Band Broadening in Straight Electro-chromatographic Column with Arbitrary Cross-section
Emilij Zholkovskij, Jacob H. Masliyah
Insitute of Biocolloid Chemistry of Ukrainian Academy of Sciences, Kiev, Ukraine and Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Canada

Band broadening of a solute due to the electroosmotic flow through a long straight channel is analyzed theoretically. Two sources of dispersion are considered: a non-uniformity of electroosmotic flow within a cross-section and incomplete equilibrium of solute adsorption at the channel walls. Three types of electroosmotic velocity non-uniformity are taken into account: (i) the non-uniformity within the double layer region and the non-uniformities due to (ii) longitudinal and (iii) transverse variation of electrokinetic potential. The analysis is conducted using a thin double layer approximation which is valid when the Debye length is much smaller than the characteristic dimensions of the cross-section. Analytical expressions are obtained to address dispersion in terms of plate height for arbitrary magnitude of varying surface potential, electrolyte type and cross-section geometry. The results are presented for different cross-section geometries: parallel planes; circle; annulus; ellipse; arbitrary circumscribed polygon and rectangle. It is discussed how the predicted plate height depends on the cross-section geometry, transverse and longitudinal electrokinetic potential distributions, electrolyte content, adsorption isotherm and adsorption rate constant.

Electric and Hydrodynamic Stretching of End-Labeled DNA Conjugates in Free Solution Electrophoresis
Sorin Nedelcu, Gary W. Slater
Department of Physics, University of Ottawa

The conjugation technique of an uncharged polymer to DNA makes possible its separation in free solution electrophoresis. The end-labeled free solution electrophoresis (ELFSE) method has been proved experimentally successful in 1999 in separating ssDNA with single monomer resolution up to 110 bases. It is the aim of this presentation to investigate in more detail the coupled hydrodynamic and electrophoretic stretch of the DNA conjugate and offer predictions on the optimization of the method. The modeling is based on earlier work of Stigter and Bustamante (Biophys. J, 75, 1197-1210, 1998) that considered separately the problems of chain extension in hydrodynamic flow and the electrophoretic stretch of a tethered polyelectrolyte. Basically, these two separate models are now used together, in the sense that the charged DNA is “tethered” to the uncharged polymer (i.e., the drag-tag), along with a proper change of the individual velocities of the segments, so as the complete problem is fully consistent. The theoretical predictions are rather rich and indicate several avenues in order to improve the ELFSE read length and separation resolution. It becomes apparent that an increase of the field strength leads to an increase in the resolution. Surprisingly, this dependency proves to be non-linear in the applied field. Explanations are put forward in order to explain the observed behavior of the DNA conjugate. Also, increasing the viscosity of the buffer solution seems to decrease the hydrodynamic friction provided by the drag-tag, while on the other hand, increasing the ionic strength of the solution decreases the persistence length of DNA and therefore increases the hydrodynamic friction of the drag-tag and eventually the read-length. The findings presented in this paper give a good insight into this non-trivial coupled electro-hydrodynamic problem.

A surfactant gradient method for micellar electrokinetic chromatography
Yukihiro Esaka, Mika Sawamura, Bunji Uno
Gifu Pharmaceutical University

Electrokinetic separation of eleven benzoates was investigated using mixed micellar solutions of CTAC and Tween 20 possessing polyether chains, where electroosmotic flow was eliminated virtually by coating the inner walls of capillaries with poly-N,N dimethylacrylamide. The benzoates being injected from the anodic side were detected with an order of magnitude of their hydrophobicity, fundamentally. The synergistic influences of attractive electrostatic interactions and hydrophobic ones gave rise to quite large retention factors of many of the benzoate anions in a pure CTAC system to result in their co-elution. Addition of an adequate amount of Tween 20 to the pure CTAC system decreased the electrostatic interaction significantly to give remarkably improved separation of the analytes, though some hydrophilic ones of the benzoates were not detected due to slight mobility under higher concentrations of Tween 20 required for sufficient separation of the relatively hydrophobic ones. A surfactant-gradient method should be useful to decrease analysis time and to improve separation simultaneously when analyte anions of a very wide range of hydrophobicity are to be separated. Under the present conditions, the cationic micelles migrate fastest compared with all of the benzoates and thus, the mixed micelles of the surfactant composition in running solution of the inlet reservoir can pass through and interact with all of the analytes before they are detected. This means we can perform surfactant gradient elution by a simple and practical method, namely, by changing the inlet reservoir including running solutions of different compositions of the surfactants during a single run. All of the eleven benzoates were separated completely within a reasonable time using a gradient program for the concentration of Tween 20 from 40 mM to 0 mM in the presence of 100 mM CTAC.

Insights into the Mechanism of Focusing Weak Bases in Capillary Electrophoresis using a Transient pH Boundary
Michael C. Breadmore1, Richard A Mosher2, Wolfgang Thormann3
1 ACROSS, School of Chemistry, University of Tasmania, GPO Box 252-75, Hobart, Tasmania 7001, Australia 2 RAM Software Solutions, Tucson, AZ, USA. 3 Department of Clinical Pharmacology, University of Bern, Murtenstrasse 35, 3010 Bern, Switzerland.

Enhancing the sensitivity of capillary electrophoresis is a major challenge. Various stacking and focusing mechanisms have been developed over the past 2 decades to allow the injection of large volumes of sample with little loss in resolution and efficiency. Transient pH boundaries have been used since the early 1990s and while the applicability of this approach for focusing ionisable analytes has been demonstrated numerous times, there have been few studies understanding how the transient pH boundary forms and moves throughout the capillary. This presentation will detail fundamental studies into the effect of varying the sample and electrolyte composition on the ability to focus weak bases in capillary electrophoresis. Computer simulations were used to provide snapshots of concentration, pH and conductivity profiles along the capillary during focusing to allow delineation of the most significant factors, including the amount of salt present in the sample. General guidelines?? for designing the best system to ensure maximum focusing efficiency with maximum resolution will also be discussed.

Mechanism of Affinity Capillary Electrophoresis Based on Mass Transfer Equation
Ning Fang, David D.Y. Chen
University of British Columbia, Vancouver, BC, Canada

Affinity capillary electrophoresis (ACE) has been used to estimate thermodynamic constants of binding interactions by linear or nonlinear regression methods. The accuracy of this approach relies heavily on the binding interaction’s mechanism which is controlled by both the nature of the interaction and the experimental conditions. The development of a highly efficient computer simulated ACE system makes it possible to demonstrate the mechanism of any given interaction under any conditions. The order of the mobilities of the complex and the two binding species in their free forms is a key factor to determine what molecules in what locations of the column are involved in the interaction, and the peak shape resulted from such interactions, of a given ACE experiment. In this work, 18 scenarios in 6 different combinations of migration orders of the free analyte, free additive, and complex formed are studied by a computer simulation program based on the mass transfer equation. From the study of the mechanisms, we conclude high additive concentration (ensuring high capacity factor) and low analyte concentration (ensuring fast fill-in of the free additive in the analyte plug) are crucial for obtaining accurate results when using the regression methods. The approach to estimate binding constants with computer simulation can be much more accurate as long as accurate and efficient simulation models can be developed, especially when the ratio of the additive and analyte concentrations is not large enough.

Numerical Analysis of Pore-scale Electroosmotic Flow and Dispersion in Random-close Packings of Hard Spheres
D. Hlushkou, A. Seidel-Morgenstern, U. Tallarek
Otto-von-Guericke-Universität, Magdeburg, Germany, and Max-Planck-Institut für Dynamik komplexer technischer Systeme, Magdeburg, Germany

We present a numerical model for analyzing steady-state electroosmotic flow (EOF) and hydrodynamic dispersion in three-dimensional porous media. The developed approach, involving solution of the coupled Poisson, Nernst-Planck and Navier-Stokes equations, and the particle-tracking method, allows to simulate electrokinetic transport and associated dispersion under most general conditions, including arbitrary value and distribution of electrokinetic potential at the solid-liquid interface, applied electrical field strength, electrolyte composition, and pore space morphology. This feature was utilized to characterize EOF velocity fields in random bulk and confined packings of hard (impermeable, nonconducting) spheres. Important aspects of pore space morphology (sphere size distribution), surface heterogeneity (mismatch in electrokinetic potentials at confining wall and sphere surface), and fluid phase properties (electrical double layer thickness) were investigated with respect to their influence on the EOF dynamics over microscopic and macroscopic spatial domains. Most relevant is the observation of a generally nonuniform pore-level EOF velocity profile in sphere packings (even in the thin-double-layer-limit) which is caused by the pore space morphology and which is in contrast to the plug-like velocity distribution in a single, straight capillary under the same conditions. The pore-scale velocity heterogeneity of EOF in a packed bed leads to Taylor dispersion and, thus, reduces the performance advantage of EOF with respect to hydraulic flow as compared to an open straight capillary. References: 1. D. Hlushkou, D. Kandhai, U. Tallarek, Int. J. Numer. Meth. Fluids 2004, 46, 507-532. 2. D. Hlushkou, A. Seidel-Morgenstern, U. Tallarek, Langmuir 2005, 21, 6097-6112. 3. D. Hlushkou, V. Apanasovich, A. Seidel-Morgenstern, U. Tallarek, Chem. Eng. Commun. 2005, accepted.

Electrohydrodynamics in Hierarchically-structured Monolithic and Particulate Fixed Beds
I. Nischang, G. Chen, U. Tallarek
Otto-von-Guericke-Universität, Magdeburg, Germany

The dependence of electroosmotic flow (EOF) velocity and hydrodynamic dispersion in capillary electrochromatography (CEC) is investigated on the variation of applied field and mobile phase ionic strengths employing silica-based particulate and monolithic fixed beds. These porous media have a hierarchical structure characterized by discrete intraparticle (intraskeleton) mesoporous and interparticle (interskeleton) macroporous spatial domains. While the macroporous domains contain quasi-electroneutral electrolyte solution, the ion-permselectivity (charge-selectivity) of the mesoporous domains determines co-ion exclusion and counter-ion enrichment at electrochemical equilibrium without superimposed electrical field. It depends on mesopore-scale electrical double layer overlap and surface charge density. This adjustable, locally charge-selective transport realized under most general conditions in CEC forms the basis for concentration polarisation (CP) induced by the externally applied electrical fields. CP characterizes the formation of convective diffusion boundary layers with reduced (depleted CP zone) and increased (enriched CP-zone) electrolyte concentration, respectively, at the anodic and cathodic interfaces in the fixed beds containing cation-selective, silica-based particles or monolith skeleton. CP originates in the electrical field-induced coupled mass and charge transport normal to the charge selective interfaces and has consequences for the EOF dynamics, hydrodynamic dispersion, and analyte retention in CEC. A secondary EDL with mobile counterionic space charge in the depleted CP zone can be induced, leading to induced charge EOF in the macroporous domains characterised by nonlinear average EOF velocities and strong local velocity components tangential to the surface which enhance lateral pore-scale dispersion, thereby decreasing (axial) zone spreading. Differences in pore space morphology of random-close sphere packings and monoliths critically affect the intensity of CP and induced charge EOF in these materials. CP is identified as a key phenomenon in CEC which also influences effective migration and the retention behaviour of charged analytes because the local intensity of CP inherently depends on applied field and mobile phase ionic strength.

Optimisation of Separations by Micellar Electrokinetic Chromatography
M. Roses, A. Tellez, E. Fuguet
Departament de Quimica Analitica - Universitat de Barcelona

Micellar electrokinetic chromatography (MEKC) is a powerful technique for micro-scale separations of mixtures of charged and uncharged analytes. The ionic micelles of the MEKC pseudostationary phase provide both ionic and hydrophobic sites for interaction of the solutes. Up to three different mechanisms (electrophoretic mobility, water-micelle distribution, and micelle-solute ion-pairing) can contribute to the separation of the mixture components. Thus, solute retention is strongly dependent of the surfactant concentration and mobile phase pH. Separation of the components of a complex mixture may be achieved by an adequate optimisation of these two factors. Literature describes several models that relate solute mobility or retention factor to surfactant concentration and mobile phase pH. In this work, these models are tested for a mixture of phenol derivatives of different acidity which retention has been measured at different mobile phase pH values and surfactant concentration (SDS). The buffer pH and SDS concentration were optimized for the separation of the mixture of phenols by means of a global resolution map based on peak purity. The map allows an easy identification of pH/SDS concentration areas with good overall resolution and the optimum pH and surfactant concentration, i.e. for a resolution as close as possible to 1. References M. G. Khaledi, S. C. Smith, J. K. Strasers; Anal. Chem. 1991, 63, 1820-1830. E. Fuguet, C. Rŕfols, J. R. Torres-Lapassió, M. C. García-Álvarez-Coque, E. Bosch, M. Rosés; Anal. Chem. 2002, 74, 4447-4455.

Negatively Cooperative Binding of Melittin to Neutral Phospholipid Vesicles
Francisco Torrens
Institut Universitari de Ciencia Molecular, Universitat de Valencia Dr. Moliner-50, E-46100 Burjassot (Valencia ), Spain

The association of basic amphipathic peptides to neutral phospholipid membranes is studied with binding and partition models. The binding of native and modified melittin (Mel) to egg-yolk phosphatidylcholine (EPC) is studied by spectrofluorimetry and size-exclussion chromatography (SEC). Results: (1) The binding isotherms for DNC-Mel and Mel to EPC. (2) The Scatchard plot for the binding of DNC-Mel and Mel to EPC. (3) Hill plots at fixed peptide concentrations. (4) The dissociation constant pK_D^h calculated from pK_D at each site with Hill coefficient h. (5) The partition coefficient GAMMA for the binding of DNC-SP to EPC and of mastoparan to dioleoylphosphatidylcholine (DOPC). (6) K_D^(h-h_o) vs. ionic strength I plot. Provisional conclusions: (1) SEC is proved useful for quantifying the polyelectrolyte-lipid association. (2) Spectrofluorimetry suggests a similar lipid-bound state for native and modified Mel. (3) Gouy-Chapman and Debye-Huckel formalisms apply the Poisson-Boltzmann equation for the influence either of a plane charged surface or between two charged spheres over the structure of the adjacent ionized liquid. (4) pK_D^h in the binding of native and modified Mel to EPC is dependent on h. (5) Coulombic repulsions drop more than peptide-lipid attractive forces with I, enhancing association.

Preferential Counterion Binding to Curved DNA Molecules
Nancy C. Stellwagen, Qian Dong, Earle Stellwagen
Department of Biochemistry, University of Iowa, Iowa City, IA, U.S.A.

DNA molecules containing A-tracts, runs of 4-6 contiguous adenine residues, are intrinsically curved and bind monovalent counterions in their minor grooves. A-tract-containing DNA oligomers also migrate anomalously slowly in free solution, consistent with excess counterion binding. In the present study, the free solution mobilities of DNA oligomers containing various numbers and types of A-tracts are compared with the mobility of a random-sequence oligomer of the same size. All solutions contained 200 mM diethylmalonate as the buffering anion, various concentrations of the test ions NH4+, Li+, Na+, K+, or Tris+, and sufficient concentrations of tetrapropyl (TPA+) or tetrabutyl (TBA+) ammonium ions to keep the ionic strength of the solution constant. The results indicate that A-tract-containing oligomers have two cation binding sites, termed the high affinity and low affinity sites. TBA+ ions are too large to bind to either site; TPA+ ions bind strongly to the high affinity site but not to the low affinity site. For the high affinity site, TPA+ >> Li+ = Tris+ = NH4+ > Na+ = K+. For the low affinity site, Li+ = Tris+ >= NH4+ > Na+ >> K+. No preferential counterion binding is observed in the low affinity site if the oligomers are first equilibrated with netropsin, which binds in A-tract minor grooves. Hence, the low affinity site is located in the A-tract minor groove, while the high affinity site is located in the major groove.

