Flavonoids, Troxerutin and Ascorbic Acid in Pharmaceuticals and Food Supplements by Capillary Electrophoresis. Jitka Siroka1, Andrea Martincova2, Klara Petru1, Marie Pospisilova1, Miroslav Polasek1, 1Charles University in Prague, Faculty of Pharmacy, Department of Analytical Chemistry, Hradec Kralove, CZECH REPUBLIC; 2Charles University in Prague, Faculty of Pharmacy, Hradec Kralove, CZECH REPUBLIC Flavonoids hesperidin (HE), diosmin (DI), rutin (RU), troxerutin (TR) are compounds of polyphenolic structure. Together with ascorbic acid (AA) they are used as active components of antioxidant mixtures in pharmaceuticals and food supplements. A simple CE method was devised for their separation and determination. The analyses were performed at 25kV in 50µm i.d. fused silica capillary of 21cm effective length (32 cm total length) maintained at 25°C by using 40mM borate buffer of pH*9.5 containing 25% methanol (v/v) as the background electrolyte. The samples were injected hydrodynamically (at 50mbar for 6s) and the detection was spectrophotometric at 280nm. The linear calibration ranges (0.05 – 0.5 mg/mL for HE, DI, RU, TR and 0.1 – 0.04 mg/mL for AA; r = 0.9994-0.9998), limits of detection 0.014 – 0.042 mg/mL, good intra- and inter-day repeatability of migration times and corrected peak areas (RSD≤ 3.6%; n = 6; n=18) were achieved. Accuracy of the method was evaluated by recovery experiments (recoveries 95.5- 99.8%). The method was applied to the analysis of 5 commercially available pharmaceuticals and food supplements. Financial support by SVV 2012-265 002 project is gratefully acknowledged. P-307 Determination of Impurities in Newly Synthesized Quaternary Ammonium Compounds by Capillary Zone Electrophoresis. Klara Petru1, Pavla Prskavcova1, Jan Marek2, Miroslav Polasek1, 1Charles University in Prague, Faculty of Pharmacy, Hradec Kralove, CZECH REPUBLIC; 2University of Defense, Faculty of Military Health Sciences, Hradec Kralove, CZECH REPUBLIC Capillary zone electrophoretic method with UV detection was developed for separation and determination of pyridine and isoquinoline in newly synthetized quaternary ammonium compounds possessing a long alkyl chain in the molecule. The analysis was performed in a fused silica capillary at 20 kV and 25 °C with UV detection at 210 nm and 254 nm. The total length of the capillary was 58 cm with the effective length of 49.5 cm and its inner diameter was 75 µm. The sample was injected hydrodynamically at 50 mbar for 6 s. The background electrolyte consisted of 70 mM acetic acid with 5% (v/v) of methanol. The pH was adjusted to 4.7 with sodium hydroxide. Imidazole was chosen as an internal standard. The calibration curves were linear for both pyridine and isoquinoline in the range 0.1 – 10 µg/ml; r2 ≥ 0.9972. The limits of detection were 60 ng/ml for pyridine and 20 ng/ml for isoquinoline. The optimized method was used to determine the content of impurities of pyridine and isoquinoline as parent compounds in newly synthesized quaternary alkylpyridinium and alkylisoquinolinium salts that could be employed in various ways (e.g., in pharmaceutical, chemical, textile and food industry).

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P-308 Urinary 1-hydroxypyrene as a Biomarker to Carcinogenic PAH Exposure. Clinton Ifegwu, Chimezie Anyakora, University of Lagos, Pharmaceutical Chemistry, Lagos, NIGERIA In order to capture the extent of exposure to polycyclic aromatic hydrocarbons (PAHs), various biomarkers have been employed. The biomarkers employed for PAHs include: PAHs genetoxic end points in lymphocytes; urinary metabolites; PAH-DNA adducts; PAH-Protein adducts. Of all these, excretory 1-hydroxypyene, a metabolite of pyrene, has been used extensively as a biological monitoring indicator of exposure to PAHs. This study attempts to assess the level of this biomarker in the body fluid of 68 exposed subjects using HPLC. The subjects screened include mechanics, drivers and fuel attendants. 1-hydroxypyrene was extracted from the urine of the subjects using solid phase extration method. The HPLC analysis was done in isocratic mode using water:methanol (12:88v/v) mobile phase. The stationary phase was X-Bridge C18 (150×4.6 mm) 5 µm column. The Wavelength was 250nm at a flow rate of 1.2 ml/mins The oven temperature was 30oC and the injection volume was 20 µl The run time was3 minutes. The level of urinary 1-hydroxypyrene detected varied for the different categories of occupation studied. About 27% of sampled fuel attendants had detectable 1-hydroxypyrene in their urine samples, but for auto mechanics it was 22%. There was no detectable 1-hydroxypyene in the urine samples of commercial drivers and students used as control. P-309 Neutral Coated Capillary Strength and Performances in CE-UV and CE-MS Applications. Anna X. Lou, Ingrid D. Cruzado-Park, Jean-Marc M. Busnel, Beckman Coulter, Inc., Brea, CA, USA Neutrally coated capillaries, which do not exhibit any significant electroosmotic flow (EOF), are commonly used in a variety of capillary electrophoresis (CE) modes. In addition to suppressed EOF, another key characteristic of such coating is their ability to reduce significantly, if not avoid completely, non-specific interactions commonly occurring between separated analytes and capillary walls, thereby promoting the achievement of very high separation efficiencies. Herein, the capabilities of a new neutral coating have been assessed. The main properties pursued were increased stability over an extended pH range (up to pH ≥10) and increased robustness to provide extended run-life (≥100 runs) under various CE modes, mainly capillary zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF). The coating chemistry is based on the formation of a double layer constituted exclusively of chemical compounds (monomer and polymer) and covalently bonded to the inner fused silica walls. To demonstrate coating efficiency and stability, a CE-UV test was initially implemented. The test buffer was made of NH4OAC which is compatible with both CE-UV and CE-MS, and the buffer pH ranged from 2.0 to 9.0. Even under extreme pH conditions, the EOF of the neutrally coated capillary was consistently less than 1.0E-07 (cm2.V-1.S-1) in more than 100 repeated runs, indicating a remarkable neutral coating strength. The neutrally coated capillaries were further tested for CZE by separating a test mix composed of 8 proteins with pI values ranging from 4.2 to 10.0 and MW values from 12.3 kDa to 29 kDa. Another test mix composed of synthetic peptides with pI ranging from 5.5 to 10.0 was also used. The standard deviation for migration time was <0.5% (n=25), and <5% (n=25) for both resolution and theoretical plates. After having proven the stability for CZE separations, its capabilities for CIEF were also evaluated. The linearity (R2) was > 0.99 for the pI values of 3 synthetic peptides (pI10, 9.5, and 5.5); and extensive capillary run-life was demonstrated. Finally, the newly developed coating was also implemented in sheathless capillary electrophoresis-electrospary ionization-mass spectrometry (CE-ESI-MS) where it allowed taking full benefit of both CE and nanoESI capabilities to achieve very high peak capacities and exquisite sensitivities. P-310 Salivary Nitrite and Nitrate in Fibromyalgia Patients. Hugo Barrague1, Nathalie Cantagrel5, Anne Marie Grimound2, Robert Salvayre3, Francois Couderc4, Elisabeth Causse3, 1CHU Rangueil/Universite de Toulouse, Toulouse, FRANCE; 2Odontology Department, CHU Rangueil Universite de Toulouse, Toulouse, FRANCE; 3Biochimie, CHU RANgueil, Universite de Toulouse, Toulouse, FRANCE; 4IMRCP, Universite de Toulouse, Toulouse, FRANCE; 5Pain Assessment and Treatment Center, CHU Rangueil Universite de Toulouse, Toulouse, FRANCE Nitric oxide plays a major role in cardiovascular protection. Its formation occurs from exogenous and endogenous nitrates involving the entero-salivary cycle. Salivary nitrates are converted into nitrites acting as anti-infectious agents against periodontal infections and fungal infections. Drugs, including those prescribed in fibromyalgia patients can disturb the biological balance of the mouth. Objectives. The aim of our study was i / to validate the quantification of nitrite / nitrate (NOx) in saliva by capillary electrophoresis/UV and ii / to quantify NOx in control subjects and in treated or untreated fibromyalgia patients. Methods: We included patients referred to Pain Centre : treated (n = 18) or untreated (n = 6) fibromyalgia patients (F 21/3 M, mean age 51.5 ± 8.54 years) and 24 healthy controls ((F 19 / M 5; 46.6 ± 14.1 years). Saliva was collected in the morning using a Salivette Kit (cotton, Sarstedt), then placed on ice until analysis. Data from the buccal examination and clinical questionnaire were recorded. The salivary ultra-filtrate were analyzed by capillary electrophoresis/UV.Results: i / The method we developed allows quantification of nitrite / nitrate (NOx) in 10 minutes. The use of an internal standard (Molybdate) improved linearity. ii / Our findings in salivary extracts show a high inter-individual variability as reported in the literature. NOx concentrations in fibromyalgia patients (n = 24) and controls were comparable (nitrites : 132.7 ± 95.5 µmol/L vs 98.0 ± 70.9 µmol/L ; nitrates : 222.0 ± 211.8 µmol/L vs 220.0 ± 326.1 µmol/L). In treated patients, no major changes in the effect of drugs (anticholinergics) we found. On the

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other hand, untreated patients exhibited significantly lower nitrite concentrations (37.0 ± 15.4 µmol/L, p <0.03). This suggests that untreated patients were at risk for developing an infectious lesion requiring clinical monitoring. An inverted profile was found in a patient with an abnormal parotid gland confirming the biological interest of salivary analyses. Conclusion: We propose a standardized method of saliva collection and an easy, inexpensive quantification of biological parameters that may help control the effect of drugs or diagnose buccal and systemic diseases. P-311 CE-LEDIF and CE-LIF (UV) Technologies for Characterization of Both Neutral and Sialylated N-linked Oligosaccharides Labeled with 2-ANSA. Audrey Rodat Boutonnet1, Richard -A Gardner2, Francois Couderc3, Pierre Naccache1, Daniel IR Spencer2, Jacques Fabre1, Arnaud Morin1, 1Picometrics, Toulouse, FRANCE; 2Ludger Ltd, Culham Science Centre, Abingdon, UK; 3IMRCP, Universite de Toulouse, Toulouse, FRANCE Glycan characterization of therapeutic proteins is important due to the role of oligosaccharides in protein stability, half-life, biological activity and efficacy. Glycans are typically characterized by releasing them from the therapeutic protein of interest and analysing them after they have been fluorescently derivatised. The most widely, currently used fluorescent label for glycan analysis by Capillary Electrophoresis (CE) is 8-aminopyrene-1,3,6-trisulfonate (APTS). Although this dye is an excellent label for the analysis of neutral oligosaccharides, it is not well adapted for the analysis of sialylated oligosaccharides. Indeed, it has three negative charges present. When analysed by CE, the accumulative amount of charges from the APTS label and the negative charges of sialic residues result in migration times that are too short for a good separation of neutral and sialylated oligosaccharides in the same run. In this study, oligosaccharides were derivatised with 2-amino-1-naphthalenesulfonic acid (2-ANSA) which contains a single sulfonic acid group, one negative charge. 2-ANSA has been shown to allow the separation of complex oligosaccharides, both neutral and sialylated, by CE as well as High Pressure Liquid Chromatography (HPLC) with high resolution. 2-ANSA labelled oligosaccharides can also be analysed by mass spectrometry (MS) to provide structural information. The properties of this dye are such that it fluoresces in the UV range. A comparative study was performed using a 7100 Agilent Technologies CE and two fluorescence light sources: a 266 nm pulsed diode laser and a 280 nm Light-Emitting Diode (LED). The high divergent light beam of the LED was focused on the capillary using an original patented optical arrangement. The LED technology provides similar results to that of a conventional Laser Induced Fluorescence (LIF) technology which is based on a laser to produce the excitation light source. P-312 Development and Validation of a Stability-Indicating High Performance Liquid Chromatographic Method for the Determination of an Antipsychotic Drug. Victor Pontes1, Maria Veronica Carranza-Oropeza2, Helena Yano1, Reinaldo Giudici2, Marina Tavares3, Maria Segunda Aurora-Prado1, 1University of Sao Paulo, Faculty of Pharmaceutical Sciences, Pharmacy/Physical and Chemical Quality Control of Medicines and Cosmetics, Sao Paulo, BRAZIL; 2University of Sao Paulo, School of Chemical Engineering, Sao Paulo, BRAZIL; 3University of Sao Paulo, Institute of Chemistry, Sao Paulo, BRAZIL Risperidone is a benzisoxazole atypical antipsychotic drug used for the treatment of psychosis including schizophrenia, some forms of bipolar disorder and psychotic depression. The aim of this work was to develop and validate a simple, and stability-indicating high performance liquid chromatographic method for the quantitative determination of risperidone in presence of its degradation products in pharmaceutical dosage forms (tablets). A Chromolith® Performance RP-18e (100 mm x 4.6 mm) column was used with mobile phase consisting of methanol:water (45:55 v/v) containing 0.5% triethylamine, pH 3.0 adjusted with ortho-phosphoric acid, the flow rate was 1.5 mL min-1. The risperidone peak was monitored at a wavelength of 235 nm, the retention time being 1.9 min. The analyses were made at room temperature (25 ± 1º C). The method was validated according to the International Conference on Harmonization and USPharmacopeia guidelines. The high determination coefficient (R2) = 0.9995 value indicated clear correlation between the investigated compound concentrations and their peak areas within the test range. The repeatability and intermediate precision, expressed by the RSD, were 0.93% and 1.05%, respectively. The accuracy, resulting from recovery experiments, was between 98.60 and 99.70 % for the sample tablets. The stability-indicating capability of the method was established by enforced degradation studies combined with peak purity assessment using the PDA detection. Risperidone standard, samples and placebo were subjected to acid and alkaline hydrolysis, oxidative, humidity and thermal stress conditions. No interference was observed from tablet excipients neither from degradation products. The method can indicate stability and can be used for the routine analysis of production samples and to check the stability of risperidone samples. Acknowledgments: FAPESP, CNPq