Simultaneous determination of myo-inositol and scyllo-inositol by capillary electrophoresis
Huey-Fen Tzeng, Jin-Yi Chen
Department of Applied Chemistry, National Chi Nan University

In this study, two capillary electrophoretic methods for simultaneous determination of myo-inositol and scyllo-inositol in Ringer’s solution were investigated. The first method coupled capillary zone electrophoresis with the indirect UV detection by using 2-naphthalenesulfonic acid as the background absorbent. This system was simple and temperature control was not necessary. Using fructose as an internal standard, the linear range of this method was 5-50 mM, and the concentration limits of detection of myo-inositol and scyllo-inositol were 1.44 mM and 0.98 mM, respectively. This method can be used to determine millimolar inositols. The second method involved the precolumn derivatization of inositols with N-methylisatoic anhydride to form N-methylanthraniloyl (MANT) esters at 25°C for 10 min, the dilution of the derivatized sample mixture, and the subsequent separation and determination by micellar electrokinetic capillary chromatography with fluorescence detection. The separation of myo-inositol-MANT, scyllo-inositol-MANT from glucose-MANT was performed at room temperature using glycine as the background electrolyte and sodium dodecyl sulfate (SDS) as the micelle. Under the optimal condition, the good separation with high efficiency was achieved in 8 min. Several parameters affecting the separation were studied, including the concentrations of SDS and organic modifiers, and the pH of electrolyte. Using glycerol as an internal standard, the linear range of the method was 0.4-6 mM for myo-inositol and 0.05-1 mM for scyllo-inositol. In the presence of glucose in the assay mixture, the concentration limits of detection of myo-inositol and scyllo-inositol were 0.105 mM and 840 nM, respectively. This is a sensitive method for inositols assays in biological samples.

Solute Transfer in Optimally Working Nano-electrospray Interface between a Liquid Phase Separation Technique and Mass Spectrometry
K. Klepárník, P. Kusý, M. Otevrel, F. Foret
Department of bioanalytical instrumentation Institute of Analytical Chemistry Czech Academy of Sciences Brno, Czech Republic

Operational conditions of liquid junction electrospray interfaces are usually chosen according to a laboratory experience by the trial and error method. This procedure, however, is not an easy task. There are many parameters including geometry and size of the interface, which must be combined to find an optimum. Therefore, the objective of this contribution is to develop a theoretical background of solute transfer through the interface under the effect of hydrodynamic flows combined with electromigration and electrospray. The theory will provide a tool for the determination of optimum conditions and interface geometry guaranteeing a stable spray and highest possible sensitivity of detection. The quantity according to which other parameters of the interface must be optimized is the magnitude of flow induced by the spray. This is determined by the spraying capillary ID and applied voltage, whose optimum value is given by the composition of the spraying liquid. Essential for a stable spray is the continuity between the flow induced by spray and pressure driven flow into the spraying capillary. When the flow into the capillary is either too small, or too high, the spray is interrupted. In the first case, due to the lack of spraying liquid and, in the second case, macroscopic droplets are formed at the tip, which destroy the spray plume. Another issue could be an excessive spreading and dilution of solutes leaving the separation and entering the spraying capillary. Navier-Stokes equations for three-dimensional steady-state flow of an incompressible viscous Newtonian liquid together with equations of diffusion-convective transport provide mathematical description of our problem. Since an exact solution to these equations in rather complex geometry of the interface is not known, the finite elements numerical method was used to model bulk hydrodynamic flows, dilution and spreading of solute zones in the interface. The theoretical results will be compared to experiments performed in a new miniaturized instrumentation for combination of capillary electrophoresis with mass spectrometry. Its capability will be demonstrated on the identification of proteins by analyses of their tryptic digests.

Effect of Polymer Architecture on Electrophoretic DNA Migration Mechanisms in Microfluidic Devices
Thomas N. Chiesl, Ryan Forster, Brian Root, Michael Larkin, Annelise E. Barron
Northwestern University Department of Chemical and Biological Engineering

We have utilized single-molecule epifluorescent DNA videomicroscopic imaging to visually verify mechanisms of electrophoretic DNA separations in microfluidic devices. In the course of our research we have observed both transient entanglement coupling and DNA reptation in polymer solutions. While these mechanisms are fairly well understood, the transition between the two has not been fully investigated. Here we present results that explore the relationship between the polymer concentration and the DNA sieving mechanism. Polymer solutions with various concentrations of LPA of different average molecular weights (500,000 g/mol, 1.3 million g/mol, and 4.5 million g/mol) ranging from 0.2% to 4.0% (w/v) were tested to analyze how the DNA moved through the solution. It has previously been hypothesized that at concentrations above C*, DNA changes its dynamic mode of migration from a transient entanglement coupling mechanism to a reptation mechanism. Our data shows the transition from transient entanglement coupling to reptation occurs over a relatively small concentration region where both mechanisms are apparent simultaneously. The full transition, where the majority of DNA undergoes pure reptation, occurs at concentrations of 10 times greater than C*. These data have been compared to polymer rheology measurements, and indicates that the concentration at which the transition occurs is most likely similar to a Ct concentration threshold (the transition to an elastically effective entangled polymer network) previously described by Candau et al.

Biomedical Applications of Vesicle Affinity Capillary Electrophoresis
E.D. Breyer1, Y. Jia1, K. Koticha1, R. Plemper2, W.V. Brown3
1 Department of Chemistry, Georgia State University, Atlanta, GA 30303 2 Department of Microbiology and Immunology 3 Veterans Medical Center, School of Medicine, Emory University, Decatur, GA 30033

Vesicle Affinity Capillary Electrophoresis (VCE) has been used in various biomedical applications such as lipoprotein transport and apolipoproteins exchange, drug transport, binding and inhibitions. This technique can also be used to gain insights in the progression of certain diseases such as cardiovascular and atherosclerosis. The system offers an in-vitro models by which biomolecular interaction can be studied and evaluated at the molecular level. Apolipoprotein binding as a function of size, charge and composition of lipid vesicles have been investigated using VCE. Protein folding, structure and functions has also been studied using this technique. Finally, in this study we also showed the feasibility of using this technique for screening drug activity and transport.

Direct injection of Macroemulsions in Micellar Electrokinetic Chromatography for the Determination of Structurally Related Hydrophobic Minor Constituents in a Hydrophobic Matrix
M. Pütz, C. Huhn, U. Pyell
Philipps-Universität Marburg, Germany Bundeskriminalamt, Wiesbaden, Germany

Essential oils containing safrole are the dominant precursor source for the clandestine production of MDMA (3,4-methylenedioxymethamphetamine). These oils contain a variety of structurally closely related analytes (including double bond isomerization products and cis trans isomers) in the presence of large amounts of safrole (up to 95% w/w). The determination of these minor constituents is of high forensic interest with respect to the origin and batch-to-batch comparison of natural MDMA precursors. It will be shown that this analytical task can be solved by employing micellar electrokinetic chromatography as separation technique. The base line separation of 10 constituents is possible with a separation electrolyte containing disodium tetraborate (7.5 mmol/L, pH 9.2), SDS (60 mmol/L), acetonitrile (20% v/v), urea (4 mol/L) and CaCl2 (0.5 mmol/L). For the determination of the minor constituents, only slightly diluted samples have to be injected into the capillary, although the main constituent is neither soluble in water nor in separation electrolyte. High amounts of SDS or organic solvents in the injection solution are detrimental regarding the efficiency of the subsequent separation. We show that a stable macroemulsion (opaque liquid) gained by the addition of the nonionic surfactant Triton X 100 and water to the oil samples can directly be injected while maintaining the separation efficiency. Assuming that sweeping and desweeping dominate the injection process, a possible mechanism of the efficiency-maintaining injection process is described based on the intermediate analyte transport by mixed micelles. Slightly diluted samples (dilution 1:100) contain safrole at a concentration of about 100 mmol/L, which is higher than the molar concentration of the micelle-forming anionic surfactant SDS in the separation electrolyte. Consequently, peak overload was observed using UV- and LIF-detection in tandem. However, there is no disturbance of the signals of closely migrating analytes even at a molar excess of safrole of 1000:1. The method developed was successfully applied to numerous reference and seized essential oils from clandestine laboratories.

Domain size-induced Heterogeneity As An Explanation for the Poor Performance of Small Domain Monolithic Columns and Other LC support Types.
J. Billen, P. Gzil, G. Desmet
Department of Chemical Engineering Vrije Universiteit Brussel

The present paper reports on a theoretical investigation of the experimental observation that the band broadening in small domain monoliths always tends to be larger than can be expected from their domain size. It was hypothesized that this effect can be explained by assuming that the monolithic synthesis process cannot avoid the occurrence of a domain-size independent uncertainty on the size and the position of the solid phase portions of the monolithic skeleton. A computational fluid dynamics study of the band broadening in a series of randomized 2-D cylinder arrays with decreasing domain size and subjected to the same (i.e., domain size-independent) absolute size and position variance was made to verify this hypothesis. Both non-porous and porous supports were considered. The considered domain sizes ranged from 12 down to 1
mm. The obtained van Deemter curves confirmed that the increased relative pore space heterogeneity and the larger tendency to form larger solid zone agglomerates in the small domain limit indeed leads to a dramatic increase of the reduced plate heights. In absolute values, it is found that the plate height curves decrease at a much smaller pace with the domain size than the linear decrease expected that is familiar in packed bed HPLC (at least provided the particles are equal to or larger than 3 micrometer). The obtained results indicate that the key to obtaining high quality small domain size monoliths will lie in the ability to reduce the standard deviation on the size and the position of the skeleton branches to below 0.2 µm. To achieve this, extensive studies of the relationship between the exact process conditions and the structural variances will become absolutely critical in the next development stages. For this purpose, microscopic visualization methods like the laser scanning confocal microscopy (LSCM) method will become indispensable research tools. Introducing more vigorous mixing methods such as ultra-sound or chaotic advection mixing producing the smallest possible mixing eddies could be one of the possible solutions to improve the size variability of small domain monoliths.

The Potential of Cyclodextrin in Developing Generic Buffers for the Separation of Steroids
Y.Shakalisava, F.Regan
Dublin City University, Dublin, Ireland

The analytical interest in the determination of steroids is maintained at a high level. This is due to their physiological functions in the body and the potential danger in disruption of the endocrine system. This work shows the potential of cyclodextrin modified capillary electrophoresis (CD-CE) in the development of a generic buffer system for the separation of steroids. Steroids are known to form an inclusion complex with cyclodextrins [1]. Fundamental knowledge of equilibrium and the association constants of analysed compounds and ligand in the system is a powerful element in the understanding of that system and in discovering ways to evaluate it. The association constants of inclusion complexes were determined for a range of structurally related steroids and sulphobutyl-β-CD. The optimal conditions for separation were chosen for several case studies. The potential of the method is demonstrated in the example of a related steroid not examined in the original work, but having similar structural or physical characteristics to those studied. The association constant and other physical characteristics of the target compound were used in the determination of the optimum conditions of separation. Statistical evaluation of data is presented. 1. N. Sadlej-Sosnowska, J. Inclusion Phenomena and Molecular Recognition in Chemistry 27 (1997) 31.

Capillary Isoelectric Focusing (cIEF): Performances Comparison of Different Analytical Systems
M. Poitevin1, A. Morin1, J.M. Busnel1, S. Descroix1, A. Varenne2, P. Gareil2, G. Peltre1, M.C. Hennion1
1 Laboratory of Environment and Analytical Chemistry, UMR CNRS 7121, ESPCI, 10 rue Vauquelin, 75005 Paris France. 2 Laboratory of Electrochemistry and Analytical Chemistry, UMR CNRS 7575, ENSCP, 11 rue Pierre et Marie Curie, 75055 Paris France

After human genome sequencing, proteomics is becoming a field of growing importance. Its goal is to identify, characterize, quantify and localize at a given moment the proteins involved in a process, as well as to study their functional roles and the variations that can occur under the effect of various stimuli. Within this context, it is necessary to dispose of bioanalytical tools of high resolution, wide dynamic range and high sensitivity. Most of the proteomic analyses are performed with a bidimensionnal separation system coupled with a characterization technique. As an alternative, capillary isoelectric focusing (cIEF), a miniaturized and automatized technique, allows the separation of proteins in a pH gradient according to their isoelectric points. While cIEF is usually carried out in aqueous medium and in coated capillaries, a previous study highlighted the interest of glycerol in the separation electrolyte for proteins, particularly hydrophobic ones, in cIEF mode and in a bare silica capillary [1]: separation of transmembrane proteins, hitherto impossible in classical cIEF, lower background noise, reduced cost and better stability. We have therefore tried to compare the performances of that analytical system to the ones of a classical cIEF system, for non hydrophobic proteins. We have initially optimized the separation conditions for the classical cIEF method, which is performed with an aqueous separation electrolyte in a coated silica capillary. First, different coating procedures have been tested, both in term of electroosmotic flow values at different pHs and in term of separation of basic proteins. We have then studied the isoelectric focalisation mechanism. The different studies described in the literature about this subject foresee that the anionic and cationic forms of the analytes migrate from each extremity of the pH gradient, to focalize under neutral form at their isoelectric point [2, 3]. Separations of model proteins in a HPC (hydroxypropylcellulose) coated capillary allowed us to observe this coalescence phenomenon. Once the focalisation mechanism studied, numerous separation parameters in a coated capillary of model proteins have been optimised: focalisation voltage, and mobilisation pressure, nature and concentration of anolyte and catholyte, ampholyte nature, and capillary length. At last, the performances of this optimised classical cIEF separation system have been compared to the ones obtained with the cIEF method in the presence of glycerol in a bare silica capillary, both for model proteins and milk proteins. [1]- J.M. Busnel, A. Varenne, S. Descroix, G. Peltre, Y. Gohon, P. Gareil, Electrophoresis, 2005, 26, 3369-3379 [2]- W. Thorman, T. Huang, J. Pawliszyn, R.A. Mosher, Electrophoresis, 2004, 25, 324-337 [3]- Q. Mao, J. Pawliszyn, W. Thormann, Anal. Chem. 2000, 72, 5493-5502.

Highly Efficient Protein Separations Using a Novel Surfactant/Polymer Coating
A.M. MacDonald, C.A. Lucy
University of Alberta, Edmonton, Alberta, Canada

Wall coatings are important in the separation of proteins by capillary electrophoresis (CE) because proteins may adsorb onto the negatively charged capillary wall. This adsorption results in band-broadening, poor reproducibility, and low sample recovery. An effective coating reduces the interactions of the proteins with the capillary wall. Coatings fall into the following broad categories: covalently bonded polymeric coatings, physically adsorbed polymers, or adsorbed surfactants, also known as dynamic coatings. The coating under study is dynamic, which is attractive due to its simplicity, versatility and cost-effectiveness. Cationic surfactants such as dodecyldimethylammonium bromide (DDAB) and other longer chained species form a bilayer on the capillary wall. These surfactants have been shown to be very stable as wall coatings.(1) However, these coatings are terrible for anionic protein separations because the proteins are attracted to the positive charge and thus stick to the coating. Capillaries coated with these double-chained cationic surfactants also have such a large electroosmotic flow (EOF) that cationic proteins can migrate off the capillary too quickly for separation to occur. The coating being examined is a mixture of a long, double chained surfactant known as DODAB (dioctadecyldimethylammonium bromide) and polyoxyethylene stearate. Polyoxyethylene (POE) is known to be a relatively inert hydrophilic polymer.(2) The stearate portion interacts with the hydrophobic chains of the surfactant while the hydrophilic oxyethylene tail protrudes into solution as can be seen in Figure 1.(3) This type of coating allows for the separation of both cationic and anionic proteins. This is an advantage over the solely cationic surfactant coatings, which are only useful for cationic protein separations. Cationic proteins yield efficiencies of over 0.9 million plates/m with this coating. Efficiencies greater than 1 million plates/m have been achieved for anionic proteins, which is higher than any values shown in the literature for similar types of coatings. The coatings used here are also much easier to prepare. This mixture of DODAB and POE stearate suppresses the EOF in the capillary by at least a factor of ten. The suppressed EOF allows for the separation of the cationic proteins. The EOF was found to be stable in the DODAB / POE coated capillary even after a one hour buffer rinse. The ultimate goal of this research is to create a tunable coating for different types of protein separations. (1) Yassine, M.Y., Lucy, C.A. Anal. Chem. 2005, 77, 620-625 (2) Whitesides, G.M. et al. Langmuir, 2001, 17, 5605-5620 (3) Warr, G.G. et al. Langmuir, 2005, 21, 2779-2788 Figure 1: 0.5-1.0 wt. % polyoxyethylene (100) tethered to a DDAB bilayer.