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P-401 Determination of Dissociation Constants of Glutathione and its Novel Analogues by Capillary Electrophoresis. Jana Kazarjan1, Merike Vaher2, Riina Mahlapuu3, Mats Hansen4, Ursel Soomets5, Mihkel Kaljurand6, 1Tallinn University of Technology, Department of Chemistry, Tallinn, ESTONIA; 2Tallinn University of Technology, Department of Chemistry, Tallinn, ESTONIA; 3University of Tartu, Department of Biochemistry, Tartu, ESTONIA; 4University of Tartu, Department of Biochemistry, Tartu, ESTONIA; 5University of Tartu, Department of Biochemistry, Tartu, ESTONIA; 6Tallinn University of Technology, Department of Chemistry, Tallinn, ESTONIA Glutathione (GSH) system is an attractive target for drug discovery because of it importance and versatility. Thiol-containing compounds have many roles in pharmacological and biochemical actions like antioxidativity, detoxification, regulation of biomolecules and signal transmission [1]. Decrease of GSH level is related to several pathological states, including cardiovascular, pulmonary, immune system diseases. Exogenous administration of GSH to compensate for the decrease of GSH levels is not reasonable because of its degradation in the plasma and the poor cellular uptake. To overcome the problems of the GSH administration, several GSH analogues have been created to increase the GSH level and support the functionality of the GSH system [2]. The main goal of the present study was to determine the pKa values for proper characterization of newly synthesized molecules. The knowledge of ionization constants is important in the understanding of certain chemical phenomena such as biological uptake, activity and transport. In the present work the capillary electrophoresis (CE) method was used for determination of pKa values of four new GSH analogues - UPF1 (Tyr(Me)-γ-Glu-Cys-Gly), UPF17 (Tyr(Me)-α-Glu-Cys-Gly), UPF50 (β-Ala-His-Tyr(Me)-γ-Glu-Cys-Gly), UPF51 (β-Ala-His-Tyr(Me)-α-Glu-Cys-Gly), as well as their homo-and heterodimers. In the CE procedure used for pKa determination, the migration velocity of the analytes were measured in background electrolytes of ionic strength (200 mM boric acid) and of varying pH ranging from 6.21 to 10.5O. The migration velocity depends on the degree of dissociation. The fully dissociated analyte migrates according to its ionic mobility, partially dissociated analyte migrates according to its effective mobility and the neutral form possesses zero mobility. CE has been proved to be an easy, fast and accurate method for the determination of physicochemical parameters, with such advantages as small amount of sample required, and no interference of impurities in the sample, moreover the accurate concentration is not necessary. From the relationship between measured mobilities and pH of the electrolyte, an S-Shaped curve was obtained for each analyte and the pKa values were determined using a non-linear regression analysis: electrophoretic mobilities plotted against the pH, can be fitted with the sigmoidal model, where the two unknows mobility of the fully ionised species and pKa are the regression parameters. [1] M. Zilmer, U. Soomets, A. Rehemaa, U. Langel, „ The Glutathione Sysytem as an Attractive Therapeutic Target”, Drug Design Reviews-Online, 2, 121-127, 2005. [2] C. Kairane, R. Mahlapuu, K. Ehrlich, K. Kilk, M. Zilmer, U. Soomets, “Diverse effects of glutathione and UPF peptides on antioxidant defense system in human erythroleukemia cells K562”, International Journal of Peptides, 2012. P-402 Chitosan Coating Capillaries for the Separation of Inorganic Anions and Basic Proteins. Sesil Uzasci, Filiz Tezcan, F.Bedia Erim, ITU, Department of Chemistry, Istanbul, TURKEY Chitosan is a linear polysaccharide produced commercially from N-deacetylation of chitin, which is a polysaccharide found abundantly in nature. As a cationic polyelectrolyte, chitosan has been used as coating material for the fused silica capillaries in capillary electrophoresis. In the present study, a permanent coating procedure was developed for the first time by the use of chitosan and/or quaternary ammonium chitosan (2-hydroxypropyltrimethylammonium chloride chitosan) together with genipin as cross-linking agent. The chromatographic characteristics of the columns were tested by the separation of basic proteins and inorganic anions (NO2−, Br−, NO3−, I-, SCN-) as model analytes. The cationic pH ranges of the columns, plate numbers and separation selectivities of analytes were compared with corresponding features in dynamically coated capillaries by chitosan and quartenary chitosan. P-403 Determination of Acid-base Dissociation Constant of Azahelicenes by Non-aqueous Capillary Electrophoresis. Milan Elias, Angelina Andronova, Andrej Jancarik, Jiri Klivar, Michal Šamal, Jaroslav Zadny, Irena G. Stara, Ivo Stary, Vaclav Kasicka, Institute of Organic Chemistry and Biochemistry AS CR v.v.i., Prague, CZECH REPUBLIC Non-aqueous capillary electrophoresis (NACE) with UV-absorption detection was employed to determine acid-base dissociation constants (pKa) of ionogenic groups of five azahelicenes. Azahelicenes represent unique helically chiral polyaromatic heterocycles [1], which are sparingly soluble or insoluble in water. Therefore, NACE in methanolic solutions of the background electrolytes (BGEs) has been employed for analysis and characterization of these analytes. Acid-base dissociation constants of azahelicenes were calculated from the dependence of their effective electrophoretic mobilities on pH by nonlinear regression analyses. The effective mobilities were determined by NACE in 10 – 15 BGEs within a wide pH range 1.6 – 11.2. NACE separations were performed in a home-made CE device equipped with UV-absorption detector operating at 206 nm. Experimental data were collected and handled by chromatography station Clarity (Data-Apex, Prague, CR) and program Origin 6.1 (OriginLab, Northampton, MA, USA), respectively. Separations were performed in fused

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silica capillary (id/od= 50/375 µm, effective/total length 201/306 mm, outer polyimide coating, internally untreated). All separations were performed at ambient temperature. Applied voltage for separations was 20 kV (anode at the injection capillary end). Analytes and electroosmotic flow (EOF) marker (dimethyl sulfoxide) were injected into the capillary at the anodic end by pressure 11 mbar for 3-10 s, except for trichloroacetate-based BGEs. In this case, due to a reverse (anodic) direction of EOF, the EOF marker was injected at the cathodic capillary end in order to detect analyte and EOF marker within a single analysis [2, 3]. The work was supported by the GACR (grant Nos. 203/08/1428, P206/12/0435, P207/10/2207 and 203/09/1766), GA ASCR (grant No. IAA400550916 and IAA400550916) and by the ASCR, Research Project RVO61388963. References [1] a) Míšek J., Teplý F., Stará I. G., Tichý M., Šaman D., Císařová I., Vojtíšek P., Starý I., Angew. Chem. Int. Ed. 2008, 47, 3188-3191; b) I. Starý, I. G. Stará in Strained Hydrocarbons (Ed.: H. Dodziuk), Wiley-VCH, Weinheim, 2009, pp. 166-176. [2] Ehala S., Míšek M, Stará I. G., Starý I., Kašička V., J. Sep. Sci. 2008, 31, 2686-2693. [3] Psurek A., Scriba G. K. E., Electrophoresis 2003, 24, 765-773. P-404 Study of Biomolecular Interactions by Partial-filling Affinity Capillary Electrophoresis. Martin Ruzicka, Karolina Strohalmova, Michael Jirasek, Martina Cizkova, Michaela Rumlova, Filip Teply, Vaclav Kasicka, Institute of Organic Chemistry and Biochemistry v.v.i., Academy of Sciences of the Czech Republic, Electromigration Methods, Prague, CZECH REPUBLIC The understanding of biomolecular interactions is important in drug research. In this work, we would like to demonstrate application of Partial-Filling Affinity Capillary Electrophoresis (PF-ACE) in the study of DNA-intercalator interaction and virus capside protein-peptide interaction. Recently synthesized compounds from the group of helquats (helical extended diquats) [1] were proposed to have a significant ability to intercalate into DNA. Rough affinity estimation indicated that their binding constants are comparable to those of ethidium bromide and thus considered to be carcinogenic agents. On the other hand, if successfully targeted onto tumor cells, intercalating compounds are widely used as anticancer drugs. A virus particle is formed by assembly of structural proteins forming virus capside. This process requires interaction of several thousand individual protein molecules, but only small epitope on molecular surface is involved. In case of Human Immunodeficiency Virus (HIV), it was demonstrated that short oligopeptide derived from peptide sequence of capside protein interaction site competes with other capside proteins for binding site resulting in blocking virus assembly [2]. Analogous effect is studied in similar Mason-Pfizer Monkey Virus (M-PMV) [3]. Evaluation of virus particles assembling was conducted by simple observation in electron microscope, not giving detailed insight into the interaction. In this work, using the PF-ACE technique, dissociation constant of the analyte-ligand complex is calculated from migration time changes of analyte depending on the length of the ligand zone in the capillary [4]. We investigate interaction between DNA oligonucleotide and well characterized intercalator ethidium bromide as a model, and subsequently analyze in the same way affinity of several newly synthesized helquates to DNA and calculate dissociation constants of the complexes. Using HIV as a model of M-PMV, we investigate binding of both effective peptide and control peptide to virus capside protein. The work was supported by the Czech Science Foundation, projects nos. 203/08/1428, P206/12/0453, P207/10/2391) and by the ASCR, Research Project RVO61388963. [1] L. Severa, D. Koval, P. Novotna, M. Oncak, P. Sazelova, D. Saman, P., Slavicek, M. Urbanova, V. Kasicka, F. Teply, New J. Chem., 34, 1063-1067 (2010) [2] J. Sticht, M. Humbert, S. Findlow, J. Bodem, M. Muller, U. Dietrich, J. Werner, H. Krausslich, Nature Structural & Molecular Biology 12, 671-677 (2005) [3] K. Bohmova, R. Hadravova, J. Stokrova, R. Tuma, T. Ruml, I. Pichova, M. Rumlova, J. Virol. 84, 1977-1988.(2010) [4] M. Nilsson, V. Harang, M. Bergstrom, S. Ohlson, R. Isaksson, G. Johansson, Electrophoresis, 25, 1829-1836 (2004) P-405 Development of Liquid Chromatography Method using Multiple Injection for Increasing the Instrumental Throughput. Jacqueline Pereira Vistuba, Luciano Vitali, Melina Heller, Ana Carolina de Oliveira Costa, Gustavo Micke, Federal University of Santa Catarina, Chemistry Department, Florianopolis, BRAZIL Amino acids are present in various types of samples including dietary supplements and pharmaceuticals. One example is the hepatoprotectives that containing the amino acids methionine and betaine, which are activators of the hepatic system. Many techniques are employed for the determination of amino acids, mainly liquid chromatography (LC) with several types of detectors including photometric, mass and fluorescence. The throughput is an important point in development of methods of quality control in pharmaceutical. Some strategies to decrease the instrumental analysis time without losing on the separation efficiency and selectivity include, use of gradient elution and separations in special columns of ultra performance liquid chromatography. However, others strategies could be used to increase the throughput like multiple injections in a single run. The aim of this study was develop a LC-MS/MS separation method by using multiple injections in order to determine the methionine and betaine content on pharmaceuticals formulations. The method using LC-MS/MS analysis was performed on chromatographic equipment LC system 1200 Series Agilent Technologies (Germany). Separation was performed on a SeQuantTM ZIC®-HILIC column (150 mm, 2.1 mm ID, 3.5 µm particle size) Merck (Darmstadt, Germany). The runs were performed in isocratic mode using a mobile phase composed of H2O:ACN (70:30). The flow rate was set at 100 µL min-1. In all instances, the injection volume was 2 µL. The column temperature was set at 30 °C. The LC system was coupled to a mass spectrometer system consisting of

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a hybrid triplequadrupole/linear ion trap mass spectrometer Q Trap 3200 Applied Biosystems/MDS Sciex (Concord, Canada). The experiments were performed using the TurboIonSprayTM source electrospray in positive ion mode. Methionine and betaine were monitored and quantified using multiple reaction monitoring. For comparison, the methods of single and multiple injections were applied. The instrumental throughput increase of 10 samples h-1 (single injection) to 33 samples h-1 (multiple injections - nine injections of different levels of the calibration curve). The parameters were similar between the calibration curves with single and multiple injections for both analytes. Moreover, the proposed method has shown good precision values. The method was applied in analysis of betaine and methionine in ten samples of hepatoprotectives. The sample preparation was by dilution contributing to increased sample throughput. Beside, the multiple injections have the potential to be used for tracking fast reactions, such as kinetic studies. Furthermore, using the mass detector to monitor can selectively reactants and products in a study involving chemical reactions. P-406 The Use of Self-assembled Multi-walled Carbon Nanotubes/Chitosan Composites as a New Stationary Phase in Capillary Electrophoresis. Yang Hua1, Ding Yao1, Gao Wen1, Cao Jun2, Li Ping1, 1China Pharmaceutical University, State Key Laboratory of Natural Medicines, Nanjing, CHINA; 2Hangzhou Normal University, College of Material Chemistry and Chemical Engineering, Hangzhou, CHINA Self-assembled multi-walled carbon nanotubes/chitosan composites (S-MWNT/C) were developed as a novel stationary phase for the simultaneous determination of organic acids and flavonoids. In order to obtain optimum performance, separation system parameters were optimized, mainly including the type and concentration of chitosan, the concentration of MWNTs and buffer PH. The result indicated that CE with immobilized S-MWNT/C could obtain the high electrochromatographic resolution, good capillary efficiency and short separation time for tested compounds compare to the untreated capillary due to the interaction between analytes and the surface of S-MWNT/C. The holistic characteristic of these capillaries were represented by scanning electron microscope (SEM) and electroosmotic flow (EOF) measurements. All the analytes showed good linearity (r2>0.9990) with a relatively wide test range. The proposed method was fully validated, and subsequently applied to simultaneously quantitative evaluation of active constituents in Lonicera fulvotomentosa. P-407 A Parametric Optimization Approach Based on D-optimal Mixture Design for Water-in-oil Microemulsion Electrokinetic Chromatographic Determination of Five Anthraquinones in Rheum Palmatum L. Yang Hua1, Ding Yao1, Gao Wen1, Cao Jun2, Li Ping1, 1China Pharmaceutical University, State Key Laboratory of Natural Medicines, Nanjing, CHINA; 2Hangzhou Normal University, College of Material Chemistry and Chemical Engineering, Hangzhou, CHINA A chemometric method combining the D-optimal mixture design and quadratic equation was proposed to search for the optimum water-in-oil electrophoretic buffer system in microemulsion electrokinetic chromatography (MEEKC) for the simultaneous quantification of five anthraquinones (aloe emodin, chrysophanol, emodin, physcion and rhein). The concentrations of Sodium dodecyl sulphate (SDS), 1-butanol and sodium acetate buffer were optimized. The total separation time and the resolutions were used to evaluate the separation performance. The obtained separation conditions were validated in term of linearity, stability, precision, repeatability, as well as recovery, and compared with that of the conventional single factor optimization. Eventually, an efficient W/O MEEKC method, where the analytes were separated on baseline, was developed. The strategy described was successfully applied to determination nalytic compounds in Rheum palmatum L. P-408 Functionalized Graphene as Separation Carrier in Capillary Electrophoresis. Yu Fu1, Hua Yang1, Yao Ding1, Jun Cao2, Ping Li1, 1China Pharmaceutical University, State Key Laboratory of Natural Medicines, Nanjing, CHINA; 2Hangzhou Normal University, College of Material Chemistry and Chemical Engineering, Hangzhou, CHINA In this study, the use of functionalized graphene (FG) can serve as the pseudostationary phase for the capillary EKC separation of alkaline compounds. The effects of the concentration of FG, the buffer composition, pH, addition of organic modifier were optimized in order to obtain better CE performance. Compared with the results obtained using an aqueous micellar solution alone, the addition of FG in buffers changed the electrophoretic behavior of tested compounds and enhanced their resolutions as result of the influence of dispersed FG in BGE. Furthermore, the proposed method has been used for detection of the ingredients of the complex plant sample with satisfactory results. P-409 FSCE Methods Development using Peakmaster Software and Ionic Mobility Calculation through Molecular Descriptors and Neural Networks. Marcone Oliveira, Universidade Federal de Juiz de Fora, Department of Chemistry, Florianopolis, BRAZIL Capillary electrophoresis (CE) is a separation technique based on differenced migration of ionic or ionizable species when they are submitted to electric field. The most used separation mode in CE is the free solution capillary electrophoresis (FSCE), which present low operational costs, reagent consume and waste generated as the main advantages. The method optimization using FSCE is many times performed by trial and error. In order to systematyze the method optimization, the univariate or multivariate approaching can be used. The