Analysis of Japanese Volcanic Hot Springs by Capillary Electrophoresis
Tomoko Asai*, Takashi Miyado**, Shin-ichi Wakida**, Kenji Chayama*
*Department of Chemistry, Graduate school of Natural Science, Konan University **National Institute of Advanced Industrial Science and Technology (AIST)

Japan is located on the Pacific rim active volcano zone, and has many hot springs from ancient times and many people use them as hot spa. Although hot springs become to be popular in these decades, they have sanitation problems concerning Legionella bacterium occurred in circulate filters and the problem of dry up of resources. The Ministry of Environment revised hot springs law to make it a duty to inform the details of hot springs concerning the water quality when adding water to decrease the temperature, or heating to add the temperature, using of circulate filters, and additions such as minerals or flavors. Now, minerals in hot springs are measured by flame photometry method, atomic absorption spectrometry, titration methods, ion exchanging method, ion chromatographic methods and so on. All of this method needs long times and great cost. On the other hand, CE is possible to separate many inorganic species at the same time and to detect these species in several minutes. Further more some indirect UV detection methods are possible to measure the species without absorption. Hot springs have various types up to landforms and areas, and include the characteristic elements by types. They are containing elements, sometimes over 1000 ppm, and elements under 1 ppm. As determination of low concentration elements are interfered with high concentration elements, measurement is difficult. However capillary electrophoresis (CE) is able to perform the high concentration determination by addition of a known quantity of high concentration elements to background solution (BGE), and high concentration elements and low concentration elements are able to determine at the same time. The aim of this study was to develop a new method for determination of inorganic cations and anions in hot spring water with capillary electrophoresis and indirect UV detection. Separations were performed with fused-silica capillary (92 cm (80 cm to the detector)×50 mm I.D.). Sodium, magnesium, potassium, calcium, lithium and barium as cation sample, and fluoride, chloride, bromide, nitrate, sulfate and phosphate as anions were examined. BGE for cations were solution containing copper (
) acetate and ethylenediamine, and BGE for anions were solution of benzoic acid, Tris, and hexadecyltrimethylammoniumhydroxide. Concentrations, pH of BGE and capillary temperature, applied voltage, and so on were examined. Electropherograms were obtained by means of indirect UV detection at 220 nm. Separation of six cations and six anions were achieved. The detection limits for cations and anions ranged from 0.12 to 1.6 ppm. The relative standard deviations (RSDs) of migration times ranged from 0.10 to 1.3%, and the RSDs of peak areas ranged from 0.99 to 2.5%. This method was applied to determine these species in different samples of hot spring water at city of Kobe in Japan (Fig.1).

Capillary-Assembled Microchip (CAs-CHIP): Two Types of Different Applications for Electrophoretic Separations
Hideaki Hisamoto Seigi Takeda, Shigeru Terabe
Graduate School of Material Science, University of Hyogo

“Capillary-Assembled Microchip (CAs-CHIP)” is fabricated by embedding the chemically-functionalized square capillaries into lattice PDMS channel having same channel dimensions as the outer dimensions of square capillaries. This new approach of chip fabrication allowed us not only the integration of various chemical functions, but also that of multiphase flow process onto a single microchip (H.Hisamoto et al., Anal. Chem., 2004, 76, 3222.). We have been applied this technique for chemical sensing such as multiple-ion sensing or valving and sensing (H.Hisamoto et al., Anal. Chem., 2005, 77, 2266.; Anal. Chim. Acta, 2005 in press.). Here we propose two types of different applications of CAs-CHIP for electrophoretic separations. First one is the usage as a pretreatment attachment for CE separation, and the other is the usage as a microchip CE by fabricating “injection cross” structure. For first application, CAs-CHIP is used for the deproteinization of sample, which is based on the diffusion-based separation under multilayer flow, before CE separation. In this case, fluorescent low molecular weight species were successfully separated from protein-mixed sample to be analyzed by CE. Concerning the application to microchip CE, injection of small amount of sample solution was successful by using “gated injection” mode, although the dead volume at the injection cross was fairly large compared to that of standard cross-type microchip. In our case, chemically-functionalized capillaries can be easily implemented. Actually, we have implemented the ODS-modified capillary as a separation capillary of microchip CE, and the separation by CEC mode was successfully demonstrated. These preliminary results suggested the wide applicability of CAs-CHIP in the field of electrophoretic separations.

Novel strategies for the control of the electroosmotic flow in capillary electrophoresis: application to the separation of peptide mixture
H. Cottet, G. Danger, J. Taillades
Equipe « Dynamique des Systčmes Biomoléculaires Complexes », Organisation Moléculaire, Evolution et Matériaux Fluorés, UMR CNRS 5073, Université de Montpellier 2, Place Eugčne Bataillon, 34095 Montpellier cedex 5, France.

The electroosmotic flow (EOF) is an important parameter in electrokinetic separations since it is directly related to the apparent selectivity and to the analysis time of the separation. The aim of this work was to study two novel strategies for controlling the EOF in capillary electrophoresis. Both strategies were compared for the separation of a model mixture containing 8 peptides. Repeatability on migration times and on electroosmotic mobility is reported. The first strategy was based on the use of polyelectrolyte multilayers. By changing the charge density of the last polyelectrolyte layer (polyanion), it was possible to control the amplitude of the cathodic EOF. The stability of the coating as a function of the charge density of the last layer was studied. Resolutions and analysis times can be modified very easily using this approach. The second strategy was based on partial coating of the capillary. First, we tried capillaries modified by polyelectrolyte multilayers with a partial last layer. Such modification can be simply obtained by a partial filling of the capillary with the last polyelectrolyte coating solution. In this first mode, the capillary is partially cationic and partially anionic. The amplitude of the EOF depends linearly on the proportion of the capillary modified by the last partial layer. However, peak distortion and high peak dispersion were obtained using such partially cationic/anionic capillaries. In a second mode, a non-covalent poly(ethyleneoxide)-coated capillary was partially modified by a polycation, resulting in an anodic EOF. Interestingly, the peak dispersion was much lower on such partially cationic/neutral capillaries. Compared to bare silica capillary, resolution of the separation can be highly improved using this second mode.

Oscillating Electrolytes in Capillary Electrophoresis
V. Hruska, M. Jaros, M. Stedry, B. Gas
Charles University in Prague, Faculty of Sciences, Prague, Czech Republic

We discovered a new class of electrophoretic systems exhibiting oscillations in concentration profiles of all ions [1]. The oscillations are a direct consequence of complex system eigenmobilities in the system. By analogy with the behaviour of dynamic systems, complex eigenmobilities implicate that the system will be unstable. The oscillation structures arise from any arbitrarily small initial disturbances and spread along the entire volume of the separation channel. The imaginary part of the system eigenmobility governs frequency and amplification of oscillating waves and the real part determines the spatial mobility of the wave pattern. Oscillation of electrolytes is an analogy to chemical oscillation; however, the driving force is not the gradient of the chemical potential but rather the gradient of the electric potential. We reveal from our calculations that the complex system eigenmobilities can only be obtained with a specific composition of the background electrolyte (BGE). E.g., for a binary BGE composed of divalent acid and strong univalent base there is a strict rule between the mobility of univalent (u1) and divalent (u2) ionic form of acid: u2 > 2.12*u1. We show that the optimum composition of the BGE has to be within the pH range of 5 to 5.5. For a computed assisted design of oscillating electrolytes we used our simulation programs Simul and PeakMaster [2] and found that the conditions for oscillations can be obtained with the BGE composed of sebacic acid and imidazole or sodium. We confirmed experimentally that this BGE exhibits oscillations. The financial support of Grant Agency of the Czech Republic, grant number 203/05/H001, is gratefully acknowledged. [1] Hruška, V., Jaroš, M., Gaš, B., Oscillating electrolytes, Electrophoresis, in print. [2] Programs Peakmaster 5 and Simul 5 are available on our Internet web-site

Determination of micelle CMC by system peaks in electrophoresis
J. Lokajova, I. Vesela, V. Hruska, E. Tesarova, B. Gas
Charles University in Prague, Faculty of Sciences, Department of Physical and Macromolecular Chemistry, Prague, Czech Republic

The micelle electrokinetic chromatography (MEKC) is important mode of capillary electrophoresis techniques. The anionic or cationic detergent as the separation medium creates charges micelles. From the point of view of mathematical description of electromigration we describe detergent as BGE constituent with special ability to create micelles when its concentration is higher than CMC [1]. We implement this modification to program PeakMaster which enables us to qualitatively predict behavior of micelle electrophoretic system. The sodium dodecylsulfate (SDS) is one of the most important detergent in MEKC. From calculations in the program PeakMaster we can see that there is non-continuity in anionic system eigenmobilities (created in the certain composition of BGE) when we get over CMC. We can use this “jump” in mobility for the determination of the CMC. The experimental work fully proved our presumptions and this method seems to offer easier CMC determination comparing to the standard methods because we observe “jump” instead of continuous change in the measured quantity. The financial support of Grant Agency of the Czech Republic, grant number 203/05/H001, is gratefully acknowledged. [1] Eva Tesarová, Juraj Ševcík, Bohuslav Gaš, Daniel W. Armstrong, Electrophoresis 2004, 25, 2693-2700.

T. Levandi, M. Kaljurand, K. Helmja
Tallinn University of Technology, Department of Chemistry, Faculty of Science, Tallinn, Estonia

In the present study, micellar electrokinetic chromatography(MEKC) as a proposed method of CE, was successfully applied for the investigation of the kinetics of metabolic conversion of ATP to ADP by ATPases, assisted in laboratory rat heart muscle. Advantage of the proposed separation protocol was no need for any sample preparation, the protein precipitation step could be omitted since the proteins are included into the CTAB micelles and does not interfere the analysis. Although, it is noteworthy that the quantification of ADP in ADP/ATP mixtures was not controlled by concentration of ATP, therefore it was possible to quantify ADP peaks even for very large substrate/product concentration ratios (there wasn´t any overlap of ADP and ATP peaks at high ATP concentrations as using conventional reverse-phase HPLC). Initial rates of the reactions were calculated from the ADP concentrations as time functions and expressed in nmol ADP per min per mg wet weight (WW). Collection of initial rate data was fitted to the functions followed from different kinetic models. For minimization of summed squares of differences between experimental data and model function the ”Excel Solver” minimization procedure was implemented which resulted in the desired parameters of the kinetics model functions. Best fit of the data was achieved by the equation that involves the sum of the Michaelis-Menten and Hill equations. A wide plateu region is formed at physiologically significant concentrations, the inflection point (Km2) followed from Hill equation is shifted upward in the curve, therefore, obviously the potential allosteric effects haven´t any significance of physiological in cardiac muscle fibres. The precision of the CE system was evaluated by an experiment using replicate (n = 3) injections of sample solution. The precision of migration times and peak areas was determined by calculating the relative standard deviation (RSD). The reproducibility of migration time and peak areas varied accordingly from 1.3 to 3.5 % and from 2.3 to 5.9 over the measured concentration range. The next step is the implementation of the proposed method to the isolated cardiomyocytes and the following investigation of their kinetic.

Optimization for Separation and Detection of Microorganisms by Capillary Electrophoresis
M. Torimura1, S. Inagaki1, H. Tao1, T. Shintani2, T. Manabe3
1 National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan 2 Textile Industrial Institute of Ehime Prefecture, Ehime, Japan 3 Department of Chemistry, Faculty of Science, Ehime University, Ehime, Japan

Establishment of more rapid and highly sensitive microorganism detection method has been expected. We developed and optimized a separation condition of microorganisms by capillary electrophoresis (CE). Optimized condition gives higher sensitivity for microorganism detection in CE. The 1st key technology for the development of this CE method is the appropriate selection of buffer additive to migrate microorganism accurately inside capillary. The 2nd one is an establishment of microorganism on-line sample stacking technique. In this study, we evaluated an effect of buffer additive on migration profile of model microorganisms and analyzed the relation of types of buffer additive and migration properties. Addition of anionic polymer to running buffer was effective to acquire good peak efficiency for some model microorganisms. On the other hand, on-line CE sample stacking techniques ware applied to living microorganism cell. Especially here, we focused on the viability of microorganism exposed to stacking condition such as low ionic strength. Under the optimized stacking condition, more than 100 times condensation of living microorganism was achieved with improvement detection sensitivity drastically. The detail of damage to the microorganisms by stacking will be discussed.

Analytical determination of individual proteins, peptides, and size of peptides required for folding into three-dimensional structures with hydrophobic core using capillary electrophoresis
J.C. Sřrensen, A.D. Sřrensen, H. Sřrensen, S. Sřrensen
Department of Natural Sciences, Biochemistry and Natural Product Chemistry, The Royal Veterinary and Agricultural University, Thorvaldsensvej, Frederiksberg C, Denmark.

Micellar electrokinetic capillary chromatography (MECC) has been developed as two different methods of protein and peptide analyses by use of either sodium dodecyl sulphate (SDS) or sodium cholate (NaCh) as surfactant. Both methods are high resolution techniques, which give supplementary informations on structure and properties of peptides and small proteins. The NaCh-based MECC method provides possibilities for analysis at gentle condition, preserving the native three-dimensional structure and bioactivities of the molecules e.g. as utilised in binding studies. To avoid association between positively charged proteins and the capillary wall, mobile coating with taurine has been found suitable as part of the NaCh-based MECC. In addition to be a high resolution technique, this separation method also reflects the net charge on the protein and peptides. The MECC techniques based on SDS as surfactant result in transformations of protein and peptides into polyanions, when the compounds have a size, which provide basis for a three-dimensional structure with a hydrophobic core. Thus, the SDS-based MECC method represents a potential for analytical determination of the size of small proteins and peptides forming polyanions with SDS. In the present study, it is found that peptides composed of less than about eight amino acids will normally not result polyanions with SDS. They are, thus, well separated from proteins and peptides with a size resulting in three-dimensional folding, which are sufficient for SDS binding and polyanion formation. Both of the developed MECC methods have been optimised as regards effects from temperature, voltage and buffer composition on the relative migration times (RMT), relative normalised peak area (RNA), resolution and number of theoretical plates per meter (N/m). Repeatability expressed as relative standard deviation varied between 0.1 and 0.6 % for RMT and 0.9 and 4.6 % for RNA. Linearity of the optimised methods showed correlation coefficients between 0.955 and 0.999 and the limits of detection (LOD) were of the size 10-5 g.

Determination of individual thiol- and disulphide containing amino acids and peptides by capillary electrophoresis and spectroscopy
J.C. Sřrensen, A.D. Sřrensen, K. Mortensen, H. Sřrensen, S. Sřrensen
Department of Natural Sciences, Biochemistry and Natural Product Chemistry, The Royal Veterinary and Agricultural University, Thorvaldsensvej, Frederiksberg C, Denmark.