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univariate approach presents the inconvenient of did not get information about interaction among variables and, many times, results about the local optimum are in fact obtained. Moreover, the univariate approach also presents the inconvenient that a higher number of experiment can be necessary. An interesting alternative to procedures described above is the utilization of theoretical models what decrease the amount of reagent and time necessary for optimization, fitting this mode of optimization method as free reagent. The PeakMaster software is a freeware software that can be used for this purpose. As the first part of this work will addressed systematic procedure to method optimization using effective mobility curve versus pH in order to select the separation mode (co or counter electrosmotic), internal standard and back ground electrolyte (BGE) components. This procedure has been successfully applied to analysis of biofuel, pharmaceutical formulations, biologic matrixes and food in analysis time of sub minute. PeakMaster software has as input pKa and ionic mobility values to analytes and BGE components to running electrolyte. An “silico” optimization, without practical experiments can be achieved through mathematic models to obtaining these constants. As a second part of this work will demonstrated that the use of molecular descriptors (polarizability, parachor, molar volume, molecular volume, etc ...) linked to the use of artificial neural networks or other multivariate calibration tool can be an alternative to obtaining the ionic mobility values. The pKa values can be obtained by simulation through software such as Pallas 3.1, which use models based on group theory to getting these constants. The results obtained up to now exhibit a good correlation and effective mobility deviations lower than 7%. The proposal model was applied to analyzes of kojic acid in cosmetics and glucosamine in pharmaceutical formulations. It is important to stress that these methodologies were optmized without previous knowing of any physical chemistry propriety pre-determined experimentally. P-410 Characterization of Binding to Cannabinoid Receptors CB1 and CB2 using Open Tubular Column. Sylvestre Dossou, Ruin Moaddel, Irving Wainer, National Institutes of Health, Baltimore, MD, USA Cannabinoids receptors (CB) have been increasingly targeted therapeutically for the treatment of anxiety, obesity, movement disorders, glaucoma, and pain. In addition, they have been shown to be potential anti-tumoral targets owing to the ability of their ligands to reduce tumor in animal models including gliomas, breast cancer and prostate cancer. To date, two subtypes of cannabinoid receptors have been identified, CB1 and CB2. CB1 receptors are abundant in the central nervous system and are also expressed in peripheral areas, including but not limited to the pituitary gland, immune cells, heart, lung, and gastrointestinal tissues. They also suppress the release of a number of excitatory and inhibitory neurotransmitters. CB2 receptors are reported to be abundant in immune cells, such as macrophages, B lymphocytes and NK cells, although they have also been detected in brain. In order to study the cannabinoid receptors, we have immobilized cellular membrane fragments of the human-derived hepatocellular carncinoma (HEP G2 cells) containing CB1/CB2 receptors on to the surface of a modified open-tubular (OT) capillary. The resulting CB1/CB2-OT column was used for characterizing both the CB1 and CB2 receptors. The binding activities of several ligands for the CB1/CB2 receptors were determined using frontal displacement chromatography and [3H]-Win 55-212 as the marker ligand. The data from this study confirm that the CB1/CB2–OT column was immobilized in a functional state and that it can be used to determine the binding affinities (Ki values) of agonists and antagonists for the CB1 and CB2 receptors. P-411 Use of Water Rich Mobile Phases in Reversed Phase Liquid Chromatographic Separations. Undugodage Perera, Barry Lavine, Oklahoma State University, Chemistry Department, Stillwater, OK, USA Water rich mobile phases in reversed phase liquid chromatography are not generally used because of the longer retention times that occur with organic modifiers such as methanol or acetonitrile. The problem of longer retention times can be addressed using hydrophobic alcohols such as pentanol or butanol in low quantities (less than 1% v/v) as organic modifiers. Advantages of using these mobile phases in reversed phase liquid chromatography for the separation of water soluble and weakly retained congeners are demonstrated through the separation of imidacloprid and its degradation products and of vanilla and its isomers. P-412 Determination of Polyimide Composition using CE-MS. Tim Causon, Markus Himmelsbach, Wolfgang Buchberger, Christian Klampfl, Johannes Kepler University, Linz, AUSTRIA Polyimides are a group of widely used synthetic materials that with a variety of different areas of application. Depending on the requirements (defined by the particular final product) the actual composition of polyimides may show quite substantial variations. Capillary electrophoresis coupled with mass spectrometric detection (CE-MS) was employed for the determination of polyimide composition following chemical degradation of the polymer sample. Polyimide is chemically decomposed to corresponding aromatic diamine and aromatic acid components using an alkali fusion reaction. Solid polymer samples are fused in a potassium hydroxide melt yielding reaction products that are diluted in acid and can be immediately analysed by CE coupled to a quadrupole time-of-flight (Q-TOF) MS. This approach involves a simple and rapid sample preparation yielding both qualitative and quantitative information regarding the chemical composition of the polymer. Application of the CE-MS approach is shown for a range of commercially available polyimide and poly(amide-imide) samples and the results used to confirm the chemical composition.

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P-413 Neutral Nonpolar Monolithic Capillary Columns with C8-, C12- and C16- alkyl Moieties for Proteins and Peptides Separation in Capillary Electrochromatography. Chanida Puangpila1, Thumnoon Nhujak2, Ziad El Rassi1, 1Oklahoma State University, Chemistry Department, Stillwater, OK, USA; 2Chulalongkorn University, Bangkok, THAILAND This study has assessed the optimal n-alkyl chain length as well as the monomer composition for the preparation of neutral monoliths best suited for the reversed-phase capillary electrochromatography (RP-CEC) separation of peptides and proteins. Using the appropriate free radical initiator, two different neutral nonpolar monolithic columns series (designated as A and B columns series) with C8-, C12- and C16- alkyl moieties were prepared by the copolymerization of the functional monomers C8-methacrylate, C12-acrylate and C16-methacrylate, respectively, with the crosslinking monomer pentaerythritol triacrylate in a ternary porogenic solvent composed of cyclohexanol, ethylene glycol, and water. In the A columns series, the composition of the functional monomers and crosslinker was adjusted to yield comparable chromatographic retention regardless of the alkyl chain length. In the B columns series, the composition of the functional monomers and crosslinker was kept constant yielding chromatographic retention, which increased as expected in the order of increasing the n-alkyl chain length. Due to their direct influences on the monolith porosity and retention energetic, the nature and composition of the monomers at a given porogen composition have largely affected the solute’s mass transfer characteristics and sorption kinetics, as assessed by the van Deemter plots and separation efficiencies. The C16-monolith provided better separation efficiency (N) for alkylbenzenes (N of 200,000, 162,000, 150,000 plates/m for C16-, C12- and C8-monoliths, respectively), and yielded better separation for tryptic peptide mapping. However, the C8-monolith provided better separation efficiency for proteins (N of 332,000, 236,000, 225,000 plates/m for C8-, C12- and C16-monoliths, respectively). For large protein molecules, the energetically “softer” C8 surface allowed faster sorption kinetics and in turn improved efficiency, while an energetically “harder” C16 surface favored better separation of the smaller size peptide solutes. P-414 Maximizing cIEF Resolution and Loading Capacity in a 100 cm Long Neutral-Coated Capillary. Ingrid D. Cruzado-Park, Anna X. Lou, Jean-Marc Busnel, Beckman Coulter, Brea, CA, USA Capillary isoelectric focusing (cIEF) is a powerful analytical technique that has been utilized successfully in the characterization and purity assessment of monoclonal antibodies. The cIEF performances already achieved are satisfactory for the vast majority of applications, however, other applications may require pushing the cIEF limits even further. In this context, targeting an improved resolution as well as an increased loading capacity may be such a goal. This poster describes the successful conversion of a cIEF method developed for a 50 µm i.d., 20/30 cm long Neutral Capillary for use into a 75 µm i.d., 90/100 cm long neutral-coated capillary. Initial cIEF method optimization in the 100 cm long capillary was performed using peptide markers with pI values of 3.4, 4.1, 5.5, 7.0, 9.5 and 10.0. Increasing the focusing time was critical in achieving complete formation of the pH gradient in such a long capillary. Due to the use of a longer focusing time, it was necessary to increase the amount of anodic stabilizer in the cIEF sample in order to minimize distortions at the acidic end of the pH gradient. The monoclonal antibody known as Remicade (Imfliximab) was used as the cIEF sample (in conjunction with pI 10.0, 9.5 and 7.0 markers) to assess the success and reproducibility of the separation method. Remicade separated into five major isoforms when using a 50 µm i.d., 20/30 cm long capillary and 12 µL of Pharmalyte 3-10 in the cIEF sample. Under these experimental conditions, the slope of the pH 7-10 gradient was -0.29 pH units/min and the most acidic major isoform was barely resolved from the pI 7.0 marker. When using a 75 µm i.d., 90/100 cm long capillary, the slope value decreased to -0.078 pH units/min. This resulted in the detection of additional Remicade species within the major isoforms, but it still failed to baseline resolve the most acidic main isoform from the pI 7.0 marker. Replacing Pharmalyte 3-10 with Pharmalyte 5-8 increased resolution between the most acidic Remicade isoform and the pI 7.0 marker from 0.8 to 3.7. Finally, the amount of Remicade loaded into the 100 cm long capillary per cIEF separation was increased incrementally from 424 ng up to 3.4 µg in order to determine its maximum loading capacity without losing cIEF resolution. P-415 Polar Monoliths for Hydrophilic Interaction Capillary Electrohromatography. Dilani Gunasena, Ziad El Rassi, Oklahoma State University, Chemistry Department, Stillwater, OK, USA Two novel monoliths designated as diol-monolith and aminopropyl-monolith (AP-monolith) have been introduced for hydrophilic interaction capillary electrochromatography (HI-CEC). Both monoliths have been recently introduced and exploited by our laboratory in immunoaffinity chromatography [1]. Since the backbone of the two monoliths carry polar and hydrophilic groups it was naturally advantageous to exploit their full potentials in other modes of interactive chromatography such as HI-CEC. The diol-monolith resulted from the in situ polymerization of glyceryl methacrylate (GMM) and pentaerythritol triacrylate (PETA) in a ternary porogenic solvent. GMM and PETA possess hydroxyl functional groups, which impart the diol monolith with hydrophilic groups. Although the monolith is neutral and void of a fixed charge on the surface, a relatively strong EOF was observed due to the electric double layer formed by the adsorption of ions from the mobile phase, producing a bulk mobile phase flow. The monolith showed a cathodal EOF over a wide range of pH and acetonitrile concentration in the mobile phase. The novel monolith can be used to separate polar compounds such as phenol derivatives, small aliphatic

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amides, carbohydrates, nucleosides, etc. The AP-monolith, which possesses amine/amide functionality on the surface was prepared by the in situ polymerization of N-(3-aminopropyl) methacrylamide hydrochloride (NAPM) and ethylene dimethacrylate (EDMA) with cyclohexanol, dodecanol and methanol as porogens. Over the pH range studied a strong anodal EOF was observed with the applied negative voltage allowing the achievement of broad selectivity for the various polar solutes investigated. The AP-monolith was further exploited in HI-CEC by modifying its surface with neutral mono- and oligosaccharides in order to obtain different type of polar monoliths having different characteristics for the separation of polar compounds. 1. D.N. Gunasena and Z. El Rassi, J. Sep. Sci., 2011, 34, 2097-2105. P-416 An Immobilized Graphene Oxide Stationary Phase for Open-Tubular Capillary Electrochromatography. Chun Wang1,2, Sergio de Rooy1, Cheng-Fei Lu1, Vivian Fernand1, Leonard Moore Jr.1, Isiah M. Warner1, 1Department of Chemistry, Louisiana State University, Baton Rouge, LA, USA; 2College of Science, Agricultural University of Hebei, Hebei, CHINA (presented by Paula Berton) The research literature currently abounds with studies of graphene-related materials as a result of the extraordinary properties of such materials. On the basis of these citations, it is clear that the range of applications for such materials is substantial. In this manuscript, we report the immobilization of graphene oxide (GO) onto a fused-silica capillary to form a novel stationary phase for use in open-tubular capillary electrochromatography for the first time. We report the successful incorporation of GO through an in situ condensation reaction with 3-aminopropyl triethoxysilane (APTS) on the inner surface of the capillary. This GO-incorporated capillary was then characterized by use of scanning electron microscopy, infrared spectroscopy, and measurements of electro-osmotic flow. The electrochromatographic features of this stationary phase have also been evaluated. Evaluation of acquired data indicates high electrochromatographic resolution and good capillary efficiency. Highly reproducible results between runs, days, and capillaries were also obtained. P-417 Poten tia l o f CE/MS for Small Carboxylic Ac ids ana lys is a s Alte rn a tive to GC/MS Reference An a lytica l Methods . Johann Far1,2, Jean-Bernard Falmagne2, François de l'Escaille2, Edwin De Pauw1, 1Analis R&D, Suarlee, BELGIUM; 2Laboratory of Mass Spectrometry, Liege, BELGIUM GC/MS (Gas Chromatography coupled to Mass Spectrometry) is the analytical method of choice for carboxylic acids analysis because of good sensitivity, low limit of detection and the possibility to compare the pattern of fragmentation with existing databases for identification. However GC requires that the analytes are volatile. If it is not the case, the use of chemicals in order to perform the derivatization is mandatory, this may induce analytical bias. CE (Capillary electrophoresis) is a technique of choice for ions separation without prior treatments. The method is fast and do not require highly technical skills. A UV detector is the most common detection method; however electrospray mass spectrometry detection is recently gaining interest, while it really helps for structural information of the detected compounds. In this poster, the preliminary results of CE/MS analysis of several carboxylic acids are presented. All carboxylic acids are analyzed without any sample pretreatment. These acids looked at are from the “Citric Acid Cycle” including pyruvate and some isotope labeled analogues but also glyoxylate, lactate, oxalate and tartrate. Moreover, the preliminary results of a sample preparation approach to remove phosphate salts are presented. Phosphate is a very common salt that is often used in biological buffers but prevents the derivatization of carboxylic acid for GC/MS analysis and reduces the reproducibility of results for both GC/MS and CE/MS analysis. P-418 Separation of Harmala Alkaloids by CZE: Optimization, Calculation of Analytical Figures and Method Transfer between Different CE Instruments. Marcos Tascon1, Nora Vizioli2, Fernando J. Benavente Moreno3, Leonardo G. Gagliardi4,1División Química Analítica, Universidad Nacional de La Plata, La Plata, Buenos Aires, ARGENTINA; 2Instituto de Química y Fisicoquímica Biológica (IQUIFIB-CONICET), Universidad de Buenos Aires, Ciudad Autonoma de Buenos Aires, Ciudad Autonoma de Buenos Aires, ARGENTINA; 3Departament de Química Analítica, Facultat de Química, Barcelona, Barcelona, SPAIN; 4División Química Analítica, Universidad Nacional de La Plata y CIDEPINT, La Plata, Buenos Aires, ARGENTINA Harmala alkaloids (HAlks) are a family of compounds with psychotropic effects on humans. Its presence in invasive algae on atlantic coast of the patagonian sea would result in economical benefits. Very low concentrations or absence of HAlks becomes these algae eatable and with potential uses in food industry. On the other hand, significant concentration of HAlks becomes the algae not eatable, but the possibility to extract them provides potential uses in the pharmaceutical industry. All the HAlks are based on a similar basic molecular structure, changing only few of the substituent without significant molecular mass differences. All of them have, at least, one -amine- ionizable group. These properties become the HAlks an interesting challenge for analytical separations and, particularly for capillary electrophoresis. In this work we separated harmine, harmaline, harmol and harmalol and two less abundant, harmane and norharmane. Firstly, the background electrolyte (BGE) pH has been optimized based on the theoretical curves of mobility as a function of pH. After establish the pH zones that provide the best selectivity, different buffering compounds have been experimentally tested. Secondly, other parameters like BGE concentration, voltage, type and proportion of organic solvents, have been optimized. Finally, using the optimized conditions, analytical figures for each studied compound have