Thiol-containing amino acids, peptides and proteins are essential biomolecules owing to their diverse biological activities including complex binding of metal ions, redox properties, and easy oxidation to disulphides. These biochemical properties are crucial for numerous metabolic reactions including xenobiotica metabolism and biological important redox systems and disulphide groups are essential for stabilisation of protein and peptide structures. In the present study, a new method of analysis has been developed for specific determination of thiol and disulphide groups in amino acids and peptides. Cysteine, N-acetylcysteine, glutathione (GSH) and other peptides have been selected as model compounds. Determinations of disulphides including oxidised glutathione (GSSG) have been performed after initial reduction of disulphides to the corresponding thiol compounds. The thiol-containing amino acids and peptides were after isolation transformed into dithiocarbamates, which have a special chromophore with a twin peak of high absorptivity in UV. These properties of dithiocarbamates have been utilised in the applied liquid chromatography methods for sensitive detection of the compounds and for their specific determination by use of a high performance capillary electrophoresis (HPCE) system equipped with a diode array unit. The applied HPCE analyses have been micellar electrokinetic capillary chromatography (MECC), which allowed efficient separations of individual dithiocarbamates. Disulphide analysis comprised treatment with dithiothreitol (DTT), which resulted in an equilibrium mixture of disulphides and thiols. With excess of DTT, GSSG was thus, transformed into GSH, which subsequently was transformed into the corresponding dithiocarbamate used in the MECC method.

Analysis of crude extracts from plant material by MECC
J.C. Sřrensen, H. Sřrensen
Department of Natural Sciences, Biochemistry and Natural Product Chemistry, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.

Phenolics and glucosinolates are structurally different groups of natural products. Both of these groups contain however, compounds which may have great influence on the quality of food and feed containing plant materials, in which both can be accumulated in appreciable levels. The biological effects of the phenolics are often related to their redox properties and/or oxidation products. The effects of glucosinolates are most often associated with their transformation products which may be formed in autolysis reactions. In all cases it is important with access to methods of analysis, which give basis for determination of the labile native biomolecules in crude extracts as well as for efficient separation of individual molecules. Minimal handling and a relatively short time span from extraction to analysis is normally favourable for analysis of labile compounds. This developed method using MECC analysis on crude extracts provide a possibility of having a short period from extraction to analysis and furthermore in a sufficient rugged method. The method can not substitute the more sophisticated methods based on group separation applied in hyphenated LC techniques prior to final analysis. However, for some compounds more reliable data can be obtained with this method. This is the case for some glucosinolates and phenolics which in this method can be analysed as the intact compounds in a micellar electrokinetic capillary chromatography (MECC) technique using cholate as surfactant. The method benefits from being a fast screening method for important low molecular weight compounds in different plants.

Method of glyphosate analysis based on HPCE of the dansyl derivative
P. Mřller, I.L. Petersen, H. Sřrensen, J.C. Sřrensen
Department of Natural Sciences, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark

Glyphosate (N-phosphonomethylglycine) is a widely used herbicide with effects in the plants on the biosynthesis of aromatic amino acids. It is assumed, that glyphosate inhibit the transformation of shikimate-3-phosphate into 5-enolpyruvyl-shikimate-3-phosphate (EPSP) by interaction with EPSP-synthase (EC Limited informations on the concentration of glyphosate in plant materials seem to be caused by analytical problems resulting from binding of glyphosate to minerals and lack of an efficient chromophore system in glyphosate. A micellar electrokinetic capillary chromatography (MECC) method has now been developed for analysis of glyphosate following group separation of crude extracts prior to production of the glyphosate dansyl derivative used for MECC analysis. Derivatisation with dansyl chloride introduced a chromophor system into glyphosate as basis for sensitive detection at 216 nm and by diode array systems also for identification. The observed limit of detection (LOD) and quantification (LOQ) for glyphosate was found to be 3 µM and 15 µM, respectively, in the MECC method with linearity up to 150 µM and a correlation coefficient of 0.9868. The number of theoretical plates per meter (N/m) in the developed system was found to be 485000 N/m. By use of spiking experiments with Brassica napus L. leaves and seed matrix systems, the recovery of glyphosate was investigated.

Synchronous monitoring of substrate and products in the myrosinase catalysed hydrolysis of glucosinolates by MECC
N. Bellostas, J. C. Sřrensen, H. Sřrensen
The Royal Veterinary and Agricultural University, Frederiksberg, Denmark

Glucosinolates are allelochemicals present in all plants of the order Capparales that are hydrolysed by endogenous isoenzymes (myrosinase; EC forming a variety of compounds with biological activity, including beneficial effects on human health. Different methods exist for the measurement of the activity of myrosinase isoenzymes, however, despite their accuracy in the quantitative determination of the amount of glucosinolate degraded, most methods are not designed for the determination of the individual products formed and the proportions in which they are produced. In the present study, a micellar electrokinetic capillary electrophoresis (MECC) method has been developed for monitoring the myrosinase catalysed hydrolysis of 2-hydroxy glucosinolates and the simultaneous formation of the corresponding degradation products (oxazolidine-2-thione (OZT) and nitriles). Glucosinolates containing 2-hydroxy groups were chosen as models owing to the difficulties in determining hydrolysis rates of this type of substrates in traditional UV-assays. Glucosibarin ((2R)-2-hydroxy-2-phenylethylglucosinolate) was chosen as the model glucosinolate and the method was afterwards validated with other 2-hydroxy glucosinolates, such as glucobarbarin ((2S)-2-hydroxy-2-phenylethylglucosinolate) and progoitrin ((2R)-2-hydroxybut-3-enylglucosinolate). The method was also tested with aromatic glucosinolates without a 2-hydroxy group in their side chains, such as glucotropaeolin (benzylglucosinolate) and gluconasturtiin (phenethylglucosinolate). Formation of the glucosinolate hydrolysis products was monitored simultaneously at 206 nm and 230 nm. This allowed estimating the extinction coefficient of the OZT derived from glucosibarin, which was found to be 18000 (M-1 cm-1) and 12000 (M-1 cm-1) at 206 nm and 230 nm respectively. The developed method has LOD of 0.04 mM (0.08-0.12 pmol) and 0.06 mM (0.12-0.18 pmol) and LOQ of 0.2 mM (0.4-0.6 pmol) and 0.3 mM (0.6-0.9 pmol) for the OZT (230 nm) and the nitrile (206 nm) respectively. Linearity of the glucosinolate concentration was examined at six concentration levels from 2.5 to 100 mM and at 206 nm a straight line (y=2089.7x + 0.0628) with a correlation coefficient (R2=0.9996) was obtained. The number of theoretical plates per meter (N/m) at the optimal system conditions was found to be 370000 for the intact glucosibarin, 410000 for the OZT and 390000 for the nitrile. The development of analytical methods able to determine the compounds produced under different conditions and the role that the type of myrosinase, cofactors and additional proteins play in the hydrolysis of glucosinolates is of great importance for the study of the biological role of these compounds.

Analysis of crude extracts from plant material by micellar electrokinetic capillary chromatography
J. C. Sřrensen, H. Sřrensen
Department of Natural Sciences, The Royal Veterinary and Agricultural University

Phenolics and glucosinolates are structurally different groups of natural products. Both of these groups contain however, compounds which may have great influence on the quality of food and feed containing plant materials, in which both can be accumulated to appreciable levels. The biological effects of the phenolics are often related to their redox properties and/or oxidation products. The effects of glucosinolates are most often associated with their transformation products which may be formed in autolysis reactions. In all cases it is important with access to methods of analysis, which give basis for determination of the labile native biomolecules in crude extracts as well as for efficient separation of individual molecules. Minimal handling and a relatively short time span from extraction to analysis is normally favourable for analysis of labile compounds. This developed method using MECC analysis on crude extracts provide a possibility of having a short period from extraction to analysis and furthermore in a sufficient rugged method. The method can not substitute the more sophisticated methods based on group separation applied in hyphenated LC techniques prior to final analysis. However, for some compounds more reliable data can be obtained with this method. This is the case for some glucosinolates and phenolics which in this method can be analysed as the intact compounds in a micellar electrokinetic capillary chromatography (MECC) technique using cholate as surfactant. The method benefits from being a fast screening method for important low molecular weight compounds in different plants.

Electrophoretic mobilities of diuretics and probenecid in methanol
Heli Sirén, Suzana Abenet, Ruth Kuldvee, Marja-Liisa Riekkola

In this study, capillary electrophoresis (CE) was used to measure electrophoretic mobilities of diuretics and probenecid in non-aqueous medium to get optimal resolution in their simultaneous separation. CE has a wide range of applications in many areas, including chemical, clinical, pharmaceutical and environmental analysis. It is well known that it can also be used in getting experimental data for calculating analytes´ physical parameters. Non-aqueous capillary electrophoresis (NACE) involves the separation of analytes in a medium composed of organic solvents. The use of non-aqueous medium allows additional selectivity options in method development and is also valuable for the separation of hydrophilic and hydrophobic compounds. Diuretics are pharmaceuticals that promote the removal from the body of excess water, salts, poisons, and accumulated metabolic products, such as urea. They serve to rid the body of excess fluid (edema) that accumulates in the tissues owing to various disease states. Having various hydrophilic and hydrophobic functional groups in their molecular structures they are a very challenging group of analytes in NACE studies. The primary objective of this work was to find an optimal electrolyte composition for separation of thiazide diuretics (chlorothiazide, benzthiazide, and hydrochlorothiazide), loop diuretics (furosemide, bumetanide) and probenecid in non-aqueous methanolic solution. On that account, actual mobilities and dissociation constants of diuretics and probenecid were determined in methanol. Actual mobilities were derived from the dependence of the effective mobilities of the analytes on the pH of the methanolic background electrolyte solution (pH(MeOH)). The pKa values of the analytes in methanol (pKa(MeOH)) were calculated by non-linear curve fitting to the measured mobility values. The pKa values could be utilised in the optimisation of the electrolyte pH for separation.

On-line Concentration of Microheterogeneous Proteins by Capillary Electrophoresis Using SDS and PEO as Additives
Y.F. Huang, M.M. Hsieh, W.L. Tseng, H.T. Chang
National Taiwan University, Taipei, Taiwan

We describe a method for analyzing large-volume protein samples using capillary electrophoresis in conjunction with laser-induced fluorescence detection (CE-LIF). To improve the stacking and separation efficiencies of proteins, we added either 0.01% sodium dodecyl sulfate (SDS) or 0.01% poly(ethylene oxide) (PEO) to the TB solutions (pH 10.0) used to prepare the protein samples. After injection of the large-volume samples (ca. 1.0 µL), the proteins migrate against the electroosmotic flow (EOF) and enter the PEO zone; this process causes them to slow down and stack at the boundary between the PEO and sample zones. As a result, the limits of detection (S/N = 3) for most proteins are sub-nM to several nM. For instance, the LOD for a-lactalbumin is 0.48 nM, which is an 84-fold sensitivity enhancement over the traditional method. By applying a short plug of 0.2% SDS prior to sample injection, a greater number of peaks, representing the microheterogeneity of the proteins, were resolved and the stacking efficiency of the proteins increased slightly. This method allowed us to detect 12 peaks when injecting a large volume of sample containing six model proteins (0.1 µM). The practical method is validated by the detection of human serum albumin in a urine sample, obtained from a normal female, without sample pretreatment; its concentration was 0.18 µM. We further demonstrate the capability of this method to detect low amounts of proteins through the detection of 45 nM hemoglobin after injecting ca. 1.0 µL of ultradilute lysed red blood cells. The experimental results indicate that our proposed method has great potential for use in diagnosis and proteomics applications.

Proteins of Calcified Endoskeleton: Characterization of Proteinaceous Organic Matrix of Sclerites from the Alcyonarian, {\itSINULARIA POLYDACTYLA }
M.A. Rahman, T. Uehara
University of the Ryukyus, Okinawa, Japan

The calcified organic substances in the skeleton contain a protein-polysaccharide complex playing a key role in the regulation of bio-calcification. However, information concerning the matrix proteins in alcyonarian and their effect on calcification process is still unknown. For this reason, we have studied the organic matrix of endoskeletal spicules from alcyonarian coral, Sinularia polydactyla, to analyze the sequences and functional properties of the proteins present. The separated sclerites from the colony were identified by scanning electron microscope (SEM). The soluble organic matrix comprised 0.04% of the sclerites weight. By recording the decline of pH in the experimental design, the inhibitory effect of the matrix on CaCO$_{3}$ precipitate was revealed. The SDS-PAGE analysis of the preparations showed seven bands of proteins with apparent molecular weights of 109, 83, 70, 63, 41, 30 and 22 kDa. The proteins were transferred to polyvinylidene difluoride (PVDF) membranes and their N-termini were sequenced. Two bands of proteins of about 70 and 63 kDa yielded N-terminal amino acid sequences. For the detection of calcium binding proteins, a Ca$^{2+}$ overlay analysis was conducted on the extract by $^{45}$Ca autoradiography. The 109 and 63 kDa calcium binding proteins were found to be radioactive. Periodic acid-Schiff staining indicated that the 83 and 63-kDa proteins were glycosylated. A carbonic anhydrase, which is thought to be play an important role in the process of calcification, revealed low level of activity. These findings suggest that the endoskeletal sclerites of alcyonarian corals have protein-rich organic matrices, which might be related to the calcification process.

Development of disposable monolithic silica extraction tip for mass spectrometry analysis of phosphoproteins
Shota Miyazaki1, Hironobu Morisaka2, Kei Morisato3, Kazuki Nakanishi4, Mitsuyoshi Ueda2
1 GL Sciences Inc., Saitama, JAPAN 2 Graduate School of Agriculture, Kyoto University, Kyoto, JAPAN 3 Kyoto Monotech Co., Kyoto, JAPAN 4 Graduate School of Science, Kyoto University, Kyoto, JAPAN

Mass spectrometry (MS), now routinely used to identify proteins, has been the preferred tool for detailed chemical structural analysis of the proteins. Therefore, proper sample preparation is a key factor to attain the highest levels of sensitivity and data quality. Phosphopeptides can be difficult to detect in MS analysis due to suppression effects and low ionization efficiency. One used step to overcome this problem is to concentrate the proportion of phosphopeptides present in a sample prior to MS analysis. In the field of HPLC, titania dioxide (TiO2) particles was studied by Dr. Nakamura, and recently it is reported that the column packed titanium dioxide particles was applied to the HPLC analysis of phosphopeptides (1-3) and monolithic silica column was proved unique features, including low pressure-drop and total porosity higher than particle column.(4) To simplify sample preparation and to facilitate identification of phosphopeptides, we developed novel procedure utilized titania-coated monolithic silica for micropurification of phosphopeptides. The monolithic-structured silica was prepared on phase separation by polymerization of tetraethoxysilane in the presence of poly (ethylene oxide). Monolithic silica was titania-coated by tetraisopropyl titanate monomer in the 2-propanol. Then, titania-coated monolithic silica was investigated physical characteristic by SEM, nitrogen absorption, and X-ray diffraction (XRD) (5). Titania-coated monolithic silica applied as separation medium for HPLC analysis of phospho-compounds (nucleotides, phospholylated glucoses). Titania-coated monolithic silica was fixed microtips (MonoTip® TiO 200 uL). To purify phosphopeptides, optimal condition was established by recovery of standard phosphopeptides and nonphosphopeptides. MonoTip® TiO was compared to IMAC columns on selectively and recovery of phosphopeptides. MonoTip® TiO was then applied to selectively purify phosphorylated peptides from a tryptic digest of bovine beta-casein prior to MALDI-TOF/MS. MonoTip® TiO had high selectively and efficiency at binding and eluting phosphopeptides. MonoTip® TiO was also eluted phosphopeptides with not only phosphate buffer (pH7.0) but dilute base (0.5-5% ammonium). Base elution enabled direct MS analysis without having to remove salt over RP microtips, thereby saving time and analyte. Moreover, mild base could be favorable for phosphopeptide analysis by ESI-MS in the negative mode. MonoTip® TiO offers advantages, in terms of both selectivity and recovery, including base elution. MonoTip® TiO is a highly efficient and versatile tool to purify phosphorylated peptides form proteolytic digests prior to mass analysis. 1. Sano A, and Nakamura H, Anal Sci. 2004, 20, 565-6 2. Pinkse MW, Uitto PM, Hilhorst MJ, Ooms B, Heck AJ, Anal chem. 2004, 76, 3935-43 3. Kuroda I, Shintani Y, Motokawa M, Abe S, Furuno M, Anal Sci. 2004, 20, 1313-93 4. Cabrera K, Lubda D, Minakuchi H, Nakanishi K, J. High Resolut. Chromatogr. 2000, 23, 93-99 5. Miyazaki S, Miah MY, Morisato K, Kuroha T, Nakanishi K, J Sep Sci. 2005, 28, 39-44

Integration of Solid-phase Microreactor and Liquid Chromatography-tandem Mass Spectrometry for Proteomics
Naoko Hirotani, Naoshi Dohmae, Hiroshi Nakayama
Biomolecular Characterization Team, Advanced Development and Supporting Center, RIKEN

We present here an automated system to identify, quantify, and precisely characterize diluted or crude protein samples. The system involves microprecolumns packed with hydrophobic media as solid-phase reactors, a microflow liquid chromatograph, and a tandem mass spectrometer and can process up to nine samples in a single run. The procedures are illustrated using serum albumin and transferrin. First, protein samples containing denaturing reagents, such as guanidium salt or urea, were introduced to the microprecolumn and the excess reagent was washed out. The adsorbed proteins were then ‘on-bead’ proteolyzed by delivering protease solution containing a blocking reagent that prevents hydrophobic interaction between the protease and the bead. Resulting peptides were eluted to a capillary analytical column and, thus separated, were analyzed by tandem mass spectrometry. Obtained elution patterns of the digests in the presence of our blocking reagent were similar to those obtained from conventional proteolysis methods in solution. Thus the efficiency of ‘on-bead’ proteolysis was considered to be remarkably high. Using our automated system reduced the likeliness of human errorĹ@or contamination and produced reproducible results. Therefore, the system should be suited for differential display or quantitative analysis as well as primary structural analysis of proteins.