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been calculated from the data obtained with three different commercial instruments in order to verify the method transfer. The method showed to be useful for the separation and quantification of these six structurally related alkaloids. P-419 Comparison of Oil-in-water and Water-in-oil Microemulsion in Microemulsion Electrokinetic Chromatography for the Analysis of Water-Soluble Vitamins. Gabriel Roth1, Claudinei Silva2, Marina Tavares2, Kevin Altria3, Erika Kedor1, Maria Ines Santoro1, Maria Aurora-Prado1, 1University of Sao Paulo, Faculty of Pharmaceutical Sciences, Pharmacy/Physical and Chemical Quality Control of Medicines and Cosmetics, Sao Paulo, BRAZIL; 2University of Sao Paulo, Institute of Chemistry, Sao Paulo, BRAZIL; 3GlaxoSmithKline, Harlow, UK Microemulsions are classified as being oil-in-water (O/W) or water-in-oil (W/O). O/W emulsions disperse in water. W/O systems do not disperse in water, but do disperse in oil. In this work O/W and W/O microemulsion electrokinetic chromatography (MEEKC) were compared to separate and determine water-soluble vitamins (thiamine hydrochloride, niacin, pyridoxine hydrochloride and cobalamin) in available multivitamin formulations. The effect of surfactant concentration, oil type, organic modifier and buffer concentration on separation were examined in order to optimize the two methods. The electrophoretic conditions included the use of uncoated-silica capillaries effective lengths 30.2 cm and 20 cm x 75 µm id, hydrodynamic injection of 0.5 psi/5 s and 0.5 psi/3 s for O/W and W/O microemulsion, respectively; a temperature of 25ºC and 214 nm UV detection. The applied voltage was +20kV for O/W and +30 kV for W/O MEEKC. Organic modifier and oil type and content were found to markedly influence on the separation selectivity for both W/O and O/W systems. Surfactant concentration rarely affected separation resolution. A highly efficient O/W MEEKC separation method with baseline resolution, was achieved by using microemulsion solution composed of 0.5% ethyl acetate, 1.2% butan-1-ol, 0.6% SDS, 15% 2-propanol and 82.7% 10 mmol L-1 tetraborate buffer pH 9.2. The W/O MEEKC was constituted of 14% SDS, 40% butan-1-ol, 20% pentanol, 2% octane, 10% 2-propanol and 14% 60 mmol L-1 TRIS buffer, pH 9.3, a baseline resolution was also achieved. The methods were validated and good linearities were obtained with determination coefficients of (R2) > 0.99. The detection and quantitation limits ranged from 2.77 to 5.35 µg mL-1 and from 8.40 to 16.20 µg mL-1, respectively for O/W MEEKC method and for W/O MEEKC method ranged from 7.97 µg mL-1 to 16.72 µg mL-1 and from 24.16 µg mL-1 to 50.67 µg mL-1, respectively. Precision (intra-day) ranged from 0.2 to 1.0% R.S.D. and from 0.49 to 1.74% R.S.D. for O/W and W/O MEEKC, respectively. No interference from the formulation (tablets) excipients was observed. These results suggest that MEEKC methods can be used for the determination of vitamins in multivitamin formulations and can be appropriate for rapid and routine analysis. Acknowledgments: FAPESP, CNPq P-420 Micella r e lec trokine tic Chromatograph y (MEKC) Sys tem s fo r the Sepa ra tion o f Mixtures o f Charged and Unch arged Compounds . Caroline Lamalle, Anne-Catherine Servais, Ines Fradi, Jacques Crommen, Marianne Fillet, Laboratory of Analytical Pharmaceutical Chemistry, University of Liege, Liege, BELGIUM The migration behaviour of charged and uncharged analytes in MEKC was investigated under different conditions. Effective mobilities – electrophoretic mobilities under the influence of the negatively charged micelles – of cations, anions and neutrals were measured at neutral, basic and acidic pH values (7.5, 11 and 2.2) using background electrolytes containing different sodium dodecyl sulfate (SDS) concentrations (0-90 mM) and acetonitrile proportions (0-75 %, v/v). The SDS concentration and acetonitrile proportion were found to have a tremendous effect on the effective mobilities and the migration order of the tested compounds. Although the SDS micelles interact more strongly with neutrals and cations, the migration of anionic compounds is also affected by the SDS concentration, indicating that hydrophobic interactions can occur between the micelles and these compounds. Since cationic, anionic and neutral solutes exhibit rather different migration behaviours, it is possible to considerably enhance the separation selectivity by properly adjusting the SDS concentration and the acetonitrile proportion in the background electrolyte. These observations confirm the interest of using MEKC not only for the separation of neutral substances but also for the analysis of mixtures of charged compounds. P-501 Development and Valida tion o f a LC-Chip /MS Method fo r the Quantifica tion o f Fluoxetine and Norfluoxetine in Rat Se rum. Virginie Houbart, Anne-Catherine Servais, Jacques Crommen, Marianne Fillet, Laboratory of Analytical Pharmaceutical Chemistry, University of Liege, Liege, BELGIUM A number of quantitative analyses in biofluids need to be performed on samples available in limited volumes, such as those performed in pharmacokinetic studies on small animals. In this context, microfluidic systems such a LC-Chip device coupled to a mass spectrometer combine small sample volume requirements and high sensitivity. In this study, we present the development of a microfluidics-based method for fluoxetine (FLX) and norfluoxetine (NFL) determination intended to be used for pharmacokinetic investigations in rat serum. Using the methodology of experimental design, LC parameters were optimized with respect to peak resolution, analysis time and sensitivity. An SPE method was then developed for the cleanup of serum samples on miniaturized 96-well plates containing a mixed-mode strong cation exchanger that provided very clean extracts with a good analyte recovery (≥ 66.0%). The total SPE-LC-Chip/MS process required only 20µL per sample and the method

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provided a good sensitivity in a total run time of 12 minutes. Finally, the developed method for FLX and NFL quantification in rat serum was fully validated. After selection of the most appropriate regression model on the basis of the accuracy profiles, method selectivity, trueness, precision, accuracy and linearity were demonstrated. P-502 Protein Analysis Based on Microfabricated Sensors with Amalgam Working Electrodes. Petra Juskova1, Hana Cernocka2, Veronika Ostatna2, Emil Palecek2, Frantisek Foret1, 1Institute of Analytical Chemistry ASCR v. v. i., Brno, CZECH REPUBLIC; 2Institute of Biophysics ASCR v. v. i., Brno, CZECH REPUBLIC Rapid and sensitive characterization of protein forms and modifications is one of the challenges in current proteomic research. Development of new sensitive and selective bioanalytical tools to address this issue and systems amenable to automation and parallelization with reduced requirements for the sample amount are of key importance. Electrochemistry with its sensitivity, relative simplicity and low power requirement represents one of the promising methods for protein analysis with high miniaturization potential. Practically all proteins produce electrocatalytic chronopotentiometric peak H at the mercury containing electrodes with sensitivities down to subnanomolar concentrations [1]. Recently we have developed fabrication process for preparation of a microarray of the amalgam working electrodes with proven ability to protein analysis [2]. Electrodes were prepared in microarray format enabling parallel operation; however, manipulation with the external connections to the reference and the counter electrodes was still complicated. Here we present the fabrication process for improved version of the electrode array integrating amalgam working electrodes with the counter and reference electrodes into one sensor. This simplified operation with the whole system, requiring only deposition of a small sample amount (microliters) onto the sensor surface. Performance of the newly developed system for universal and/or selective biological specimen detection was tested for protein analysis using constant current stripping analysis. This work was supported by The Grant Agency of the Czech Republic (P301/11/2055, P206/12/G014) and institutional support RVO: 68081715. References [1] Palecek, E; Ostatna, V. Electroanalysis, 2007, 19, 2383-2403. [2] Juskova, P; Ostatna, V; Palecek, E; Foret, F. Analytical Chemistry, 2010, 82, 2690-2695 P-503 A Novel Phase Separation Detection of Emulsion using a Parallel, Continuous, and Real-time Electrical Impedance Sensing Chip. Yung-Sheng Lin1, Wei-Lung Chou2, Chih-Hui Yang3, Chih-Yu Wang4, Keng-Shiang Huang5, 1HungKuang University, Department of Applied Cosmetology and Master Program of Cosmetic Science, Taichung, TAIWAN; 2HungKuang University, Department of Safety Health and Environmental Engineering, Taichung, TAIWAN; 3I-Shou University, Department of Biological Science and Technology, Kaohsiung, TAIWAN; 4 I-Shou University, Department of Biomedical Engineering, Kaohsiung, TAIWAN; 5 I-Shou University, The School of Chinese Medicine for Post-Baccalaureate, Kaohsiung, TAIWAN Emulsion stability is an important factor for good emulsion products such as drink, food, and cosmetics. This study developed a new impedance chip to detect phase separation of emulsion. This impedance chip contained 8-pair indium-tin oxide microelectrode arrays which could detect 8 samples simultaneously. This impedance chip has advantages of small sample volume needed, parallel, continuous, and real-time detection. In this study, we estimated effects of the surfactant concentration on squalene/water emulsion stability by eye observation, conductance probe, and impedance chip. Three concentrations of tween 20 surfactant were employed for exanimations (9, 17, and 29 wt %). Results indicate that the phase separation rate was faster in a lower surfactant concentration. However, the emulsion in 29 wt % tween 20 was stable for over three days regardless of detection methods. The reaction time (tr) to complete the measured phase separation process differed in three methods by the aqueous phase height, conductance, and impedance, respectively. For the 9 wt % tween 20, tr are 24 hours, 20 minutes, and 5 minutes in tests of eye observation, conductance probe, and impedance chip, respectively. For the 17 wt % tween 20, tr was also shorter in the method of impedance chip than the other two methods. Therefore the proposed impedance chip has a quick reaction response and provides an alternative method to detect emulsion stability. P-504 Development of Biomolecules Patterning Method in Extended Nanochannel Utilizing Low-temperature Bonding Toward Single Cell Analysis. Kentaro Shirai1, Kazuma Mawatari2, Takehiko Kitamori2, 1The University of Tokyo, Department of Bioengineering, Kitamori Laboratory, Tokyo, JAPAN; 2The University of Tokyo, CREST, Tokyo, JAPAN Recently, absolute quantification method of proteins in single cells is required. The extended nanospace fabricated on quartz glass microchips, which has been investigated by our group, has extremely small volume as femtoliter to attoliter which is much smaller than single-cell picoliter volume by 3 to 6 orders. Mass transport, reaction and detection in nanochannels can greatly enhance sensitivity for low-abundance analytes. For realizing the concept, surface patterning of biomolecules in nanochannel is critical since analytes should be captured at defined area for detection. However, glass substrates, which should be used as material of microchip for nanofluidic research in terms of optical property and rigid structure at nm-scale, are usually bonded by thermal bonding, and biomolecules are destroyed by heat during bonding process. If glass substrates can be bonded at low temperature, pattern prepared before bonding can be used without loss of

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function. This study aims to develop a patterning method of biomolecules in extended nanochannels by using low temperature glass bonding. Critical requirements for patterning in nanochannels are, firstly, to generate chemical pattern, and secondly, to keep enough surface cleanliness for acquiring high bonding energy. We have solved these two points by using vacuum ultra violet (VUV) light. Aminopropyltriethoxysilane modified on quartz glass substrate was degraded by reactive oxygen generated by VUV light through photomask. The substrate was bonded with another one including microchannels and nanochannels at 100 °C under 5000 N after oxygen plasma surface activation. After bonding, thiol-modified DNA was covalently bonded by crosslinker. Distinct pattern was observed with well-regulated width when texasred-labeled complimentary target DNA was hybridized. Binding specificity was confirmed because non-complimentary DNA showed no increase of intensity. In hybridization reaction, fluorescent intensity saturated from upstream to downstream, and reaction reached to equilibrium within 20 seconds. Compared with microchannels case reported as several minutes, it was decreased by 1 order because of short molecular diffusion distance in nm-scale depth. For investigation of hybridization kinetic parameters in nanochannels, hybridization dissociation constant was calculated from time course measurement of hybridization at different concentration by assuming Langmuir first-order kinetic model. Dissociation constant was 0.9 x 10^-8 M which was comparable to bulk value. The results indicate that established method can be used for investigation of kinetic parameters of biomolecule interaction in nanochannels. This method can be applied to sensitive and fast immunoassays utilizing immobilized probes as templates for generation of primary antibodies, and protein quantification of single cells. L-505 Dielectrophoretic Field-flow Microchamber for Fractionation of Electroporated Cells. Jaka Cemazar, Tadej Kotnik, University of Ljubljana, Faculty of Electrical Engineering, Laboratory of Biocybernetics, Ljubljana, SLOVENIA We describe the development and testing of a setup that allows for dielectrophoretic field-flow fractionation (DEP-FFF) of irreversibly electroporated, reversibly electroporated, and nonelectroporated cells based on their different polarizabilities. The chamber was constructed from a single Pyrex wafer with interdigitated Au electrodes, a spacer, and a top cover glass, making the events in the chamber observable under most optical microscopes. The dimensions were optimized based on numerical computations of the electric field, its gradient and the fluid-flow velocity profile. The electrodes were fabricated using photolithography. A double-sided self-adhesive tape of 100 µm thickness was used as a spacer, with an opening of 8 cm length and 2 cm width cut in its middle to form a channel of 100 µm height, and with water-resistant acrylic glue of the tape holding the glass plates together and providing a tight seal. The glue loses its adhesive properties above 70°C, allowing for easy disassembly of the chamber in hot water and its thorough cleaning. We first optimized the flow rate and electric field parameters for efficient DEP-FFF separation of moderately heat-treated CHO cells (50°C for 15 min) from untreated ones, with the former used as a uniform and stable model of electroporated cells. We then used CHO cells exposed to electric field pulses with amplitudes from 1200 to 2800 V/cm, yielding six groups containing various fractions of nonporated, reversibly porated, and irreversibly porated cells, and we tested their fractionation in the chamber. DEP-FFF at 65 kHz resulted in distinctive flow rates for nonporated and each of the porated cell groups. At lower frequencies the efficiency of fractionation deteriorated, while at higher frequencies the separation of individual elution profiles was further improved, but at the cost of cell flow rate slowdown in all the groups, implying undesired transition from negative into positive DEP, where the cells are pulled towards the electrodes at the bottom of the chamber. Our results demonstrate that fractionation of irreversibly electroporated, reversibly electroporated, and nonelectroporated cells is feasible at a properly selected frequency. P-506 Major Milk Protein Separation by Microchip Electrophoresis. Fabiano Freire Costa1, Maria Aparecida Vasconcelos Paiva e Brito1, Marco Antonio Moreira Furtado2, Marcone Augusto Leal de Oliveira2, Marta Fonseca Martins1, Isabella Silvestre Barreto Pinto2, 1Embrapa Cow Milk Research Institute, Juiz de Fora, BRAZIL; 2Federal University of Juiz de For a, Juiz de Fora, BRAZIL The separation and quantification of individual milk proteins are an important consideration in dairy research. SDS-PAGE, HPLC, capillary electrophoresis have all been successfully applied for the separation and/or quantification of proteins in various milk proteins systems. The disadvantages of almost all separation techniques is the time required for the sample preparation, the physical separation of the proteins and the final integration and quantification of the individual protein components. Recently a novel microchip technique has been developed for separation and quantification of proteins, DNA and RNA. This technique has been reported to be a high-throughput, automated alternative with protein separation and quantification within 30 min. The objective of this study was to evaluate the separation potential of microfluid chip technique in milk with buffer solution added. The equipment was an Agilent 2100 Bioanalyzer system and the Protein 80 kit (Agilent Tecnologies, Waldbronn, Germany). The buffer solution (pH 3.0) consisted of 6.0 M urea, 20 mM trisodium citrate dehydrate, 0.1 M citric acid and 0.05% (w/w) hydroxypropylmetyl cellulose was added in milk (1/4 of milk). The results showed an excellent separation for whey proteins (α-lactalbumin and β-lactoglobulin) in the presence and absence of buffer solution. The separation time for the whey proteins was consistent with the molecular weights of the individual proteins, with α-lactalbumin eluting first and β-lactoglobulin last. Better