Evaluation of Capillary Isoelectric Focusing in Glycerol-water Media with a View to Hydrophobic Protein Applicatons
A. Varenne, J.M. Busnel, S. Descroix, A. Morin, G. Peltre, Y. Gohon, P. Gareil
Ecole Nationale Supérieure de Chimie de Paris, Ecole Supérieure de Physique et Chimie Industrielle de la ville de Paris, et Institut de Biologie Physico-Chimique, Paris, France

Capillary isoelectric focusing (CIEF) is often considered as the most powerful monodimensional technique for protein separation. CIEF is usually performed with neutral coated fused silica capillaries in aqueous anticonvective media. Glycerol, a very viscous solvent (viscosity of 945 mPa.s at 25 °C), known to help stabilizing any kind of proteins and solubilize hydrophobic ones, was tested as an alternative to commercial gels. Viscosity and electroosmotic mobility were measured as a function of gel or glycerol content in water, and a 30:70 (v/v) glycerol-water medium appeared as a good compromise for performing CIEF in a bare fused silica capillary without imposing too high a viscosity. To demonstrate the feasibility of this new CIEF system, a standard mixture of nine model proteins was separated according to their isoelectric point. Linear correlation was obtained between literature aqueous pIs and migration time, which enables still using this system for aqueous pI determinations. Moreover, better resolution was achieved with this system than with the conventional aqueous CIEF system, as two of the model proteins could not be separated in the latter system. Glycerol-water CIEF in bare silica capillary was next applied to the separation of horse radish peroxidase (HRP), a complex mixture of protein presenting numerous isoforms. A better resolution was obtained with this new system than the conventional CIEF system, and the concordance of the pI values indicated the adequacy of this new system [1]. Separation of milk proteins was also successfully performed with the new CIEF system. Finally, as anticipated from the results obtained for the separation of bacteriorhodopsin, a membrane protein, glycerol-water CIEF performed in bare silica capillary appears to be a promising alternative to conventional aqueous CIEF for hydrophobic protein characterization, under their native form [1]. Furthermore, with regard to a transposition into microchip format, this new CIEF system presents the additional main advantage of avoiding any coating of the microchip channel. [1] JM. Busnel, A. Varenne, S. Descroix, G. Peltre, Y. Gohon, P. Gareil, Electrophoresis, 2005, 26, 3369-3379.

CE-SDS Gel Applications in Development of Recombinant Vaccines and Therapeutic Proteins
R.R. Rustandi, M.W. Washabaugh, R.D. Sitrin, Y. Wang
Merck Research Laboratory, West Point, USA

Capillary Electrophoresis (CE) SDS gel technique offers many advantages over the traditional labor intensive SDS PAGE slab gel technology. CE SDS gel method using Beckman PA800 with UV detection has been shown to be linear with respect to protein concentration, quantitative, robust, and rugged. We have used the system for standard purity and integrity analysis in the development of biologic products. Although it is possible to develop a generic CE-SDS gel method for protein based biopharmaceuticals, such as monoclonal antibodies, it is often necessary to perform method development for other proteins. Some technically challenging issues will be discussed in applying the CE-SDS gel technique for specific protein-based vaccines and therapeutics. Furthermore, the extended use of the CE-SDS gel technique to support bio-process development, to monitor product stability, and to provide information on ID and product characterization in some example programs will be presented including method optimizations as well as technical and practical considerations.

Photo-Switchable Proteins and Capillary Electrophoresis
Maxim Berezovski1, Andrew Woolley2, Sergey N. Krylov3
1 The Campbell Family Institute for Breast Cancer Research, University of Toronto. 2 Department of Chemistry, University of Toronto. 3 Department of Chemistry, York University

We are seeking to find general strategies for the photo-control of peptide and protein structure - and thereby activity. In doing so, we gain fundamental knowledge about protein conformation, folding, and dynamics. In this case, Capillary Electrophoresis is a powerful tool for the analysis of photoactive biochemical systems. For example, the protein GCN4 is a transcriptional activator from yeast. We demonstrated photo-modulation of DNA binding by GCN4 protein containing designed azobenzene. We can use the effect to control its biomolecular activity in vitro and in vivo.

Application of nano-LC-MS/MS on a Polymethacrylate Monolithic Capillary Column for Analysis of Peptides
R. Maleki Seifar*, Xie Hong**, Zhiqiang Gao**
*Technical University of Delft, Delft, The Netherlands ** Institute of Bioengineering and Nanotechnology, Singapore

A nanoscale liquid chromatography hyphenated with nanoelectrospray ionization mass spectrometery (nano-LC-MS/MS) was applied for separation and analysis of peptides from a tryptic digest of Cytochrom C. Poly(laurylmethacrylate-co-ethylenedimethylacrylate) monolith was used as reversed phase stationary phase in nano-LC. In situ UV polymerization technique was used to prepare the monolithic column from a homogeneous mixture of lauryl methacrylate (LMA), ethylenedimethylacrylate (EDM) and 2,2-dimethoxy-2-phenylacetophenone in presence of a porogen solvent containing 1-propanol and 1,4 butane diol.

Improvement of Signal Sequence for Separation of Protein
Y. Tsuchiya, K. Morioka, J. Shirai, K. Yoshida
National Institute of Animal Health, Kodaira, Tokyo, Japan

Although protein has come to be mass-produced by the low price in recent years using bioengineering technology, it is a still difficult problem to separate the purpose protein and cell ingredients. To solve this, most effective breakthrough is the secretory production of recombinant protein. Signal sequence plays an important role in the translocation of newly synthesized proteins across the membrane of the endoplasmic reticulum (eukaryotic cells) or across the cell membrane (prokaryotic cells). These sequences are composed of three structurally and functionally distinct regions: a positively charged N-terminal region, a central core region, and a C-terminal region recognized by signal peptidase. We have a human lysozyme (HLY) secretory system in yeast (Saccharomyces cerevisiae) and insect cell by use of a synthetic fusion gene for chicken lysozyme signal peptide (CLSP) and HLY. By using this system, structural requirement of each region of CLSP have been elucidated, which show the importance of positive charge in N-terminal region, hydriphobicity in central core region, turn-formation in C-terminal region. Following on these, in present study, the N-terminal region and central core region of CLSP were altered to improve the secretion amount of HLY in yeast or insect cell. The amino acid sequence of the mutant signal peptides prepared in this study and their effects on HLY secretion are summarized in Table 1. In order to raise the amount of HLY secretion, basic residues (Arg) were introduced into the N-terminal region of CLSP and the sequence of central core region of CLSP was changed into continuation of Leu. Hybrid genes coded these signal peptides connected with HLY, were expressed in yeast (S. cerevisiae) or in insect cell (BmN4). Each recombinant cell was cultured for 4 days. HLY activity in culture medium was measured photometrically by the method of Mörsky. One unit corresponded to 0.33µg of HLY/mL of culture medium under our assay conditions. Additional positive charge in the N-terminal region raised the amount of HLY secretion in yeast, but not in insect cell. About the significance of positive charge in the N-terminal region, previous report has confirmed it in prokaryote, but denied it in eukaryote. Indeed, yeast is the eukaryote, but not the multicellular organism. For the mechanism in which signal peptide enters into membrane, there may be some differences by the unicellular (yeast, or bacteria) and the multicellular organism (insecta, or mammals). In contrast with the N-terminal region, the increase of hydrophobicity in the central core region of CLSP, remarkably enhanced the HLY secretion, similar to previous reports. The structural requirement of this region may be common over species.

Multidimensional Proteome Analysis of Myxococcus Xanthus using Monolithic Columns
C. Schley1, M. Altmeyer2, R. Müller2, C. G. Huber1
1 Instrumental Analysis and Bioanalysis, Saarland University, Saarbrücken, Germany 2 Pharmaceutical Biotechnology, Saarland University, Saarbrücken, Germany

Biologically active compounds produced by microorganisms play an important role in the pharmaceutical industry. The main goal is the production of new and modified natural products, respectively, as well as their optimization. The proteome analysis of these organisms can help to get a better comprehension of the biosynthesis of these secondary metabolites and its regulation in the microorganisms. Protein samples of biological origin are by nature highly complex and therefore, their analysis requires separation techniques with high resolving power and high peak capacity, respectively. During the past few years, the separation of whole-protein lysates proteolytic digests by multidimensional liquid chromatography, which can be readily interfaced on-line to mass spectrometry, has become a real alternative to two-dimensional polyacrylamide gel electrophoresis for high-throughput protein identifications. We utilized an offline two-dimensional liquid chromatography method which integrates a 250 x 4 mm strong cation-exchange column in the 1st dimension and a 60 x 0.2 mm poly-(styrene/divinylbenzene) (PS-DVB)-based monolithic column in the 2nd dimension followed by electrospray ionization tandem mass spectrormetry (ESI-MS/MS) to analyze the proteome of Myxococcus xanthus bacteria, which are known to be very potent secondary metabolite producers. Thirty six cation-exchange fractions of a Myxococcus xanthus lysate were collected during a 27-min cation-exchange separation. The fractions were concentrated via evaporation and subsequently re-injected for 2nd dimension fractionation and identification, comprising desalting in a 10 x 0.2 mm i.d. monolithic PS-DVB trap-column and high-resolution separation in a 60 x 0.2 mm i.d. monolithic PS-DVB separation column. The fragment ion spectra generated by ESI-MS/MS were subsequently analyzed via MASCOT database search. In this analysis, 1312 peptides were identified with a Mascot score greater than 27 and could be associated with 631 unique proteins. Most interestingly, the expression of 12 different polyketide and non-ribosomal polypeptide synthetases was revealed, which play an important role in the production of secondary metabolites. Quadruplicate analysis of each SCX fraction revealed that 247 proteins (39% of all proteins) were identified in all four runs, whereas 67 (11%), 94 (15%), and 223 (35%) were found in three, two, and one of the four analyses, respectively. The orthogonality of the separation system was clearly demonstrated in a two-dimensional plot of the retention times obtained in the individual chromatographic modes.

Parallel Isoelectric Focusing Chip
Alexander Suvorov
Protein Fleet Inc

We report here the theory and experimental results of new 2D device for fast IEF and MW separation. The IEF part is a dielectric membrane with separate conducting channels filled by immobiline gels of varying pH (we use novell “parallel IEF”). The MW part is separate open channels with gradient gels for MW separation. This device work in native and denaturative conditions. The 2D device is a consumable chip designed for protein_ expression analysis and biomarker screening. Measuring approximately 40 mm

 20 mm, it is a single-use product which separates samples in 20 minutes. This product is intended for researchers who want to track protein regulation through protein_expression profiling. The 2D device high resolution can resolve an individual protein’s isoelectric point to 0.05 pH units. This makes computer analysis determining the isoelectric point of a protein precise and prevents typical 2D gel errors arising from variable gels and software vagaries.

Trypsin-immobilized Monolithic Silica with Pipette-tip Shape for a High-throughput Digestion Tool
Shota Miyazaki1, Shigenori Ota1, Hideyuki Uzu1, Kei Morisato2, Mitsuyoshi Ueda3, Kazuki Nakanishi4
1 GL Sciences Inc., Saitama, JAPAN 2 Kyoto Monotech Co., Kyoto, JAPAN 3 Graduate School of Agriculture, Kyoto University, Kyoto, JAPAN 4 Graduate School of Science, Kyoto University, Kyoto, JAPAN

In the field of functional proteomics, a pretreatment of sample has been regarded as one of the most important procedures to produce reliable analytical results. Three essential techniques for the preparation are an affinity purification for the removal of matrixes from bio-samples, a pre-concentration for trace substances, and a tryptic digestion of proteins. Published techniques of trypsin proteolysis can be classified in two ways: an in-solution digestion and a solid-phase digestion. The submerged digestion was indicated to be time-consuming process, where 4-24 hours at 37°C was typically required for its reaction. In addition, an excess amount of trypsin to the substrates, such as the ratio of the range from 20:1 to 100:1 (w/w), was recommended. The solid-phase digestion method was known to cover the shortcomings of those disadvantages. The digestion by using trypsin-immobilized material led to an enhancement of its reaction efficiency, a reduction of the contamination derived from the self-digestion of trypsin, and a better reproducibility. The use of the trypsin column was cost-effective and allowed the high throughput analysis. However, since the flow rate influenced the digestion efficiency , the optimization of analytical conditions was complicated. Our group previously presented on monolith silica with pipette-tip-shape (MonoTip®) for a solid-phase tool suitable for the pretreatment of bio-samples having various characteristics. In the previous work, C18-bonded monolith-structured silica (MonoTip® C18) was applied to the concentration, desaltination and removal of detergents from sample. Here, we newly developed an enzyme-immobilized monolithic silica in a pipette tip (MonoTip® Trypsin), which was suitable for the digestion of proteins. The surface of monolithic silica fixed into the tip was chemically modified with trypsin via an amino group. Trypsin-immobilied monolith was successfully led to a rapid digestion of reduced and alkylated proteins with merely a few times pipetting operation for the pretreatment procedure of chromatographic analysis. Novel solid-phase digestion tool using monolithic silica allowed the high-throughput trypsin proteolysis, and was effective and suitable for the analyses of bio-substances in proteomics. [1] S. Miyazaki et. al. Journal of Chromatography A vol. 1043, No. 1, pages 19 (2004)

Simplified and Automated Approach for Protein Identification Using Capillary Electrophoresis-Tandem Mass Spectrometry
C. K. Ratnayake, I.D. Cruzado-Park, M. H. Simonian
Beckman Coulter, Inc., Fullerton, CA, USA

Capillary electrophoresis - mass spectrometry is a powerful and widely accepted tool for the identification of proteins. Fractionation of complex protein mixtures according to the pI of proteins helps separate basic proteins from acidic proteins. Separation by pI enables favorable mass spectrometric analysis for identification. These protein mixtures can be further fractionated by hydrophobicity. This results in a less complex protein mixture with a large dynamic range of concentration. When the target proteins are available in femtomole quantities, it is difficult to digest them with trypsin because of the unfavorable enzyme kinetics. As a solution to this problem, protein fractions with trace amounts of protein can be pooled and then subjected to trypsin digestion. Here, we present a two-dimensional approach for peptide mapping that utilizes one capillary, thereby simplifying the automated separation of complex mixtures of low abundant protein digests. The first dimension concentrates, desalts, and fractionates the protein digest by liquid chromatography followed by a second-dimension of separation and subsequent analysis by CE-ESI-MS.

Study of Use of Ammonium Peroxodisulfate in Methacrylate-based Monolithic Columns for Electrochromatography
A. Cantó-Mirapeix, E.F. Simó-Alfonso, C. Mongay-Fernández
University of Valencia, Burjassot, Spain

The preparation of methacrylate ester-based monolithic columns was studied. Methacrylate spheres movement was avoided by means of a pre-treatment of columns. Capillary wall was modified by using 3-(Trimethoxysilyl)propyl methacrylate, as previously described by Hjertén (1). In bibliography, most of monolithic columns use azobisisobutyronitrile (AIBN). Nevertheless, the lack of availability of this reagent in the market led us to the use of new initiating agents of free radicals. The combination of ammonium peroxodisulfate and N,N,N’,N’-tetramethylethylenediamine (TEMED), as a polymerization initiating system, was studied for the preparation of monolithic columns. The use of these reagents is described in bibliography for the preparation of gels for electrophoresis and capillary gel electrophoresis (CGE), and also for the preparation of monolithic columns of 300 µm I.D. used in liquid chromatography (2). Monoliths were prepared by polymerization of butyl methacrylate (BMA) with ethylene glycol dimethacrylate (EDMA) as cross-linker, using a ternary porogen solvent (1-propanol, 1,4-butanediol and water), and [2-(methacryloyloxy)ethyl] trimethyl ammonium chloride (META) for the generation of an electroosmotic flow (EOF). Columns obtained with this procedure resulted in polymers with a very small pore size, as proved the high pressures needed to remove solvents and not polymerized monomers or oligomers from monoliths. According to the studies of Hjertén and col. (3), ammonium sulfate was added to the polymerization mixture in order to increase the pore size and reduce the flow resistance. Performance of these columns was studied for their use in electrochromatography. Different compound families were used as test substances. Acknowledgements Work supported by Project CTQ2004-06302/BQU (MEC of Spain and FEDER funds) and Agilent Technologies Spain, S.L. (Las Rozas, Madrid). The support of the Generalitat Valenciana (GRUPOS2004-16) is also acknowledged. (1) S. Hjertén, J. Chromatogr. 1985, 347, 191. (2) P. Holdsvendová, P. Coufal, J. Suchánková, E. Tesarová, Z. Bosáková, J. Sep. Sci. 2003, 26, 1623. (3) Ch. Ericsson, J.-L. Liao, K. Nakazato, S. Hjertén, J. Chromatogr. A 1997, 767, 33.

Molcularly Imprinted Capillary Monoliths
J. Oxelbark1, E. De Lorenzi1, J. Courtois2, K. Irgum2, E. Schillinger3, B. Sellergren3
1Department of Pharmaceutical Chemistry, University of Pavia, Pavia, Italy 2Department of Chemistry, Umeĺ University, Umeĺ, Sweden 3Institut für Umweltforschung, Universität Dortmund, Dortmund, Germany

Bupivacaine imprinted capillary monoliths were produced by grafting an imprinted polymer layer to a core monolith. The core monolith was made from trimethylol propane methacrylate. To this monolith, an imprinted or non-imprinted polymer layer was grafted in situ, via thermal- or UV-initiation, consisting of methacrylic acid and ethyleneglycol dimethacrylate. Presence of template bupivacaine yielded the MIP (molecularly imprinted polymer), while absence yielded the reference NIP (non-imprinted polymer). Photo initiated polymers were obtained in 0.1mm i.d. capillaries, and thermally initiated polymers were obtained in 0.32mm i.d. capillaries. For each format, MIPs and NIPs were produced, thus allowing for assessment of imprinting factors. The imprinted polymers aim at a high water compatibility, and imprinting factors were determined in different mixtures of acetonitrile and phosphate buffer. The capillary formats were compared to a crushed and sieved bulk polymer, prepared under the same conditions, but packed into a 4.6mm i.d. column.

Improved chemical modification methods on Chromolith Performance™ columns
Chun Yang, Takeshi Hara, Tohru Ikegami, Ken Hosoya, Nobuo Tanaka
Department of Polymer Science and Engineering, Kyoto Institute of Technology

A monolithic silica columns (100 × 4.6 mm) was bonded with octadecyldimethyl-N.N-diethylaminosilane and checked by reversed-phase liquid chromatographic measurements. It shows reproducible hydrophobic and shape selectivities for neutral compounds and shares homogeneity with commercial columns named as Chromolith Performance™ from Merck Inc. Successive endcappings with hexamethyldisilizane and N-(Trimethylsilyl)imidazole were carried out to investigate the effects of the two reagents and different reaction conditions. Experiments on more columns indicate endcapping of N-(Trimethylsilyl)imidazole acetonitrile is more efficient than that of hexamethyldisilizane in toluene. After endcapped by N-(Trimethylsilyl)imidazole, all columns provide better separations for the of basic and chelating compounds. TMSI endcapping also gives reduced retentions and improved performance on commercial Chromolith Performance™ columns. Conclusively N-(Trimethylsilyl)imidazole is a sufficient reagent giving high performance and a stable endcapping on Chromolith Performance™ columns.

Optimization of Continuous Bed Restricted Access Media for Microseparations
A. Maruška, V. Bendokas, R. Jarmalavicienė, O. Kornyšova
Department of Chemistry, Faculty of Natural Sciences, Vytautas Magnus University, Kaunas, Lithuania

The restricted access media (RAM) packings are a class of tailor-made stationary phases with which macromolecules (e.g. proteins, nucleic acids, etc.) are eluted within the void volume of a column without destructive accumulation, while small molecules (e. g. drugs, drug candidates, etc.) can be retained on the stationary phases and be separated. The restriction of macromolecules for accessing the hydrophobic ligands of the packings is based on size-exclusion and elimination of adsorption of proteins due to the presence of an external hydrophilic surface. This allows the application of such materials for direct injection and chromatographic analysis of biofluids. High efficiency and miniaturization are the main characteristic features of modern analytical techniques. The continuous (monolithic) bed technique has been shown to be a very attractive method for synthesis and packing of the stationary phase into small-bore (25-320 µm) capillaries. The monolithic adsorbent formation is performed by filling in a solution of monomers and an initiator into fused silica capillary and polymerizing in situ. The main task of this study was synthesis and evaluation of polymeric continuous bed restricted-access adsorbent. Capillary format, coated with different allyldextrane concentrations, continuous RAM beds were synthesised, chromatographically evaluated and compared to uncoated RP capillary columns. Minor changes of hydrophobicity, efficiency and flow rate were noticed. Increasing of vinyl groups in RAM resulted in efficiency decrease, hydrophobicity and flow rate didn’t change. The inverse size exclusion chromatography was used for porosimetric investigation of the capillary format non-particulate beds. It was determined that increase in allyldextrane concentration results in pore and channel volume decrease. The increase of the amount of vinyl groups in RAM resulted in rapid pore size mean reduction. Capillary columns were applied for chromatography of biological fluids. Direct injection of bio fluid was performed with continuous bed RAM capillary column using isocratic conditions.

Boronic acid functionalised monoliths for affinity chromatography and electrochromatography
Michael C Breadmore, Oscar G Potter, Emily F Hilder
Australian Center for Research on Separation Science

Macroporous polymer monoliths were synthesised for use as stationary phases for affinity electrochromatography and were applied to the separation of small biomolecules. Macroporous monoliths prepared by copolymerisation of 2-hydroxyethyl methacrylate, ethylene glycol dimethacrylate and p-vinylphenylboronic acid with pore sizes ranging from the low hundreds of nanometres up to several micrometres were synthesised, however these were found to exhibit no boronate affinity interaction. An alternative approach of attaching the phenylboronic acid group to the surface of a Poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate), poly(GMA-co-EDMA) via direct reaction with p-hydroxyphenylboronic acid was found to provide significant retention of nucleosides relative to their deoxy counterparts. This interaction could be enhanced further by preparation of a photografted polyGMA layer prior to modification with p-hydroxyphenylboronic acid, with these monoliths exhibiting twice as much affinity.

Everest : a novel monomeric C18 bonded phase for improved resolution and sensitivity in LC-MS/MS analysis of peptides
Bart Denoulet, Reno Nguyen, Ning Mu
Grace Davison, Brandstraat 12, 9160 Lokeren, Belgium Grace Vydac, 17434 Mojave Street, Hesperia, CA 92345, USA

The separation of protein enzymatic digests by RP-HPLC is a standard method in protein research and in the development of well-characterized biotechnology pharmaceuticals. Resolution on silica-based RP columns is determined by the nature of the silica, as well as, the silanes and the chemistry that is used to bond them to the stationary phase. Typically, 300 Ĺ C18 chemistries have carbon coverage ranging from 2.8 to 3.6 µmol/m2. The performance of a novel, monomeric bonded 300 Ĺ C18 adsorbent (VYDAC® EVEREST™) with carbon coverage >4 µmol/m2 is described. The resolution and sensitivity of this stationary phase was assessed using an LC-MS/MS analysis of several tryptic digests including: digests of individual proteins (serum albumin and green fluorescent protein); a complex sample comprised of a dozen proteins of different abundances; ribosomes, and whole bacterial proteomes. The tryptic peptides were run on nano/capillary or microbore C18 columns. LC-MS/ MS analyses were performed on an ABI Q TRAP®, an ABI QSTAR®, and a Thermo Finnigan LCQ Deca using a solvent system comprised of acetonitrile/water with 0.1 to 0.5% formic acid, or with 0.05% TFA, with subsequent database searching using Mascot and/or SEQUEST. With the complex sample of a dozen proteins, the score from Mascot was higher for a number of protein identifications on the novel phase compared to a small pore (100 Ĺ) C18 material. This corresponds to higher individual peptide scores, which are usually indicative of better MS/MS. The novel phase also performed well with ribosomes and complex proteome samples, based on SEQUEST results.

Validation and Application of an HPLC -Tandem Mass Spectrometric Method for Simultaneous Quantification of Lopinavir and Ritonavir (Kaletra®) in Human Plasma Using Semi-Automated 96-Well Liquid/Liquid Extraction
Perry G. Wang, Jake Wei, Grace Kim, Min Chang, Tawakol El-Shourbagy
Drug Analysis Abbott Laboratories

Kaletraâ is an important antiretroviral drug, which has been developed by Abbott Laboratories. It is composed of lopinavir and ritonavir. Both have been proved to be HIV protease inhibitors and have substantially reduced the morbidity and mortality associated with HIV-1 infection. We have developed and validated an assay, using liquid chromatography coupled with atmospheric pressure chemical ionization tandem mass spectrometry (LC/MS/MS), for the routine quantification of lopinavir and ritonavir in human plasma, in which lopinavir and ritonavir can be simultaneously analyzed with high throughput.

The sample preparation consisted of liquid-liquid extraction with a mixture of hexanes: ethyl acetate (1:1, v/v), using 100 ml of plasma. Chromatographic separation was performed on Waters Symmetry C18 column (3.9x 50mm, 5µ) with reverse-phase isocratic using mobile phase of 70:30 (v:v) acetonitrile : 2 mM ammonium acetate aqueous solution containing 0.01% formic acid (v/v) at a flow rate of 1.0 ml/min. A Waters sentry C18 guard column (3.9x20mm, 5µ) was connected prior to the analytical column, and a guard column back wash was performed to reduce the analytical column contamination using a mixture of tetrahydrofuran (THF), methanol and water (45:45:10, v:v:v). The analytical run was 4 min. The use of a 96-well plate autosampler allowed a batch size up to 73 study samples. A triple-quadrupole mass spectrometer was operated in a positive ion mode and multiple select ion monitoring was used for drug quantification. The method was validated over the concentration ranges of 19-5300 ng/mL for lopinavir and 11-3100 ng/mL for ritonavir. A-86093 was used as an internal standard. The coefficients of variation (CV) were
<6% for both lopinavir and ritonavir. Mean accuracies were between the designed limits (±15%). The robust and rapid LC/MS/MS assay have been successfully applied for routine assay to support bioavailability, bioequivalence, and pharmacokinetics studies.>

P. Puig, F.W.A. Tempels, G. Wiese, G.W. Somsen, G.J. de Jong
Utrecht University, Utrecht, The Netherlands

Capillary electrophoresis (CE) has a high separation efficiency but when low detection limits are required, the sensitivity is often insufficient. In order to increase the sensitivity of CE, chromatographic preconcentration can be used prior to the CE separation. Solid-phase extraction (SPE) can be coupled to CE in different ways (at-line, on-line, in-line and off-line) [1-2]. In this study we used the on-line coupling of SPE and CZE to analyze the cephalosporins ceftiofur and cefoperazone in plasma. Ceftiofur and cefoperazone are beta-lactam antibiotics used for the treatment of infectious diseases in veterinary medicine. Determination of these compounds in plasma is important for the control of drug administration. The system comprises a trapping column of 5 mm x 0.5 mm filled with reversed phase material (C18) and two valves for introduction of the sample and desorption of the compounds from the trapping column. A T-split was used to hydrodynamically inject a part of the desorption plug into the CE capillary. The breakthrough volume of the compounds was about 1 ml for both cefoperazone and ceftiofur and allows a washing step after sample introduction. The elution volume was 1.8 microliter with desorption efficiencies of 75% for cefoperazone and 90% for ceftiofur. For injection of 250 microliter of standard solutions, the detection limits are 5 ng/ml. For 100 microliter of diluted plasma (1:3), detection limits are still in the high ng/ml range. [1] F. W. A. Tempels, J. Teeuwsen, I. K. Kyriakou, G. Theodoridis, W. J. M. Underberg, G. W. Somsen, G. J. de Jong, J. Chromatogr. A 1053 (2004) 263-268 [2] A. Macia, F. Borrull, C. Aguilar, M. Calull, Electrophoresis 24 (2003) 2779-2787

Drug Monitoring of the Virustatic Drug Ganciclovir by Capillary Electrophoresis
S. Saleh, J. Boos, G. Hempel
Klinik und Poliklinik für Kinder- und Jugendmedizin, Pädiatrische Hämatologie/ Onkologie, Albert-Schweitzer-Str. 33, 48149 Münster, Germany

Ganciclovir is a potent antiviral drug showing activity against several viral infections and is the first choice therapy in immunocompromised patients. The need for therapeutic drug monitoring is particularly important in patients having acute or chronic renal impairment and in younger children and infants. Our aim is to asses the pharmacokinetics of ganciclovir in children from small plasma volumes and very few plasma samples. We choose capillary electrophoresis (CE) because of the small sample volume required and the high separation speed. Using the CE Beckman system P/ACE with a UV-detector the following conditions were tested and optimized: capillary length(37-57cm), injection time (30-50s), voltage (10-15 kV), buffers strength(40 to 150 mM borax) sodium dodecyl sulphate (SDS) concentration and buffer pH (8,8 - 9,4). Acyclovir and 2-chloroadenosine were tested as the internal standard. Sample preparation procedures investated tested included deproteination using perchloric acid, trichloroacetic acid or acetonitrile as well as ultrafiltration techniques. The best peak shape was achieved by using the ultrafiltration technique, bubble cells capillary (47 cm), pressure injection for 30 s, 100 mM borax buffer pH 8.9 containing 90 mM SDS and a voltage of 15 kV. Detection was carried out at 254 nm. The limit of quantification is 0.5 mg/l enabling us to quantify clinically relevant plasma concentrations. Validation of the method is ongoing.