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results for major caseins separation were achieved with added buffer solution. According to eluting time, β-casein eluted first, αs-casein second and κ-casein last, even though κ-casein has the lowest reported molecular weight. Using purified proteins standard solutions or typical milk system, all major milk proteins could be separated with high resolution with added buffer solution. These results indicated that the microchip technology may provide a rapid and alternative method for the separation of the major milk proteins. Key words: proteins, milk, electrophoresis and chip P-507 Sensitive Detection of Nonfluorescent Molecules using Differential Interference Contrast Thermal Lens Microscope for More Efficient Separation using Extended-nano Chromatography. Hisashi Shimizu, Kazuma Mawatari, Takehiko Kitamori, The University of Tokyo, School of Engineering, Department of Applied Chemistry, Tokyo, JAPAN A novel absorption-based detection method, differential interference contrast thermal lens microscope (DIC-TLM) was employed for detection of nonfluorescent molecules in a nanofluidic separation system. Recently, the scale of analysis has been reduced to single cell level and separation analysis of ultrasmall volume sample is required. Therefore, we have developed a chromatography using 100-nm scale nanochannels (extended-nano channels) [1]. However, detection is quite difficult in such the scale, especially in case that analytes are not fluorescent. Contrary, we have developed a thermal lens microscope (TLM) for sensitive detection of nonfluorescent molecules. Furthermore, we developed a DIC-TLM by introducing a principle of wave optics and realized detection of ~100 molecules in nanochannels for the first time. In this paper, the DIC-TLM was used as a detector of extended-nano chromatography system and its detection performance was evaluated for future nanofluidic applications. The principle of DIC-TLM is based on light absorption. Briefly, a nonfluorescent sample is introduced into nanochannel and heated by an excitation laser. Then, a local refractive index change is induced in a nanochannel and the change is detected by interferometry using a probe laser. The experimental setup composed of a pressure-driven nanofluidic control system, fused silica chip and DIC-TLM. Fluids in nanochannel were controlled by air pressure (> 1 MPa) with 100 ms resolution, which enabled an injection of ~1 fL sample. Then, the injected samples were separated and detected by a DIC-TLM. The nanochannel was 2.3 um wide, 350 nm deep and 1.1 mm long. The wavelengths of excitation and probe laser were 488 and 633 nm. Firstly, time response of the DIC-TLM was optimized for injected sample by changing integration time of signal. Next, flow rate and bandwidth were determined to investigate separation efficiency. These results clarified that a theoretical plate height in extended-nano chromatography could reach ~1,000,000 /m and higher separation efficiency than conventional HPLC is expected. This is because eddy diffusion and radial dispersion are negligibly small in nanochannel. To summarize, the DIC-TLM was successfully employed as a detector of extended-nano chromatography. Their combination suggested the possibility of more efficient separation than conventional HPLC, as well as separation of fL-aL volume. We believe that the DIC-TLM would be a key technology for future single cell and molecule analyses. [1] Ishibashi, R. et al., Small, 2012, in press. P-508 Continuous Microfluidic Separation of Live and Dead Yeast Cells Using Reservoir-Based Dielectrophoresis (rDEP). Saurin Patel, Xiangchun Xuan, Department of Mechanical Engineering, Clemson University, Clemson, SC, USA Separating live and dead cells is critical to the diagnosis of early-stage diseases and to the efficacy test of drug screening etc. We develop a novel dielectrophoretic approach to continuously separating yeast cells by viability inside a microfluidic reservoir, which is termed reservoir-based dielectrophoresis (rDEP). This approach exploits the cell dielectrophoresis that is induced by the inherent electric field gradient at the reservoir-microchannel junction to selectively trap dead yeast cells and continuously separate them from live ones right inside the reservoir. It has unique advantages as compared to existing dielectrophoretic approaches such as the occupation of zero channel space and the elimination of any mechanical or electrical parts inside microchannels etc. Such an rDEP cell sorter can be readily integrated with other components into lab-on-a-chip devices for applications to biomedical diagnostics and therapeutics. P-509 CNT-Enhanced Electrochemical Detection in Capillary Electrophoresis and Microchip - Capillary Electrophoresis. Fabiane Galdino1, Carlos Garcia2, 1Institute of Chemistry and Biotechnology, Federal University of Alagoas, Alagoas, BRAZIL, 2Department of Chemistry, The University of Texas at San Antonio, San Antonio, TX, USA The analysis of biologically important compounds like catechol, dopamine or 8-oxoguanine typically requires the combination of high separation efficiency and sensitive detection. The latter is particularly challenging because the detection electrodes must balance sensitivity, dynamic range, and stability. Although carbon nanotubes are a viable alternative, their integration with microfluidic devices is time consuming and requires microfabrication facilities that are not always available. Aiming to enable the versatile integration of carbon-based nanomaterials with microfluidic devices, this poster describes a simple design, where the detection electrode is deposited on a groove of the flat substrate, used to cover the channel. To construct the electrode a thin groove (2 cm long) was made on either glass or plastic. This channel was first filled with carbon ink and subsequently coated with carbon nanotubes. After alignment, this layer was reversibly sealed against a PDMS layer containing the

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microchannels of the device (split-T injector, 60 mm separation channel, and solution reservoirs). Experiments were performed using 25 mM MES/His buffer (pH=6.5, for neurotransmitters) or 20 mM borate (pH=9.5, for 8-oxoguanine) as the electrophoretic buffers. For the analysis of neurotransmitters (10s electrokinetic injection, ESEP=1500V, and EDET=+0.6 V), clear peaks were observed for dopamine and cathecol at 100 s and 150s, respectively. For the analysis of 8-oxoguanine (10s electrokinetic injection, ESEP=1500V, and EDET=+0.4 V), a well-defined peak was observed at 50s. Besides providing very good stability, preliminary experiments also demonstrated the possibility of integrating enzymes, therefore enabling the possibility to develop microfluidic biosensors. P-510 Sheathless Capillary Electrophoresis: A Novel Infusion Platform for NanoESI Study from Intact Proteins to High Molecular Mass Noncovalent Complexes. Rabah Gahoual1, Jean-Marc Busnel2, Yannis-Nicolas Francois1, Emmanuelle Leize-Wagner1, 1Laboratoire de Dynamique et Structure Moléculaire par Spectrométrie de Masse (LDSM2), CNRS – UMR7177, University of Strasbourg, Strasbourg, FRANCE; 2Beckman Coulter Inc., Brea, CA, USA In 1994, M. Mann and M. Wilm caused a true revolution with the development of the nanoelectrospray ion source[1,2]. As compared to the conventional electrospray source, flowrate reduction allows a significant enhancement of the sensitivity. Nevertheless, problems of robustness were noted and somehow hindered a wide applicability of the nanoESI concept. Indeed, aqueous medium and presence of salt may cause loss of sensitivity and spray instability. Here, we have evaluated the suitability of a sheathless capillary electrophoresis-electrospray ionization mass spectrometry (CESI-MS) platform with a porous tip as the nanoelectrospray emitter to analyze with high sensitivity intact proteins and non-covalent complexes. In a first step, a systematic study of the ionization profile of an intact protein (Myoglobin) has been realized to evaluate the robustness and the enhancement of sensitivity provided by the use of CESI-MS as a nanoelectrospray infusion platform. In a second step, the study of a model protein, β-ring, was realized in native conditions with both CESI and conventional ESI to determine the ability of the CESI interface to maintain noncovalent complexes such as protein-protein and protein-ligand interactions. Finally, thermodynamic titrations followed by mass spectrometry have been performed for protein-ligand complex. The ultimate aim of this project is to take advantage of the CESI-MS capabilities for the study of high molecular mass protein complexes which are only available in scarce amounts in vivo. For this study, native hemocyanin from the deep sea crab Carcinus maenas have been analyzed. Hemocyanin, the crustacean respiratory pigment responsible for O2-transport, exists as hexamers, dodecamers and 18-mers of 75 kDa subunits. [1] Wilm M. S. and Mann M., "Electrospray and Taylor-cone theory, Doles beam of macromolecules at last" International Journal of Mass Spectrometry 1994, 136(2-3), 167-180. [2] Wilm M. and Mann M., B. "Analytical properties of the nanoelectrospray ion source." Analytical Chemistry 1996, 68(1): 1-8. P-511 ESI Interface-integrated Microdevice for an Application of LVSEP to Microchip Electrophoresis–Mass Spectrometry. Mami Oketani1, Takayuki Kawai2, Kenji Sueyoshi1, Takuya Kubo1, Fumihiko Kitagawa3, Koji Otsuka1, 1Kyoto University, Kyoto, JAPAN; 2National Institute of Advanced Industrial Science and Technology, Osaka, JAPAN; 3Hirosaki University, Aomori, JAPAN Microchip electrophoresis–mass spectrometry (MCE–MS) has many advantages, e.g., a high resolution, high identification capability, short analysis time, small samples/reagents consumption, and so on. However, there still remain some disadvantages including a low detectability due to the small amount of the injected samples and requirement of the complicated voltage control. To overcome these drawbacks, an electrospray ionization (ESI) interface-integrated microfluidic device was developed to apply large-volume sample stacking with an electroosmotic flow (EOF) pump (LVSEP), which provides a highly effective preconcentration without loss of the effective separation length by an easy experimental procedure and simple voltage control, to MCE–MS. The device made of poly(dimethylsiloxane) was equipped with an ESI orifice. The orifice was located nearby the intersection of the sheath and separation channels arranged as a V-shape configuration. The separation channel was modified with poly(vinyl alcohol) to suppress EOF to employ LVSEP. In experimental, 25 mM ammonium carbonate (pH 8.3, 30% (v/v) methanol) and 1 nM sulforhodamine B in deionized water were used as a background solution (BGS) and sample solution (S), respectively. In LVSEP-microchip zone electrophoresis (MCZE)–MS on the fabricated device, a whole channel and two buffer reservoirs are filled with S with low ionic strength (I) and BGS with high I, respectively. After applying the voltage, the BGS is introduced into the channels by the temporarily enhanced EOF due to the low I of the S. The anionic analytes are concentrated by a field-amplified sample stacking and then approach the cathodic reservoir by the fast EOF. The exchange of the S with low-I for the BGS with high-I suppresses the EOF in the separation channel, so that the anionic compounds can move toward the anode by their own electrophoretic migrations. Finally, the anions are separated by MCZE and led to the orifice by the sheath flow. To evaluate the performance of the ESI interface-integrated microdevice, both LVSEP-MCZE and MCZE–MS were investigated. A generation of the Taylor cone and electrospray at the ESI orifice was observed by the microscopic imaging. A stable MS signal was obtained in MCZE–MS for at least 5 min. Under an LVSEP-MCZE condition, both the stacking of the anionic compounds and the inversion of the moving direction were confirmed by the fluorescence imaging.

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These results indicated the applicability of the developed device to the LVSEP preconcentration and subsequent ESI-MS detection. P-512 Improving the Analytical Performance of Toner-based Electrophoresis Microchips by using Colored Toner. Ellen Flavia Moreira Gabriel1, Emanuel Carrilho2, Claudimir L. do Lago3, Wendell Karlos T. Coltro1, 1Universidade Federal de Goias, Instituto de Quimica, Grupo de Metodos Eletroforeticos, Goiania, BRAZIL; 2Universidade de Sao Paulo, Instituto de Quimica de Sao Carlos, Sao Paulo, BRAZIL; 3Universidade de Sao Paulo, Instituto de Quimica, Sao Paulo, BRAZIL Polyester-toner (PT) platform has been recently employed to microfluidic applications due to its low cost and easy of processing when compared to other popular substrates. Previous reports have been focused on the fabrication by just using monochromatic laser printers. According to data previously reported, PT devices have exhibited lower electroosmotic flow (EOF) and separation efficiency as well as poor injection-to-injection repeatability when compared to PDMS and glass microchips. Furthermore, it has been observed that the toner layer contributes in almost 90% for the peak broadening. To overcome this problem, we investigated the use of a color laser printer to produce PT microchips. Colored PT devices were fabricated using a color LaserJet printer (model HP CP1515n, Hewlett Packard) with 1200-dpi resolution. PT microchips were printed in CMYK standards (cyan, magenta, yellow and key-black). Based on spectroscopic data, it has been found that colored toner contains a mixture of polymer (basically a polyester) and silica. This composition has enhanced the EOF magnitude in ca. 20%. Besides the chemical composition, the colored toner layer has exhibited lower thickness and roughness, when compared to the toner from monochromatic laser printers, these parameters directly affects in the electrophoretic separation efficiency. The channel depth produced by both monochromatic and color laser printers has been estimated to be 7 ± 1 µm and 4 ± 1 µm, respectively. On the other hand, the toner surface roughness values were calculated to be around 1.20 ± 0.50 µm and 0.24 ± 0.05 µm for monochromatic and color printers, respectively. The analytical performance of the proposed devices was evaluated by using a capacitively coupled contactless conductivity detector (C4D) to monitor separations of a mixture containing alkaline-metal cations. The detection was carried out by applying a sinusoidal waveform of 400-kHz-frequency and 8 Vpp amplitude at 4.3 cm from the injection point. All the cations were successfully separated with baseline resolution and excellent run-to-run repeatability. Separation efficiencies for all cations ranged from 14,000 to 52,000 plates/m in all colors investigated. The relative standard deviation for the injection-to-injection reproducibility (n=10) was below 2%. We believe this improvement is associated with the toner chemical composition, the printed shape and also the surface roughness. Future studies will focus on the quantitative analysis of inorganic cations in soft drink and drug samples. This work has been supported by CNPq and INCTBio. P-601 CE-LIF Analysis of Riboflavin (Vitamin B2) in Popular Beverages: Black, Green, Sage and Rosemary Teas. Filiz Tezcan, F.Bedia Erim, Istanbul Technical University, Department of Chemistry, Istanbul, TURKEY Riboflavin (RF), commonly called vitamin B2, is a water-soluble vitamin. RF is an essential vitamin in human nutrition. Since RF cannot be synthesized in the human body, it should be consumed from the daily diet. Meat, eggs, liver, cheese, yeast and some leafy vegetables are the main source of RF. The daily dietary intake is recommended as around 1.2 mg/day. In this study, a capillary electrophoresis method coupled to laser-induced fluorescence detection (CE-LIF) was optimized for the determination of riboflavin. High sensitivity was obtained by utilizing the native fluorescence of riboflavin, which overlaps the excitation and emission wavelengths of argon laser, i.e., 488 and 520 nm, respectively. Detection of RF was performed in less than 5 min, employing 30 mM phosphate buffer at pH 9.8. The linearity of the method was checked between the concentration ranges from 0.01 - 5 µM for RF with 0.999 regression. The limit of detection was determined as 2.86 nM. The method was applied to the determination of RF in black, green, sage and rosemary tea extractions. Boiled water was poured on the plants and was left on the water bath for 5 minutes to extract riboflavin. Riboflavin contents of tea samples were found between 0.5-10.4 µg/g. Sage samples have the highest riboflavin contents compared the others. To our knowledge, this is the first report on the riboflavin contents of black, sage and rosemary teas. P-602 Determination of Diterpenoids of Salvia Species by MEKC. Nevin Oztekin1, S. Evrim Kepekci2, Gulactı Topcu2, F. Bedia Erim1, Selda Baskan Kahraman1, 1Istanbul Technical University, Department of Chemistry, Istanbul, TURKEY; 2Bezmialem University, TURKEY Salvia is the largest genus of plants in the mint family, Lamiaceae, with approximately 700-900 species distributed throughout the Old World and the Americas. Around 90 species are grown in Turkey and half of them are endemic. Salvia species contain many abietane diterpenes and many of them have been isolated from the plant species. Horminone and 7-O-acetylhorminone are two bioactive terpenoids isolated from Salvia species. Some important biological activities of these compounds, like cytotoxic, antiviral, antioxidant, antibacterial, particularly antituberculous, have been shown. The present study describes a MECK method to separate these two terpenoids which are structurally very close to each other. The optimal separation electrolyte contains 50 mmol/L SDS and 25% methanol at pH 11.5. LOD values of the method for horminone and 7-O-acetylhorminone

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are 3.27 and 4.52 µg/mL, respectively. The MEKC method that we have developed was applied to the quantitative analysis of horminone and 7-O-acetylhorminone in two Salvia species, namely Salvia chionantha and Salvia kronenburgii. P-603 UHPLC-(+)ESI-MS/MS Monitoring of Melatonin and its Isomers from Grape to Wines. Federico Jose Vicente Gomez1, Ismael Gatica Hernandez1, Julio Raba2, Soledad Cerutti2, Maria Fernanda Silva1, 1IBAM, Universidad Nacional de Cuyo, CONICET, Quimica Analitica, Lujan de Cuyo, ARGENTINA; 2INQUISAL Universidad Nacional de San Luis CONICET, San Luis, ARGENTINA Melatonin (MT, N-acetyl-5-methoxitryptamine) is a neurohormone discovered more than 50 years ago in the mammalian pineal gland. This molecule, a biogenic indolamine, performs an important role in the regulation of both circadian and seasonal rhythms. Furthermore, melatonin is a “suicide” antioxidant, which is not regenerated and does not promote further oxidation reactions. Its presence in higher plants was recently discovered and the knowledge of its function in vivo is very limited. Several studies have shown the occurrence of melatonin and related compounds in grapes and wines. The latter provides evidence of the opportunity to enhance the nutraceutical properties of premium wines. The analysis of MT in foods represents a highly challenging task due to its low concentration range, the difficulty in the selection of the extraction solvents because of its amphipathic nature, and the fact that it reacts quickly with other food components. In this work, the development of a methodology for the analysis of melatonin and its isomers in plant tissues, grape skins, musts and finished wines based on ultra-high performance liquid chromatography (UHPLC) coupled with electrospray ionization in positive mode ((+)ESI) and associated with tandem mass detection (MS/MS) is proposed. Sample processing factors, preparation/cleanup procedures and chromatographic/detection parameters were evaluated. Thus, C8 cartridges were chosen for the extraction of the analytes. Because of their chemical properties, a hydrophilic interaction liquid chromatography (HILIC) column was selected, being water (A) and acetonitrile (B), both with 0.1% formic acid, the chosen mobile phases for the elution gradient (6 min run cycle). As for mass detection, two transitions from the fragmentation of both MT and the isomer´s pseudomolecular ion ([M+H]+ = 233) were selected: 233 > 216 (higher abundance for the isomer) and 233> 174 (higher abundance for MT), which correspond to the losses of the NH3 and CH3CONH2 groups of the aliphatic portion of the molecule. The limits of detection (LoD) and quantitation (LoQ) were in the low ng/L range. Recovery, repeatability, reproducibility, and matrix effect studies were performed. Melatonin was detected in plant and grape extracts while its isomer was found in musts and finished wines. From the studies mentioned, it was possible to conclude that there were many external factors influencing the levels of MT and its isomer in the different samples, including saccharomyces strain during the fermentation process, harvest schedule and biotic/abiotic stress. P-604 High-throughput Determination of Phenolic Compounds in Extra Virgin Olive Oil using Dispersive Liquid-liquid Microextraction-CZE. Romina Paula Monasterio, Maria de los Angeles Fernandez, Veronica Carolina Soto-Vargas, Maria Fernanda Silva, Universidad Nacional de Cuyo, Instituto de Biologia Agricola de Mendoza (IBAM-CONICET), Facultad de Ciencias Agrarias, ARGENTINA The beneficial effects of the consumption of extra virgin olive oils (EVOOs) on human health are well-known and related to the characteristic fatty acid composition, and the presence of minor components, such as phenolic compounds (antioxidant properties). These compounds extend olive oils’ shelf life by delaying oxidation reactions and improve some sensory properties including pungency, astringency, bitterness and flavor. Chemically, olive oil consists mainly of glycerols, which account for more than 98% of its total weigh. In addition, it contains about 2% of other, nearly 250 minor components including phenolic compounds. The amounts and composition of these compounds in EVOO depends on several factors such as olive cultivar, degree of maturation and agronomic and technological aspects of production. A simple methodology using dispersive liquid–liquid microextraction (DLLME) combined with CZE has been applied for the simultaneous determination of phenolic compounds of nutraceutic interest (caffeic, gallic, vanillic, syringic, p-coumaric acids and oleuropein, apigenin, luteolin, 3-hydroxytyrosol and tyrosol) in EVOO. The methodology developed shows significant advantages over the low throughput-current methods that use large volumes of organic solvents. Also, the evaporation step is time consuming and may affect the stability of the analytes. On the other hand, trace amounts of analytes cannot be detected due to the small mass used. Optimum DLLME conditions were as follows: extraction of 20 g of EVOO with 300 uL boric acid 30 mM pH= 9.50 and 300 uL carbon tetrachloride, extractant and dispersive solvents, respectively. The BGE that provided complete resolution of the ten analytes was boric acid 30 mM, pH= 9.50. The short time of extraction, centrifugation and electrophoretic steps allow the selective determination of phenolic compounds in oil with satisfactory sensitivities, recoveries and relative standard deviations (RSD), compatible with levels present in the sample. The optimized methodology was applied for the analysis of experimental and commercial EVOOs. The new approach may help for the classification of olive oils according to varietal origin.

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P-605 Simultaneous Analysis of Main Fatty Acids of CLA Precursors in Forage by Capillary Zone Electrophoresis. Renata de Jesus Coelho Castro1, Fausto de Souza Sobrinho2, Marco Antonio Sundfeld Gama2, Patrícia Mendonca de Castro Barra1, Rosemar Antoniassi3, Marcone Augusto Leal de Oliveira1, 1Federal University of Juiz de For a, Juiz de Fora, BRAZIL; 2Embrapa Dairy Cattle, Juiz de Fora, BRAZIL; 3Embrapa Agribusiness and Food, Rio de Janeiro, BRAZIL Forages are the major components of ruminant diets, being a source of energy and nutrients for milk and meat production. Among the fatty acids found in forages, two (C18:2 n-6 and C18:3 n-3) have received particular attention due to their roles as precursors for the synthesis of conjugated linoleic acid (CLA), a class of health enhancing compounds predominantly found in dairy products. The objective of this study was to develop an alternative method for quantification of C18:2 and C18:3 in forages through the capillary electrophoresis (CE) technique, using Brachiaria ruzizienses as the reference forage. The analytical procedure involved CE with indirect detection zone in the UV at 224 nm, capillary external coating of Teflon and analysis time around 10 minutes. A rotational central composite design (star design) with triplicate at the central point was used to optimize the composition of the electrolyte system in order to separate the C16:0/C18:2 pair. The following electrolyte composition was obtained: 15.0 mmol L-1 NaH2PO4/Na2HPO4 buffer (pH~6,8), 8.0 mmol L-1 Brij 35, 4.0 mmol L-1 SDBS, 1.5% v/v n-octanol and 43,5% v/v ACN. Additionally, three methods of lipid extraction were tested: 1. Hara & Radin, 2. Micro Folch and 3. Bligh &Dyer; the best results were achieved by using the method of Hara and Radin. After optimizing the electrolyte system and choosing the lipid extraction method, two calibration curves were made using different concentrations of selected fatty acids and C13:0 as the internal standard. After checking the homoscedasticity behavior and no occurrence of lack of fit of the models, a response factor (RF) was calculated and applied in the quantification of selected fatty acids in the forage samples. The optimized CZE method was then compared to gas chromatography (the AOCS official method for fatty acid analysis) through the paired t-test. There was no difference between methods within a 95% confidence interval (p-value = 0.937 ). Our results indicate that the optimized CE method proposed herein can be used to quantify the most abundant fatty acids in forage samples. This alternative method has some advantages over the traditional GC method: less time-consuming, no derivatization step or specific separation columns required, and lower analytical cost. P-606 Simultaneous Determination of Seven Hydrophilic Bioactive Compounds in Polygoni Multiflori Radix by Short End Injection Micellar Electrokinetic Chromatography. Kameng Lao, Xiao-jia Chen, Jing Zhao, Shao-ping Li, State Key Laboratory of Quality Research in Chinese Medicine, and Institute of Chinese Medical Sciences, University of Macau, MACAU Pressurized liquid extraction and short-end injection micellar electrokinetic chromatographic methods were developed for simultaneous determination of seven water soluble components including one alkaloid (hypaphorine), one stilbene (2,3,5,4’-tetrahydroxystilbene 2-O-β-D-glucoside), five polyphenols (proanthocyanidin B1, proanthocyanidin B2, catechin, epicatechin, gallic acid) in water extract of Polygoni Multiflori Radix. The influence of neutral additives and relevant parameters such as pH, concentration of phosphate, SDS and HP-β-CD, capillary temperature and applied voltage were studied. Optimum separation was obtained within 14 min by using 50 mM phosphate buffer containing 90 mM SDS and 2% (m/v) HP-β-CD (pH=2.5) at 15 kV and 20 oC. All calibration curves showed good linearity (r2>0.9978) within test ranges. The LOD and LOQ were lower than 2.7 μg/mL and 5.5 μg/mL, respectively. The RSDs for intra- and inter-day of seven analytes were less than 3.2% and 4.6%, and the recoveries were 97.0%-104.2%. The validated method was successfully applied to quantitative analysis of seven investigated compounds in Polygoni Multiflori Radix samples collected from different regions of China, which is helpful for its quality control. P-607 Study of the Orthogonal Chromatographic Systems Applied to the Flavonoid Glycosides Analysis. Alessandra V. Jager1, Monica R. Mazalli1, Marina F. M. Tavares2, Fernando G. Tonin1, 1University of Sao Paulo, Faculty of Animal Science and Food Engineering, Sao Paulo, BRAZIL; 2University of Sao Paulo, Institute of Chemistry, Sao Paulo, BRAZIL Great attention has been paid to natural substances with antioxidant activity due to increasing incidence of serious pathologies such as cancer, cardiovascular diseases and inflammation, which are in part attributed to harmful effects of free radicals. Flavonoids are widespread naturally occurring antioxidants present in vegetable crops such as herbs, fruits, vegetables, grains, seeds and derived foods such as juices, wines, oils, etc. Due to the immense structural diversity, the analysis of this class of secondary metabolites constitutes a real analytical challenge. In this context, the development of chromatographic systems with different retention behavior can contribute to solve problems related to selectivity, thus contributing to the development of selective methods applicable in quality control of products and foods or even in fundamental studies on the secondary vegetable metabolism. In this work, the retention of 15 flavonoid glycosides in three different stationary phases, C18, PFP and ZIC-HILIC was evaluated. The retention data were obtained with aqueous mobile phase modified with MeOH, ACN, THF and their mixtures, maintaining the solvent strength of diverse mobile phases approximately constant. The biggest difference in behavior was observed for ZIC-HILIC column regardless the mobile phase utilized, and the compounds in this column showed a pronounced retention even with a large amount of solvent,