Seperation of the Enantiomers and Diastereomers of the Aziridine Derivatives in Aqueous and Non-Aqueous Chiral Capillary Electrophoresis
Ulrike Holzgrabe, Yaser Bitar, Björn Degel, Tanja Schirmeister
Institute of Pharmacy, University of Würzburg, Würzburg, Germany

Highly substituted aziridine derivatives are attracting pharmacological interest as intermediates of protease inhibitors (e.g. for AIDS treatment). Due to the two stereogenic centres the two aziridines tested here are mixtures of two diastereomers and their corresponding enantiomers. Appling the negatively charged beta-cyclodextrin (HDMS or HDAS) as chiral selectors in separation systems consisting either of an aqueous phosphate buffer or of non-aqueous acidic methanol were compared at the similar pH values of the background electrolytes. The single isomers of HDAS and/or HDMS in acidic methanol (NACE) have been successfully applied to the racemic compounds resulted in rather good resolution of the four stereoisomers. With HDAS the resolutions were higher and migration times shorter. The four isomers of the aziridines could be also separated in one run by means of the aqueous separation system. Optimized conditions were a 1-2 mM randomly sulphated beta-cyclodextrin in 50 mM phosphate buffer at pH 2.5 as background electrolyte, a temperature of 20°C and an applied voltage of 10 kV. This method allowing the detection and determination of the trans-diastereomers of aziridine in the cis-isomers could be validated according to the International Conference of Harmonization (ICH) guidelines as well as European Pharmacopoeia with respect to specificity, linearity, range of concentration, limit of quantification and detection, precision, accuracy and robustness. This aqueous system turned out to be more robust that the NACE.

Optimization for Quantitative Determination of Four Flavonoids in Epimedium by CZE-DAD using Central Composite Design
S.P. Li, J.J. Liu, Y.T. Wang
University of Macau

Herba Epimedii (family Berberidaceae), Ying-Yang-Huo in Chinese, is a famous Chinese herbal medicine. Flavonoids are thought to be the major active components in it. A capillary zone electrophoresis (CZE) separation were developed for simultaneous determination of four flavonoids including icariin, epimedin A, epimedin B and epimedin C in Epimedium. The effects of the experimental variables on CZE had been optimized by using central composite design (CCD). The best separation of four flavonoids could be obtained using 50 mM borate buffer (pH 10.0) containing 22% acetontrile as modifier, while separation voltage was 15 kV and temperature was at 25 oC. The method developed is accurate, simple and reproducible, which could be used for quality control of Epimedium and its medical preparations.

Analysis of Serotonin in Brain Microdialysates using Capillary Electrophoresis with a half trillion theoretical plates and Native UV Laser-Induced Fluorescence Detection
N. Benturquia13, F. Couderc2, V. Sauvinet1, C. Orset1, S. Parrot1, B. Renaud1, L. Denoroy1
1- Laboratoire de Neuropharmacologie et Neurochimie, Universite Claude Bernard Lyon 1, Lyon, France 2- IMRCP, Universite Paul Sabatier, Toulouse, France 3- Picometrics, Ramonville, France

The analysis of serotonin (5-hydroxytryptamine or 5-HT) in brain dialysates via capillary electrophoresis (CE) system coupled to a commercially available laser-induced fluorescence detector allows for the study of the changes of extracellular serotonin concentration that are induced by the R and S isomers and racemic mixtures of citalopram. In this assay, a pH-mediated in-capillary pre-concentration of samples was performed, followed by separation by capillary zone electrophoresis and measurement of the native fluorescence of 5-HT using excitation at 266 nm with a solid-state laser. The separation conditions for the analysis of 5-HT in standard solutions and microdialysates were optimized using an 80 mmol/L citrate buffer (pH 2.5) containing 20 mmol/L hydroxy-propyl-ß-cyclodextrin (HP-ß-CD) at a +30 kV potential. In addition to optimizing the separation, this buffer optimized the fluorescence intensity. The overall performance of the separation was estimated in terms of the resolution between different indoleamines and in terms of theoretical plate number. We found that the method allows for separation of 5-HT with a a very important stacking of 5-HT resulting in a theoretical plate number of 500 billion and a detection limit of half trillion of 0.25 nmole/L. This method, has been validated on both pharmacological and analytical bases, and allows for in vivo brain monitoring of 5-HT using a simple, accurate CE-LIF measurement in microdialysate samples and does not require that the compound of interest be derivatized. This separation method allows the user o study the changes in the concentration of extracellular serotonin concentration that are induced by the R and S isomers as well as racemic mixtures of citalopram in rat brain.

Study on Separation of Aristolochic Acid I and II by Micellar Electrokinetic Capillary Chromatography
Wei Li, Zheng Chen, Yiping Liao, Huwei Liu
College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, China

Over the last 10 years, numerous cases of intoxications caused by ingestion of medicine or food containing Chinese herbs, leading to end-stage renal failure, have been reported to be associated with aristolochic acid (AA), which is the main component in Aristolochia plants. In this study, a rapid micellar electrokinetic chromatography (MEKC) method using 40 mM sodium borate buffer containing 50 mM sodium dodecyl sulfate (SDS) as surfactant was developed for the analysis of AA in Aristolochia plants. Baseline separation of AA-I and AA-II was achieved within 3 minutes with high separation efficiency, satisfactory sensitivity, repeatability and recovery. Resolution between AA-I and AA-II is above 5 and great performance with higher than 200000 theoretic plate number was obtained. The linearity was satisfactory with a correlation coefficient (r) greater than 0.998. The detection limits of two AAs were found to be both 1.0 µg • mL-1 (corresponding to 10 µg • g-1 dryed plants). The repeatability of this MEKC method was both below 0.6% RSD (n=5) for migration time and less than 1.8% RSD (n=5) for peak area, respectively. The recoveries of the AA-I and AA-II were 97.6% and 76.1% (n=4) with the RSD of 3.0% for AA-I and 3.8% for AA-II, demonstrating that the simple preparation method could provide acceptable extraction efficiency in a short period. Two kinds of AA in 35 herbal samples of Aristolochia plants were successfully determined. It indicated that there was high variability in the contents of AAs due to the difference of species and regions. In generally, the contents of AAs in Caulis Aristolochiae Manshuriensis samples were higher than those in Aristolochia fangchi Wu samples. Furthermore, AA-I and AA-II were not detected in the medicinal plants collected in Zhongshan, Qujiang, Gaoyao, and Zhaoqing in Guangdong province.

Surfactant Enhanced Liquid Phase Microextraction of Some Drugs of Abuse in Hair Combined with HPLC
Zarrin Eshaghi, Ali Sarafraz Yazdi
PhD student in analytical chemistry(Zarrin Eshaghi) Professor in analytical chemistry(Ali sarafraz Yazdi)

The aim of this study was to evaluate the performance of a technique for simultaneous testing of hydrophilic abuse drugs in hair. The analysis of, codeine and methadone in morphine hair included incubation in methanol (5 hours, 50 şC ), Surfactant Enhanced Liquid Phase Microextraction (SE-LPME) and HPLC analysis. This study has demonstrated that SE-LPME constitute a real alternative to the other liquid phase microextraction methods, for pre-concentration and extraction of hydrophilic drugs in biological samples and has shown the advantages of these optimized methodologies over the traditional microextraction techniques. For these drugs recoveries in the range of 57.5-93.7 were obtained from hair. The drugs were enriched by a factor of 61-128 during SE-LPME.

Silica Sol-gel Monolithic Matrices Encapsulated with Biomolecules for Drug Development
Masaru KATO12, Kumiko Sakai-Kato3, Toshimasa Toyo''oka1
1 University of Shizuoka 2 PRESTO, JST 3 Musashino University

Recently, the sol-gel encapsulation method has attracted much attention for the development of desirable biomolecule-doped matrices for biosensors. Biomolecule are entrapped into a porous, silica matrix that is formed via a low-temperature sol-gel reaction. The encapsulated biomolecules can retain their structure and biological activity for a prolonged period. This sol-gel encapsulation method enabled to reuse expensive biomolecule reagents multiple times. Furthermore, the encapsulation method often improved the stability of the immobilized biomolecules. From these reasons, this technology has been used in various fields, and expected to contribute to the effectiveness of analytical systems and the application to the high-throughput screening systems. In this presentation, we introduce various results where biomolecules were immobilized on a capillary-, microchip-, and microarray-based analytical systems using the sol-gel reaction.

Method-Development of a Cytotoxicity Assay of Bacteria by means of Capillary Electrophoresis
U. Holzgrabe, V. Hoerr
Bayerische Julius-Maximilians-Universität, Würzburg, Germany

In 1999, Armstrong et al. developed capillary electrophoresis methods that make it possible to identify, separate and characterize microorganisms simultaneously [1]. In a series of papers, different fields of use, i.e. the identification of bacterial pathogen in urine and the analysis of customer products with active bacteria as constituent [2, 3] are described. Within the frame of a greater project and in dependence on this progress, a method capable of separating the different agglomerates of Micrococcus luteus, Neisseria cinerea, Streptococcus luteus and Pseudomonas fluorescens was established which should be the basis of a method for the determination of the potency of antibiotics using syto 9 and propidium iodide as live/dead marker [4]. Analysis was performed on a Beckman P/ACE System MDQ equipped with a 488 nm laser induced fluorescence (LIF) detector containing a 520 nm bandpass filter and a 655 nm longpass filter. The separation was carried out using a fused silica capillary (total length: 31.2 cm, detection length: 21.0 cm, internal diameter: 100 µm) and a constant voltage of 10 KV. Tris (0.78 mM), boric acid (0.78 mM), Na2EDTA (0.018 mM) with 0.0125 % poly (ethylene) oxide was applied as a buffer system at pH 8.7. In a first attempt Pseudomonas fluorescens was used for method development. Besides the results obtained with capillary electrophoresis will be compared with those using a fluorometer. [1] Armstrong, D. W.; Schulte, G.; Schneiderheinze, J. M.; Westenberg, D. J.; Anal. Chem. 1999, 71, 5465-5469. [2] Armstrong, D. W.; Schneiderheinze, J. M.; Anal. Chem. 2000, 72, 4474-4476. [3] Armstrong, D. W.; Schneiderheinze, J. M.; Kullmann, J. P.; He, L.; FEMS Microbiol. Lett. 2001, 194, 33-37. [4] Hoerr, V.; Stich, A.; Holzgrabe, U.; Electrophoresis 2004, 25, 3132-3138.

Identification of Unknown Compounds Detected in Mepivacaine Local Anesthetic Sterile Isotonic Solutions by Liquid Chromatography -Tandem Mass Spectrometry (LC-MS/MS)
Luwang Andy Zhu1, Qinhua Cindy Ru1, Jennifer Killen1, Cynthia H Shields2, Chester Buckenmaier2, Michael Liebman1
1 Windber Research Institute, Windber, PA 15963 2 Walter Reed Army Medical Center, Washington, DC 20307

Local anesthetics are used in a variety of field, austere and combat environments. It is a clinical impression that local anesthetics persistently stored at high temperatures are less effective when used for regional or neuraxial anesthesia1. Four kinds of local anesthesia sterile isotonic solutions including mepivacaine, bupvicaine, tetracaine and ropivacaine have been stored at room temperature, 42 şC and 57 şC in the controlled temperature ovens with a recording thermometer at 15, 30, 45, 60, 90 and 120 days of storage. Every sample has been assayed in triplicate to help reduce measurement error, and the results of high performance liquid chromatography (HPLC) did proved that the long term high temperature storage of sterile isotonic solutions led to the degradation of anesthetics. In addition, in the sterile isotonic solution of mepivacaine, an unknown peak was detected by HPLC assay, and it was suspected to be some by-product of the degradation. Its structure was identified via liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Refernces: 1. C.C. Buckenmaier, E.H. Lee, C.H. Shields, J.B. Sampson, J.H. Chiles, Regional Anesthesia and Pain Medicine, 28: 321-327 (2003).

Determination of Nine Sulphonamides by Capillary Zone Electrophoresis using Large Volume Stacking
A.M. Garcia-Campańa, J.J. Soto-Chinchilla, L. Gámiz-Gracia, C. Cruces-Blanco
Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Granada, Spain

Sulphonamides are widely used in farm animal feedstuff for prophylactic and therapeutic purposes. Their residues in food are of concern due to their potential carcinogenic character, possibility of development of antibiotic resistance as well as severe allergic reactions. The separation of nine sulphonamides (namely: sulphadiazine, sulphadimethoxine, sulphamethoxazole, sulphapiridine, sulphametazine, sulphachlorpiridazine, sulphameter, sulphamerazine and sulphamethizole) has been carried out with CZE-DAD in eight minutes, using paraminobenzoic acid (PABA) as internal standard. The separation conditions were as follows: electrophoretic buffer, phosphate 45 mM, pH 7.3 with 10% methanol; voltage, 25 kV; temperature, 28 şC; separation capillary, 64.5 cm, 75 µm ID. A bubble-cell was employed, enhancing three-fold the signal. Moreover, the large volume stacking (LVS) technique was used, for eliminating interferents and for pre-concentrating the sample. The LVS comprised three steps, namely: (i) Hydrodynamic filling up of the capillary with sample; (ii) Switch the voltage from positive to negative, with the aim of pre-concentrate the sample plug in the inlet of the separation capillary. This step concludes when the voltage riches the 95% of the separation voltage; (iii) Application of the separation voltage. With this LVS, a pre-concentration of about 30-fold is reached. The standard calibrations ranged 5-200 ng/ml, except for sulphapiridine which ranged 25-600 ng/ml, allowing the quantification of these products below the limit maximum of residues for these compounds (100 ng/g in the EU). The method is being applied to the determination of sulphonamides in chicken tissue and milk. Acknowledgement The National Institute of Agricultural and Food Research and Technology (INIA, Ministerio de Agricultura, Pesca y Alimentación, Projects Ref. CAL03-096-C2-2 and CAL03-087-C2-1) supported this work. LGG is grateful to the Plan Propio of University of Granada for a research contract and JJSC thank to La Caixa for a grant.

Determination of Azo Colorants by Capillary Zone Electrophoresis and Ion-pair Liquid Chromatography
Shu-Ping Wang, Nai-Yun Liang
Providence University

Most of azo dyes are produced by synthesis, which are widely used as colorant agents in living life. Although these compounds do not possess instant toxicity, they still may be accumulated and caused cancer subsequently. In this study, the separation and determination methods of thirteen azo colorants by capillary zone electrophoresis (CZE) and ion-paired liquid chromatography (IPC) will be proposed. The separation of IPC was optimized by addition of 5 mM tetra-n-butylammonium hydrogen sulfate. The linear range of 0.1~60.0 µg/mL, correlation coefficient from 0.9978 to 0.9999, detection limit above 0.1 µg/mL, and shorter analytical time were achieved. The characteristic of CZE is using of dual CD. Moreover, the mechanism of migration behavior in separation using different CD was also discussed. The optimal electrolyte buffer was 46 mM sodium tetraborate buffer solution (pH 9.5), 4 mM β-CD, 2 mM a-CD, and 50 mM urea. Under this condition, linear range of 0.3~160.0 µg/mL, correlation coefficient from 0.9961 to 0.9994, and detection limit above 0.23 µg/mL were obtained. Finally, both optimum analytical methods of CZE and IPC were applied to analysis of cosmetic products, and the comparison of the two methods was performed. There was no significant difference indicated in results by means of paired t-statistical test at 90% confidence level. The more importance in this work, the mechanism of migration and retention behavior of various functional groups in separation was discussed.