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when compared to the other stationary phases. The statistical hierarchical cluster analysis showed that it is possible to obtain different chromatographic behavior with the same stationary phase only changing the mobile phase composition. It was also observed that these changes in behavior are more pronounced when comparing chromatographic systems with the presence and absence of THF. Therefore, the greatest differences in chromatographic behavior was found when comparing different stationary phases, and the order of similarity retention behavior found was C18, PFP, ZIC-HILIC, i.e. the greater orthogonality is obtained when comparing retention data of systems in which C18 and ZIC-HILIC stationary phases were used. It is believed that the results obtained in this work may contribute to the development of analytical methodologies applied to the analysis of flavonoid glycosides, as well as to the development of two-dimensional chromatographic systems where the largest possible orthogonality between different dimensions is expected. P-608 Separation and Analysis of Free Amino Acids in CE/LIF or CE/LEDIF of Lyophilized Milk for Two Species of African Gourd Seeds: Cucumeropsis Mannii and Citrullus Lanatus. Josiane Makaya1, Varravaddheay Ong-Meang2, Audrey Boutonnet3, Francois Couderc2, Michel Mvoula Tsieri1, Jalloul Bouajila2, 1Equipe Pluridisciplinaire de Recherche en Alimentation et Nutrition, Institut de Developpement Rural, Universite Marien Ngouabi, Brazzaville, CONGO; 2IMRCP, Universite Paul Sabatier, Universite de Toulouse, Toulouse, FRANCE; 3Picometrics, Toulouse, FRANCE In Sub-Saharan Africa, Cucumeropsis mannii Naudin (CM) and Citrullus lanatus (CL) were used for their oleaginous seeds consumed as thickeners of a traditional soup. They are vegetable sources rich in proteins and amino acids of high quality. There is strong interest for enhancing local agroressources in Africa. In this work, the capillary electrophoresis technique (Agilent 7100 CE) was used to separate labeled amino acids of CM and CL with fluorescein isothiocyanate (FITC). They were detected by laser-induced fluorescence (CE-LIF, 488 nm) and LED (CE-LEDIF, 470 nm). The experimental conditions of labeling and separation were optimized. The approximation of the electropherograms (CL versus CM, CE-LIF versus CE-LEDIF) showed similar qualitative AA profiles. Eleven of the 16 AA currently detected were identified by comparison with standard AA bound to FITC under the same experimental conditions. Eight AAs have been quantified (in mg/g extract): arginine (CL: 3.96 ; CM: 6.89), valine (CL: 2.66; CM: 1.12), threonine (CL: 5.31; CM: 9.81), tryptophan (CL: 10.61; CM: 5.69), phenylalanine (CL: 17.59; CM: 20.9), alanine (CL: 11.86; CM: 14.85), glutamic acid (CL: 8.77; CM: 6.10), acid aspartic (CL: 1.43; CM: 2.00). The aging study of seed milks aims to determine the optimum storage conditions in terms of nutritional conservation. The electropherograms of the extracts were i) compared with each other according to their age and ii) compared to those of biogenic amines which are degradation products of some AA. The results showed the occurrence of putrescine for milks 3 days old kept at 25°C while its presence is detected after 7 days storage at 4°C. These new results should be useful in efforts to complement or replace cow milk (which is very expensive) or soya milk (less appreciated because of eating habits) by milk from available local agroressources. P-701 Interest of LED for Analysis of Polysaccharides Labeled with APTS using CE/fluorescence. Bernard Feurer1, Audrey Rodat Boutonnet1, Pierre Naccache1, Jacques Fabre1, Francois Couderc2, 1Picometrics, Toulouse, FRANCE; 2IMRCP Universite de Toulouse, Toulouse, FRANCE 9-Aminopyrene-1,4,6-trisulfonate (APTS) is a dye that is frequently used for the analysis of mono or oligo saccharides (Fraysse et al Electrophoresis. 2003, 24, 3364). APTS is routinely used in Capillary Electrophoresis separations with a 488nm laser. Process to label sugars is very well known, it consists in a reductive amination of the reductive function of the mono or oligosaccharides. These reactions can be done on very small samples (as small as 5µL). However, 488nm does not reach the maximum excitation wavelength of the APTS. In this poster we use different excitation wavelengths of LEDs (450nm and 480nm) to monitor the mono or oligo saccharides and report the lowest detected (LOD) concentration of mono or oligo saccharides. We show that the LOD also depends on the optimization of the excitation wavelength and on the stability of the light source P-702 Analysis of Alkaloids in Peganum Harmala L by Capillary Electrophoresis-UV/VIS. Ghada Bouajila, Varravaddheay Ong-Meang, Naziha Chabir, Francois Couderc, Mehrez Romdhane, Jalloul Bouajila, UMR IMRCP 5623, Toulouse, FRANCE Various extracts (Petroleum ether, Dichloromethane, ethyl acetate, Ethanol and water) of the leaves, stems and roots of Peganum Harmala L were prepared. In all the cases the extracts of leaves give the highest yield. The stems and the roots give variables yield. The objective is to evaluate the composition of the extracts by capillary Electrophoresis with UV-Visible detection. We targeted work on two alkaloids known for biological activities which are harmine and harmaline. An optimization of the composition of electrolyte, pH, time of injection as well as the potential difference was carried out in order to identify and to separate the maximum of products and to quantify these two molecules in the prepared extracts. The quantification of these compounds was obtained for the extracts. Others made up were detected under the same conditions optimized to detect two alkaloids.

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P-703 Glutathione Modified CdTe Quantum Dots as a Label for Study of DNA Interactions with Platinum Cytostatic Drugs. Marketa Ryvolova1, Kristyna Smerkova1, Jana Chomoucka2, Vojtech Adam1, Jaromir Hubalek2, Rene Kizek1, 1University in Brno, Department of Chemistry and Biochemistry, Faculty of Agronomy Mendel, Brno, CZECH REPUBLIC; 2Brno University of Technology, Central European Institute of Technology, Brno, CZECH REPUBLIC Cancer, a plaque of 21st century, is a leading cause of a death in developed countries [1]. There are several treatment strategies including chemotherapy based on using of platinum cytostatics. Cisplatin, carboplatin and oxaliplatin represent three generations of Pt-based compounds applied successfully in clinical practice. As a consequence of the employment of Pt-based cytostatics in the cancer treatment, it became necessary to study the mechanism of their main activity - formation of Pt-DNA adducts. The mechanism of their formation is still unclear, but nanomaterials as progressively developing branch, can offer a tool for study the interactions of these drugs with DNA. The fluorescence intensity of QDs can be significantly quenched by the presence of metals [2]and therefore in this work, fluorescent CdTe nanocrystals - quantum dots (QDs, emission maximum 525 nm) were employed to study interactions of platinum cytostatics (cisplatin, carboplatin and oxaliplatin) with DNA fragment (500 bp, c = 25 ug/ml). First, fluorescent behavior of QDs in the presence of platinum cytostatics was monitored and major differences in the interaction of QDs with tested drugs were observed. It was found that the presence of carboplatin (c = 0.25 mg/ml) had no significant influence on QDs fluorescence (average decrease of 1%); however cisplatin and oxaliplatin at the same concentration quenched the fluorescence significantly (average decrease 20%). Subsequently, the amount of platinum incorporated in DNA was determined by QDs fluorescence quenching. Carboplatin-DNA adducts quenched the QDs fluorescence by 0.2% and cisplatin-DNA adducts by 0.6%. Best results were reached using oxaliplatin (9.4%). Linear trend (R² = 0.9811) was observed for DNA platinated by three different concentrations of oxaliplatin (0.250, 0.125 and 0.063 mg/ml). In average, 20% of Pt-drug was incorporated into DNA. Correlation with differential pulse voltammetric measurements provided linear trend (R² = 0.9511). As a conclusion, especially in the case of oxaliplatin-DNA adducts, the quenching was the most significant compared to cisplatin and non-quenching carboplatin. Considering the fact that the tested drugs have different way of action, the quenching of QDs fluorescence could be a new approach to study their influence on DNA. Acknowledgement Financial support from CYTORES GA CR P301/10/0356 and CEITEC CZ.1.05/1.1.00/02.0068 is greatly acknowledged. References [1] Jemal, A., Siegel, R., Ward, E., Hao, Y. P., et al., CA-Cancer J. Clin. 2009, 59, 225-249. [2] Zhang, L. J., Xu, C. L., Li, B. X., Microchim. Acta 2009, 166, 61-68. P-704 Glycosylation Marker Based Evaluation of Autoimmune Disease Treatment using Capillary Electrophoresis. Csaba Varadi1, Bertalan Mesko2, Andras Guttman1, 1University of Debrecen Medical and Health Sciences Center Research Centre for Molecular Medicine, Horvath Laboratory of Bioseparation Sciences, Debrecen, HUNGARY; 2University of Debrecen, Debrecen, HUNGARY Most autoimmune diseases are associated with a significant increase in the IgG glycoforms that lack terminal galactose residues (FA2), thus terminate with N-acetyl-glucosamine (GlcNAc). The level of agalactosylation can usually be correlated with the severity and progression of the disease. The aim of this study was to investigate the changes in the relative amount of galactosylated biantennary glycans of IgG1 in rheumatoid arthritis (RA) and inflammatory bowel disease (IBD), especially in the view of transcriptomics data on galactosyl transferase experession. The glycosylation patterns of sex and age matched samples were analyzed and compared to understand the correlation between IgG N-glycan profiles, galactosyl-transferase expression and the pathogenesis of these autoimmune diseases. IgG1 was isolated from blood using Protein A affinity cartridges and the N-glycans were released by peptide-N-glycanase F (PNGase F). The free glycans were then fluorescently labeled with aminopyrene-trisulfonate (APTS) and analyzed by capillary electrophoreiss with laser induced fluorescence detection (CE-LIF). In this study we show and discuss the comparative data on the relative amount of sialylated, galactosylated and core-fucosylated glycans with the mRNA expression levels of the respective glycosyltranferases. P-705 Detection of Poliovirus by Fluorescent Labeled Nanobodies – Initial Approach for the Development of a Biosensor. Hadewych Halewyck1, Iulia Oita2, Lise Schotte1, Ellen Merckx1, Ann Massie1, Bert Thys1, Yvan Vander Heyden2, Bart Rombaut1, 1Vrije Universiteit Brussel, Department of Pharmaceutical Biotechnology and Molecular Biology, Brussels, BELGIUM; 2Vrije Universiteit Brussel, Department of Analytical Chemistry and Pharmaceutical Technology, Brussels, BELGIUM Nanobodies or VHHs are a novel class of single-domain antigen-binding fragments derived from heavy chain antibodies naturally occurring in camelids. The discovery of nanobodies opened exciting perspectives for the production of new anti-poliovirus drugs. Moreover, we believe that these nanobodies can be used for the development of a biosensor for polio which allows to follow the spread of the virus within the environment and the individual. Till now, from the poliovirus-binding nanobody pool produced by our lab and described in Thys et al.1, two poliovirus serotype 1 neutralizing nanobodies were identified as potentially efficacious and interesting for the production of anti-poliovirus drugs. Here we use these nanobodies to develop an efficient method for identification of poliovirus in biological samples. We showed that these anti-poliovirus nanobodies can be

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successfully labeled with fluoresceine isothiocyanate (FITC) and analyzed with capillary electrophoresis with laser induced fluorescence detection (CE-LIF) without losing their affinity for their target. Furthermore poliovirus can be easily detected without the interference of the biological sample matrix. In conclusion, the combined use of nanobodies and CE-LIF can lead to fast and accurate detection of small amounts of poliovirus in clinical samples. Reference List: 1. Thys, B., L. Schotte, S. Muyldermans, U. Wernery, G. Hassanzadeh-Ghassabeh, and B. Rombaut. 2010. In vitro antiviral activity of single domain antibody fragments against poliovirus. Antiviral Res. 87:257-264. P-706 High-Sensitive Analysis of DNA Fragments by Electrokinetic Supercharging-Capillary Gel Electrophoresis: Impact of Buffer Viscosity, Electrode Configuration, and Electrolyte Carry-over. Xiaoxue Ye1, Junji Inoue1, Zhongqi Xu2, Mihoro Yamada1, Satomi Mori1, Takeshi Hirokawa1, 1Hiroshima University, School of Engineering, Hiroshima, JAPAN; 2Donghua University, College of Chemistry Chemical Engineering and Biotechnology, Shanghai, CHINA Electrokinetic supercharging (EKS [1]) is defined as a technique that combines electrokinetic sample injection (EKI) with transient ITP (tITP). We explored three factors strongly affecting the efficiency of EKS in the CGE analysis of DNA fragments; viscosity of separation electrolyte (SE), electrode configuration and electrolyte carry-over. The viscosity of SE influences the tITP preconcentration of analytes, because it may affect the mobility of leading co-ion, and cause the insufficient stacking of the analytes having larger mobility than that of the leading co-ion. Therefore the SE with a low viscosity based on low-molecular-weight hydroxypropylmethylcellulose (HPMC), TBE and mannitol [2] was chosen, in which the leading co-ion (borate polyanion) was allowed to maintain sufficient mobility. Another factor was the impact of electrode configuration on the injected amount of the analyte during EKS. In our previous work [3], it was demonstrated that analytes occurring in an effective electric field can only be introduced into the capillary, while those remaining outside of the field could not contribute due to slow diffusion. An advanced electrode configuration modified by a ring electrode gave increased loading of the analytes and notably improved detectability of EKS-CGE system. The last factor is a small amount of SE attached to the electrode and capillary, which could be transferred to the sample solution (electrolyte carry-over) and might greatly affect the efficiency of EKS step. This is because the attached SE may decrease the transport number of the analytes. In order to avoid this effect, there should be efficient cleaning step before sample introduction. Considering these factors, high detection sensitivity was achieved by EKS-CGE system: Highly diluted standard DNA sample of φX174/HaeIII digest was used to evaluate the qualitative and quantitative abilities of the proposed strategy. The LOD was down to 7.7 pg/ml for the 72 bp fragment, apparently improved more than 10,000-fold in comparison with a conventional UV detection method. References: 1. T. Hirokawa, H. Okamoto, B. Gas, Electrophoresis, 24, 498-504 (2003). 2. F.T. Han, B.H. Huynh, Y.F. Ma, B.C. Lin, Anal. Chem. 71 2385-2389 (1999). 3. T. Hirokawa, T. Koshimidzu, Z. Q. Xu, Electrophoresis, 29, 3786–3793 (2008). P-707 Tandem Lectin Affinity Chromatography Monolithic Columns with Surface Immobilized Concanavalin A, Wheat Germ Agglutinin And Ricinus Communis Agglutinin-I for Capturing Sub-glycoproteomics from Breast Cancer and Disease Free Human Sera. Subhashini Selvaraju, Ziad El Rassi, Oklahoma State University, Department of Chemistry, Stillwater, OK, USA In this study, a liquid-phase separation platform consisting of tandem lectin affinity chromatography was introduced for the selective capturing of sub-glycoproteomics that are affected in cancers, e.g., breast cancer. The platform is comprised of three monolithic columns with surface immobilized lectins including concanavalin A (Con A), wheat germ agglutinin (WGA) and Ricinus communis agglutinin-I (RCA-I). While WGA and Con A have specificities directed toward the core portion of N-glycans on the glycoprotein surface, RCA-I specifically interacts with the non-reducing terminal moieties of the outer chain structures of N-glycans. The effects of the order in which the three lectin columns were arranged in the tandem columns format were evaluated. The most suitable order proved to be WGA→ Con A → RCA-I (denoted as WCR) as far as the number of captured proteins was concerned. The WCR tandem columns allowed the capture of 113 and 112 proteins from disease free and breast cancer sera, respectively, corresponding to 75 and 65 non-redundant proteins, respectively. Using mass spectral count ratios and Q-Q plots yielded a panel of 23 non-redundant differentially expressed proteins (i.e., a panel of 23 candidate markers), which should in principle be more representative of a pathophysiological state than a single marker candidate. P-708 Application of CESI-MS for the Detection and Identification of Modified Peptides in a Complex Mixture. Bettina Sarg1, Klaus Faserl1, Marcia R Santos2, Herbert Lindner1, 1Innsbruck Medical University, Biocenter, Division Clinical Biochemistry, Innsbruck, AUSTRIA; 2Beckman Coulter Inc., Brea, CA, USA In the recent past, the Proteomics Standards Research Group (sPRG) of the Association of Biomolecular Resource Facilities (ABRF) set off on a program, which is to produce a standard that can be used for mass spectrometry methods development to detect and characterize post-translational modifications (PTMs) in complex peptide mixtures. Traditionally, LC-ESI-MS and/or MALDI-MS are the analytical techniques of choice for the analysis of such samples. However, it is well documented that analysis of i.e. phosphopeptides using LC-

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ESI-MS is not without challenges. Poor ionization efficiency, low separation efficiency and neutral losses etc. often impedes detection and characterization. In this study we have applied CESI-MS for the separation and analysis of the sPRG PTM peptide standard. Using a polyethyleneimine (PEI) coated capillary (positively charged capillary) excellent separation efficiency and analysis speed of capillary electrophoresis can be fully exploited yielding in the identification of all acetylated, mono-, di-, and trimethylated peptides and most of the nitrosylated and mono-phosphorylated peptides in less than 15 minutes. When a bare fused silica capillary is applied, di-, tri-, and tetra-phosphorylated peptides, which are less positively or negatively charged, can be fully separated from all other positively charged peptides. Compared to the analysis using the PEI coated capillary, the analysis time is elongated to 30 minutes and enables the identification of almost all post-translational modified standard peptides in a single run. The effectiveness of the CESI-MS method was compared with nanoLC-ESI-MS. P-709 Process of Analyte Elimination by Chitosan-coated Magnetic Nanoparticles for CE-UV Analysis of Unknown Biochemical Samples. Meissam Noroozifar1, Zafar Iqbal2, Edward P.C. Lai2, 1University of Sistan & Baluchestan, Zahedan, IRAN; 2Carleton University, Ottawa, Ontario, CANADA Chitosan-coated magnetic nanoparticles are a class of nanoparticles that have attracted much attention because of their advantageous characteristics, such as biochemical inertness, non-toxicity, bioconjugating ability, and potential for eliminating a variety of biochemical analytes. Chitosan with two different molecular weights (low and high) have been used for the coating of magnetic nanoparticles. They were tested as new sorbents at the analytical scale for in-vitro binding of biochemical compounds by capillary electrophoresis (CE) under a wide range of sample matrix characteristics. The chitosan-coated magnetic nanoparticles are good for the selective elimination of certain biochemical analytes. Their structure and surface chemistry were investigated to obtain results that show that they can eliminate aromatic compounds to simplify the sample matrix for better identification of unknown peaks and higher accuracy in CE analysis. Experimental conditions are optimized for modeling their adsorption behavior using isotherm equation. Keywords/Topics: (bio-)pharmaceuticals: capillary electrophoresis P-710 On-line Sample Preconcentration Prior to CE-ESI-MS/MS: Quantitation of Drugs of Abuse in Bioanalysis. Isabelle Kohler1, Julie Schappler1, Martin Greiner2, Serge Rudaz1, 1University of Geneva, Geneva, SWITZERLAND; 2Agilent Technologies, Waldbronn, GERMANY The determination of xenobiotics in body fluids is of great importance in numerous fields, including clinical and forensic. Capillary electrophoresis (CE) hyphenated to electrospray ionization-tandem mass spectrometry (ESI-MS/MS) is particularly well adapted for the quantitation of targeted analytes in bioanalysis due to numerous advantages, such as high separation efficiency, high sensitivity and selectivity, short analysis time, and low solvent and sample consumption. Because of the complexity of biological matrices, a sample preparation is often mandatory to lower matrix interferents during the ionization process. However, the sample preparation is the most time-consuming step of the analytical procedure and can be considered a primary source of analytical errors. Urine is a matrix of choice due to the ease of collection, the long detection time window of xenobiotics and the absence of protein, avoiding any tedious sample preparation. Nervertheless, urine has to be diluted prior to injection to lower the concentration of urinary salts which can be deleterious for the electrophoretic process. The loss of sensitivity inherent to urine dilution can be balanced with the implementation of an on-line preconcentration technique. In this study, a high sensitive CE-ESI-MS/MS method was developed for the quantitation of drugs of abuse in urine. A pH-mediated stacking procedure with a small preplug of NH4OH 7% (m/v) was implemented to allow a substantial sample loading capacity (20.5% of the capillary length) with a simple 10-fold dilution of urine sample. The CE-ESI-MS/MS method was fully validated with cocaine and methadone selected as model compounds. The quantitative performance was evaluated with selectivity, response function, lower limit of quantitation (LLOQ), trueness, precision, accuracy and linearity. Cocaine was validated over the range of concentrations of 10-1000 ng/mL and methadone of 25-1000 ng/mL, including the usual concentrations detected in urine from drug consumers. CE-ESI-MS/MS with on-line sample preconcentration was finally successfully applied to the analysis of real toxicological urines where both analytes were detected and quantified. P-711 Sequence-Bas ed Sepa ra tion o f Sing le -Strand ed DNA b y Capilla ry Elec trop hores is . Xueru Zhang, Linda McGown, Resselaer Polytechnic Institute, Troy, NY, USA DNA separation and analysis has been studied for many years for important applications in biology, medicine and forensics. In most cases, DNA is separated by length followed by sequencing of same-length fragments if needed. There do exist several methods for DNA separation based on sequence but these generally require differences in native conformation, or in conformation or stability of strands that are hybridized with complementary strands that target specific, known sequences. Our research focuses on direct, sequence-based separation of unknown DNA that does not have any of the requirements of the existing methods. In previous work our group successfully separated four single-stranded DNA (ssDNA) 76-mers that differ by only a few A/G bases using capillary electrophoresis with buffers containing guanosine-5’-monophosphate (GMP)1, 2. To

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further investigate the separation ability and mechanism, in the present work we designed a set of ten ssDNA 76-mers, including the four used in the previous work, four new strands in which the G were randomly replaced by A, T or C to create G-free sequences, a scrambled sequence variant of one of the original strands, and a random strand with no relation to the original set. We performed the separation using the GMP buffer as before and examined the effects of varying experimental conditions. To understand the separation mechanisms, we then attempted the separation using AMP, CMP or UMP instead of GMP. We also investigated the effects of potential interactions among the ssDNA strands in the sample. The results suggest that the separation involves differential interactions of nucleotides with pyrimidines and purines in particular sequence arrangements in the ssDNA. References: 1. Dong, Y.; McGown, L. B., Electrophoresis 2011, 32 (10), 1209-1216. 2. Case, W. S.; Glinert, K. D.; LaBarge, S.; McGown, L. B., Electrophoresis 2007, 28 (17), 3008-3016. P-712 Enhanced Performance of En trop ic Trap Arrays us ing End-Attached Mice lles . Max Fahrenkopf, Erik Ydstie, Jim Schneider, Carnegie Mellon University, Pittsburgh, PA, USA Gel-free DNA separation methods are currently designed to either separate short DNA (typically 26 to 500 bp) with high (single-base) resolution or separate long DNA (typically 5000 bp to 48500 bp) with moderate (5000 bp) resolution. Here we present a computational investigation of integrating the rapid micelle electrokinetic chromatography technique known as micelle end-labeled free solution electrophoresis (Micelle ELFSE) integrated into other microfabricated devices such as an entropic trap array which should serve to synergistically increase separation performance and partially bridge this gap between short and long DNA separation performance. Micelle ELFSE utilizes end-attached non-ionic surfactant micelles that bring about a length dependent electrophoretic mobility on DNA oligomers in gel-free buffers. These micelles are tunable in size allowing one to optimize the separation performance of specific DNA target lengths. Additionally the separation device itself can be optimized with respect to geometry, such as channel dimensions and electric field strength. Entropic trap arrays are DNA separation devices that have repeating wide and narrow channel heights such that DNA is repetitively forced through a narrow constriction during electrophoresis. DNA oligomers of different lengths are separated as they cross an energy barrier upon entering confinement with a rate that depends on DNA size. Micelle ELFSE and entropic trapping are compatible separation methods since long DNA elutes first in both cases. Here, we demonstrate that use of end-attached micelles greatly improves the performance of entropic trap arrays. Three relevant time scales were identified for DNA electrophoresis through an entropic trap that account for DNA approaching the narrow constriction, entering the constriction, and crossing the constriction. We utilize Brownian dynamics simulations to investigate the sensitivity of each of these times scale to DNA tagged with a micelle. Simulations indicate a five-fold increase in resolution of the ELFSE-entropic device over simulations of the entropic trap alone. We also discuss proper choice of micelle size and electric field for greatest enhancement. P-713 High Throughput, Mutliplexed Detec tion o f mic roRNA An alo gs u s ing Mice lla r Elec tro kine tic Chromatograph y. Johnathan Goldman, James Schneider, Bruce Armitage, Carnegie Mellon University, Pittsburgh, PA, USA Short, non-coding microRNAs (miRNA) have been implicated in an increasing number of cancers and miRNA detection has emerged as a promising means of early-stage cancer diagnosis. The characteristic features of miRNA present many challenges for detection. miRNA are present in extremely low levels in cell extracts making them difficult and time-consuming to detect by conventional means. miRNA are only 22 nucleotides in length, and therefore make marginally stable duplexes with complementary oligonucleotide probes. Complicating matters even further is the observation that many miRNA differ by only a single base in sequence leading to false-positive readings. Available methods, such as microchip arrays and bead-based assays, suffer from poor reproducibility, long assay times, and high cost while implementing enzymatic modification that presents inefficiencies given the short miRNA length. Here, we present a highly-sensitive miRNA detection method capable of detecting multiple targets in a rapid capillary electrophoresis separation without the need for target modification. Detection relies on sandwich hybridization of two probes to the miRNA target: a high affinity, highly selective synthetic DNA analogue (γ-substituted PNAs) and a DNA oligomer pre-loaded with a fluorescent intercalating dye (YOYO-1). Targets bound by both probes are separated from non-target DNA/RNA through interaction with surfactant micelles in the capillary that lowers their electrophoretic mobility in a length dependent manner. By simply extending the length of the DNA oligomer attached to the miRNA target, we can detect multiple miRNAs in a single separation. Our system is currently capable reliably identifying 3 different targets in a 5 minute separation with nanomolar sensitivity. Theory suggests that up to 13 targets may be detected in this 5 minute window, a dramatic improvement upon current multiplexing methods. We find that high selectivity, over a wide range of single-base mismatches, is realized for run performed at slightly elevated temperature (40°C). We also discuss recent efforts to increase sensitivity by orders of magnitude using stacking and sweeping methods in inhomogeneous buffer systems.

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P-714 Glycation o f Album in . Ivan Miksik, Statis Pataridis, Zdenka Stastna, Pavla Sedlakova, Katerina Lacinova, Institute of Physiology, ASCR, Prague, CZECH REPUBLIC Posttranslational modifications are significant reactions occur on proteins. One of these modifications is non-enzymatic reaction between oxo-group(s) of sugars and amino-group(s) of protein – glycation. This reaction plays an important role in the chronic complications of diabetes mellitus, or in the process of aging of organism, i.e. it has an important role on pathophysiology and “normal” physiology of animals. In the presented work we studied glycation of albumins (human serum albumin, bovine serum albumin and albumin from chicken egg white). Methodologically we used nano-liquid chromatography coupled to Q-TOF mass spectrometer. In vitro modified proteins were cleavaged by trypsin and arising peptides were separated on C18 nano column with trap-column. Peptides and their modifications were analysed by high-resolution Q-TOF mass spectrometer MaXis with precision determination of mass below 2 ppm. We found some modifications of proteins. Besides well known carboxymethyllysine new ones were determined – create mass shift 78, 132 and 218. Origin of these modifications is discussed and possible structure is presented. All found modifications were allocated to the structure of proteins and reactivity to various oxo-compounds was also examined. Acknowledgments - This work was supported by the Czech Science Foundation (Grants Nos. P206/12/0453 and 203/08/1428), the Academy of Sciences of the Czech Republic, Research Project AV0Z50110509 and support for long-term conceptual development of research organization RVO:67985823, which is gratefully acknowledged. P-715 Development and Validation of Capillary Zone Electrophoresis-laser Induced Fluorescence (CE-LIF) Method for Measurement of D-Serine Levels in Cell Lines, Plasma, Brain and Peripheral Tissues. Nagendra Singh, Ruin Moaddel, Irving Wainer, National Institutes of Health, Laboratory of Clinical Investigation, Baltimore, MD, USA D-serine (D-Ser) is a physiological co-agonist of the N-methyl D-aspartate (NMDA) type of glutamate receptor and a key excitatory neurotransmitter receptor in the brain. It is one of the few D-amino acids found in the mammalian brain, plasma and peripheral organs. The endogenous source of D-Ser is dependent on conversion of L-Ser to D-Ser catalyzed by serine racemase (SR). Changes in endogenous D-Ser levels have been associated with number of CNS disorders, like alzheimer’s, schizophrenia, depression and Complex Regional Pain Syndrome (CRPS). It has also been reported that the expression of SR is influenced by administration of the ketamine, a drug currently used for the treatment of CRPS and depression; which might also affect the endogenous D-Ser levels. Thus measuring the changes in D-Ser levels in brain, plasma and other body tissues might yield useful information for understanding the mechanisms associated with the clinical effects of ketamine. A novel and sensitive enantioselective capillary electrophoresis-laser-induced fluorescence (CE-LIF) method was developed for the analysis of D-Ser in cellular matrices, plasma, brain and peripheral tissues. The assay involves derivatization with FITC followed by CE-LIF using 0.5 mM hydroxyl propyl-b-cyclodextrin in borate buffer [80 mM, pH 9.3]. The method was able to resolve D-Ser and L-Ser with an enantioselectivity (a) of 1.03 and a resolution (Rs) of 1.37. Linearity was established from 0.25 to 100 µM. The assay was also able to enantioselectively resolve 6 additional amino acid racemates. The method was applied to the determination of intracellular D-Ser concentrations in PC-12, C6, 1312N1, and HepG2 cell lines. This method was used to determine the concentration-dependent increases in D-Ser and associated EC50 values produced by L-Ser and the concentration-dependent decreases in D-Ser and associated IC50 values produced by glycine, a competitive inhibitor of SR. Further the assay was used to study the effect of the neuronal nicotinic acetylcholine receptors (nAChRs) antagonists, methyllcaconitine, mecamylamine, ketamine, dehydroxynorketamine, hydroxynorketamine and norketamine on intracellular D-Ser levels in 1321N1 and PC-12. In addition, the CE-LIF method will be applied to the measurement of D-ser levels in the plasma, brain and peripheral tissue of Wistar rats receiving chronic admininstrations of low-dose ketamine and to determine treatment-related changes in D-ser concentrations in patients receiving ketamine for the treatment of depression and CRPS. P-716 Synthesis and Characterization of a Nuclear Membrane Affinity Chromatography Column for the Study of Human Breast Cancer Resistant Protein (BCRP) using Nuclear Membranes Obtained from the LN-229 Cells. Kaia-Liisa Habicht, Chester Frazier, Irving Wainer, Ruin Moaddel, National Institutes of Health, Baltimore, MD, USA Glioblastoma multiforme (GBM) is one of the most aggressive forms of human astrocytoma as only ~10% of patients survive 5 years post diagnosis. A major factor in the poor prognosis is due to the multi-drug resistance to drugs used in cancer treatment, which has been shown to be carried out by ATP Binding Cassette efflux transporters, of which P-glycoprotein is the best characterized member. More recently, the breast cancer resistance protein (BCRP) has recently gained importance due to its widespread tissue distribution and role in the clinical MDR phenotype. BCRP expression has been reported in GBM cell lines and clinical specimen. It was previously demonstrated that BCRP was expressed both in purified nuclei and in the soluble cytoplasmic fraction of multiple glioblastoma cell lines. In fact, the presence of BCRP on the nuclear membrane surface was demonstrated on the LN229 cell line by confocal microscopy. Therefore, the immobilization of the BCRP from the nuclear membranes could be advantageous to characterize the BCRP protein and to determine whether the nuclear membrane BCRP has different binding characteristics. To this end, we have immobilized the nuclear

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membrane fragments onto an immobilized artificial membrane stationary phase (IAM), resulting in the nuclear membrane affinity chromatography (NMAC) column. Initial characterization was carried out on the radio flow detector, as well as the LC-MSD, using frontal displacement chromatography techniques. Etoposide, a substrate for BCRP, was initially tested, to determine the functional immobilization of BCRP. Frontal displacement experiments with multiple concentrations of etoposide were run and the binding affinity was determined to be 4.54 uM, which is in close agreement with literature. The BCRP was fully characterized on the NMAC column and this demonstrates that for the first time the nuclear membranes have been successfully immobilized. The BCRP was fully characterized and the results will be discussed.