A Stability-indicating High Performance Liquid Chromatographic assay for the determination of Orlistat in capsules.
A. Mohammadi1, I. Haririan2, N. Rezanour3, R.B. Walker4
1Department of Food and Drug Quality Control, Faculty of Pharmacy, Medical Sciences University of Tehran, Tehran, Iran 2Department of Pharmaceutics, Faculty of Pharmacy, Medical Sciences University of Tehran, Tehran, Iran 3Research and Development Laboratory of Iran Daru Pharmaceutical Ind.Co. Tehran, Iran 4Department of Pharmaceutics,Faculty of Pharmacy, Rhodes University, Grahamstown, South Africa

Orlistat, N-Formyl-L-leucine(1S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxetanyl]methyl] dodecyl ester,is a novel, non-systemically absorbed, antiobesity agent which selectively inhibits the absorption of approximately 30% of fatty components from the diet [1].There is a dearth of analytical methods reported in the literature. HPLC methods for the quantitative determination of orlistat in plasma using tandem mass spectrometry have been reported [2,3]. To our knowledge, no stability - indicating analytical method for the determination of orlistat in dosage forms has been published.A stability - indicating HPLC method was developed and validated for the quantitative determination of orlistat in capsule dosage forms. An isocratic separation was achieved using a Perfectsil® target ODS - 3, 250 mm x 4.6 mm i.d., 5 µm particle size column with a flow rate of 0.7 mL/min and using a UV detector to monitor the eluate at 210 nm. The mobile phase consisted of methanol: acetonitrile: trifluoroacetic acid (82.5: 17.5:0.01,v/v/v).The drug was subjected oxidation, hydrolysis, photolysis and heat to apply stress conditions. Complete separation was achieved for the parent compound and all degradation products in an overall analytical run time of approximately 15 min with the parent compound orlistat eluting at approximately 9 min. The method was linear over the concentration range of 0.02 - 0.75 mg/mL(r = 0.9998) with a limit of detection and quantitation 0.006 and 0.02 mg/mL, respectively. The method has the requisite accuracy, selectivity, sensitivity and precision to assay orlistat in capsules. Degradation products resulting from the stress studies did not interfere with the detection of orlistat and the assay is thus stability-indicating. References: [1] L.J. Aronne, J.Amer.Diet.Assoc.98 (1998) A13. [2] P.K. Bennett, Y.T.Li, R. Edom, J. Henion, J.Mass Spectrom.32 (1997) 739-49. [3] R. Wieboldt, D.A. Campbell, J. Henion, J Chromatogr. B. Biomed. Sci. Appl.708 (1998) 121-9.

Separation and Quantitation of the Four Stereoisomers of Itraconazole in Pharmaceutical Formulations by Electrokinetic Chromatography
M. Castro-Puyana, A.L. Crego, M.L. Marina
Department of Analytical Chemistry, Faculty of Chemistry, University of Alcalá,Alcalá de Henares (Madrid), Spain.

Itraconazole is a triazole antifungal drug with a broad spectrum of activity against most human fungal pathogens. This compound has three chiral centers; however, the drug is in fact a mixture of two racemates, i.e., four stereoisomers, all of them with the cis configuration. Different studies have indicated that the stereoselective metabolism of itraconazole can be an important modulator of biological effects of the drug. Nevertheless, it is used clinically as a stereoisomeric mixture. Since enantiomers differ in their biological activity, the development of analytical methodologies for the enantiomeric separation of itraconazole has received a considerable attention. Due to its high separation efficiency and flexibility, Capillary Electrophoresis (CE) has experienced an enormous growth in the field of chiral separations. The purpose of this study was to develop a method for the separation of the four stereoisomers of itraconazole by CE. Electrokinetic chromatography (EKC) using a cyclodextrin (CD) as chiral selector was employed. The novel developed method enabled for the first time the separation of the four stereoisomers of itraconazole by CE. After a screening of different neutral CDs, the best separation was carried out using heptakis-2,3,6-tri-O-methyl-β-CD. The use of a 100 mM phosphate buffer (pH 2.5), 30 mM in heptakis-2,3,6-tri-O-methyl-β-CD together with an applied voltage of 30 kV and a temperature of 20şC enabled the separation of the stereoisomers of itraconazole with high resolutions (Rs > 3.0) and an analysis time of about 30 min. The chiral CE method was validated and applied to the quantitative analysis of all stereoisomers in pharmaceutical formulations of itraconazole.

Capillary electrophoresis method for testing of chiral purity and related substances of etomidate
M. Hammitzsch, R. Nageswara Rao, G.K.E. Scriba
University of Jena, School of Pharmacy, Department of Pharmaceutical and Medicinal Chemistry, Philosophenweg 14, 07743 Jena, Germany

A chiral capillary electrophoresis assay for the simultaneous testing of the optical purity and of related substances of the intravenous anaesthetic etomidate has been developed and validated. The method was optimized using two chiral selectors, ß-cyclodextrin and sulfated ß-cyclodextrin. Optimal conditions were found after examining the resolution power of native ß-cyclodextrin and ß-cyclodextrin derivatives. Best resolution was achieved by setting the concentrations of ß-cyclodextrin at 14 mg/ml and of sulfated ß-cyclodextrin at 1.0 mg/ml, using a 200 mM phosphate buffer, pH 2.1, and applying a voltage of 28 kV at 16°C. All impurities, i.e. the S-enantiomer of etomidate, R-metomidate and the free acid, could be well detected at the 0.2 % level required by the European Pharmacopoeia. Validation was performed with respect to specificity, linearity, range, limit of quantification and detection, precision and accuracy. Statistical experimental design was applied for robustness testing.

Estimation of the composition of recombinant human erythropoietin mixtures using capillary electrophoresis and multivariate calibration methods
E. Giménez, F. Benavente, A.C. Olivieri1, J. Barbosa, V. Sanz-Nebot
Universitat de Barcelona, Barcelona, Spain 1 Universidad Nacional de Rosario, Rosario, Argentine

Erythropoietin (EPO) is a glycoprotein hormone produced by the kidney that stimulates erythropoiesis. Recombinant human EPO (rHuEPO) is used to treat certain forms of anemia but it has become especially popular among endurance athletes due to its use as a performance-enhancing agent [1]. EPO exists as a mixture of glycoforms that differ in composition, nature and charge of the carbohydrate moieties attached to the polypeptide core. Current pharmaceutical rHuEPO products available for clinical use differ in their pattern of glycosilation. This is the case of epoetin beta which it is synthesized in Chinese hamster ovary cells (CHO) as epoetin alfa, but it contains a greater proportion of more basic glycoforms [3]. Capillary Electrophoresis (CE) has been successfully used to separate rHuEPO glycoforms [3]. The CE methodology proposed by the European Pharmacopoeia, using putrescine dynamic coating has become extensively used for quality control of rHuEPO because a different electrophoretic profile obtained for a new product can be easily and directly compared with a reference standard. However, this task can be more difficult to perform when a mixture of erythropoietins is analyzed, because glycoforms corresponding to each glycoprotein migrate in the same electropherogram region, and even in some cases their electrophoretic profiles are highly overlapped. The present work is focused on the possibilities of multivariate calibration using partial least squares (PLS) in order to characterize mixtures of EPO based on the analysis of the electrophoretic profiles obtained using the CE methodology above mentioned. PLS calibration model was developed and validated using mixtures of alfa and beta rHuEPO [4]. Due to the limited reproducibility of migration times and peak areas [3], glycoforms were identified according to their effective electrophoretic mobility values and the normalized overall area of each glycoform band was used as multivariate data. PLS-1 model was used for determination of the percentages of rHuEPO alfa and beta in the rHuEPO provided by the European Pharmacopoeia. The result was in good concordance with the specifications given by the manufacturer, and it shows the potential of using multivariate data analysis for a quantitative unambiguous determination of the composition of glycoprotein mixtures that differ in their glycosilation pattern, such as the mixture of recombinant and endogenous EPO contained in a positive athlete’s sample in doping control. [1]Lasne, F., Martin, L., Crespin, N. and de Ceaurriz, J., Anal. Biochem., 311 (2002) 119. [2]Storring, P. L., Tiplady, R. J., Gaines Das, R. E., Stenning, B. E., Lamikanra, A., Rafferty, B., Lee, J., Br. J. Haematol., 100 (1998) 79. [3]Sanz-Nebot, V., Benavente, F., Giménez, E. and Barbosa, J., Electrophoresis, 26, (2005) 1451. [4]Damiani, P.C., Moschetti, A. C., Rovetto, A. J., Benavente, F., Olivieri, A. C., Anal. Chim. Acta, 543 (2005) 192

Kinetics and Stereoselectivity of CYP2D6 Propafenone Hydroxylation and N-dealkylation Determined by Enantioselective Capillary Electrophoresis
M. Afshar, W. Thormann
University of Bern, Bern, Switzerland

An enantioselective capillary electrophoresis (CE) method was developed and used to identify the ability of the human CYP2D6 enzyme in catalyzing the transformation of propafenone (PPF) to 5-hydroxy-propafenone (5OH-PPF) and N-despropyl-propafenone (NOR-PPF). The CE assay is based upon liquid-liquid extraction at alkaline pH followed by analysis of the reconstituted extract by CE in presence of a pH 2.0 running buffer composed of 100 mM sodium phosphate, 19 % methanol and 0.6 % highly sulfated β-cyclodextrin. For each compound, the S-enantiomers are shown to migrate ahead of their antipodes, and the overall run time is about 30 min. In vitro incubations of individual PPF enantiomers and racemic PPF with CYP2D6 SUPERSOMES were investigated. With the exception of the dealkylation in presence of R-PPF only, which can be described by the Michaelis-Menten model, all CYP2D6 induced reactions were found to follow autoactivation (sigmoidal) kinetics. The formation of NOR-PPF is stereoselective and is reduced significantly when racemic PPF is incubated. Clearance values obtained for the hydroxylation are not stereoselective. This paper reports the first investigation of the PPF hydroxylation and dealkylation kinetics by the CYP2D6 enzyme and represents the first report in which enantioselective CE data provide the complete in vitro kinetics of metabolic steps of a drug.

Elucidation of the Stereoselectivity of the Lorazepam Glucuronidation in Man
A. Baldacci, W. Thormann
University of Bern, Bern, Switzerland

Lorazepam is a 3-hydroxy-1,4-benzodiazepine that is chiral and undergoes enantiomerization at temperatures above 0 °C. In man, about 75 % of the administered dose of lorazepam is excreted in the urine as its 3O-glucuronide. As the 3O-glucuronidation reaction occurs at the chiral center of the molecule, two diastereoisomers can theoretically be formed, molecules that can no longer interconvert. The stereoselective formation of lorazepam glucuronides in humans and in vitro was investigated. Micellar electrokinetic chromatography (MEKC) analysis of an extract of the non-hydrolyzed urine of a volunteer who ingested 2 mg lorazepam suggested the presence of the two different lorazepam glucuronides in the urine. The formation of the same two diastereoisomers was also observed in vitro employing an incubation of lorazepam with human liver microsomes in presence of uridine 5’-diphospho-glucuronic acid as coenzyme. Both results revealed a stereoselectivity, one diastereoisomer being formed in a higher amount than the other. After enzymatic hydrolysis using beta-glucuronidase, these peaks could not be detected any more. Instead, lorazepam was monitored. Analysis of the extract prepared from the enzymatically hydrolyzed urine by MEKC in presence 2-hydroxypropyl-beta-cyclodextrin revealed the enantiomerization process of lorazepam (observation of two peaks of equal magnitude connected with a plateau zone). The data presented provide for the first time the evidence of the stereoselectivity of the lorazepam glucuronidation in man.

Characterization of the stereoselective biotransformation of ketamine to norketamine via determination of their enantiomers in equine plasma by capillary electrophoresis
M. Knobloch, R. Theurillat, O. Levionnois, P. Larenza, M. Mevissen, U. Schatzmann, W. Thormann
University of Bern, Bern, Switzerland

Ketamine is an intravenous analgesic and dissociative anesthetic drug widely used in clinical practice of man and animals. Ketamine is chiral and is typically administered as racemate. A robust capillary electrophoresis (CE) method for the simultaneous determination of the enantiomers of ketamine and its main metabolite, norketamine, in equine plasma has been developed. It is based upon liquid-liquid extraction of ketamine and norketamine at alkaline pH from 1 mL plasma followed by analysis of the reconstituted extract by reversed polarity CE in presence of a pH 2.5 Tris/phosphate buffer containing 10 mg/mL highly sulfated beta-cyclodextrin as chiral selector. The limit of detection for all enantiomers is 10 ng/mL. After i.v. bolus administration of 2.2 mg/kg racemic ketamine, the assay is demonstrated to provide reliable data for plasma samples of ponies under isoflurane anesthesia, of ponies premedicated with xylazine and of one horse that received romifidine, L-methadone, guaifenisine and isoflurane. In animals not premedicated with xylazine, the ketamine N-demethylation is demonstrated to be enantioselective. The concentrations of the two ketamine enantiomers in plasma are equal whereas S-norketamine is found in a larger amount than R-norketamine. In the group receiving xylazine, data obtained do not reveal this stereoselectivity. Enantioselective data of ketamine and norketamine in equine plasma obtained after bolus and long term i.v. administration of ketamine are used for pharmacokinetic modelling.

Capillary Electrophoresis Characterization of the Liposomal Distribution of a Neutral Hydrophobic Drug involving Selective Electroosmotic Injection and on Line Preconcentration
Pierre Gareil1, Thomas Le Saux1, Anne Varenne1, Nathalie Bargmann-Leyder2, Laurent Duhau2
1 Laboratory of Electrochemistry and Analytical Chemistry, ENSCP, UMR CNRS 7575, Paris , France 2 Sanofi-Aventis, Analytical Sciences Department, Centre de Recherche de Paris, Vitry-sur-Seine , France

Liposomes are aggregates composed of one or more phospholipidic bilayer(s) surrounding an internal aqueous cavity. This particular structure enables to entrap wide variety of molecules in the bilayer(s), in the internal cavity or at the interface of the two compartments. This property has led to growing interest for liposomes in a variety of domains including biology (cell models) and pharmaceutics (drug partitioning model, drug targeting). Analytical methods able to monitor analyte incorporation into liposomes with reduced operational time and sample consumption are therefore strongly needed. This work describes an original integrated capillary electrophoresis (CE) protocol for such a purpose. The method consists of a selective electroosmotic introduction of the free analyte incorporating an internal calibration of sample volume followed by on-line preconcentration and transport, applying the sweeping concept. It was designed to preserve the liposomal distribution, avoid deleterious effects of liposome adsorption onto the capillary wall and to enhance detection sensitivity of the free analyte. This protocol was first developed with three homologous model compounds (alkyl parahydroxybenzoates) and validated with respect to frontal analysis continuous capillary electrophoresis and ultrafiltration coupled to reversed phase liquid chromatography (UF-RPLC). It has been then successfully applied to the determination of the free form of a biologically active ingredient in a real pharmaceutical formulation. The results obtained by the integrated CE method were again in good concordance with those provided by UF-RPLC. Finally the newly developed integrated protocol brings major advantages over UF-RPLC, in terms of easiness of implementation, automation, speed and reliability.

Determination of pKa Values by CZE with Dynamically Coated Capillary: a Performing Tool in Physicochemical Profiling of Drug Candidates
Y. Henchoz1, L. Geiser1, P.A. Carrupt2, J.L. Veuthey1
1 Laboratoire de Chimie Analytique Pharmaceutique, EPGL, Université de Genčve, Bd d’Yvoy 20, CH-1211 Genčve 4, Suisse 2 LCT-Pharmacochimie, EPGL, Université de Genčve, Quai Ernest-Ansermet 30, CH-1211 Genčve 4, Suisse

Evaluating physicochemical properties of new chemical entities (NCE’s) at an early stage of drug development is a new strategy to reduce attrition rates due to poor biopharmaceutical properties. Among these properties, ionization constants (pKa) are needed to predict ADME (absorption, distribution, metabolism and excretion) behaviour, in particular to understand pH-related permeation mechanisms and solubility characteristics. Capillary zone electrophoresis (CZE) permits the measurement of pKa values in a simple automated way. Moreover, this method does not require a good sample purity and needs only a low amount of solvent and sample. A plot of the effective mobility measured versus pH of the background electrolyte (BGE) enables the determination of pKa values. Due to the tremendous increase of NCE’s, there is a particular demand for rapid physicochemical profiling. A strategy was investigated to reduce the time needed for the pKa determination by CZE. For this purpose, short-end injection on a dynamic coated capillary was used. The short-end injection technique reduced drastically the analysis time while the dynamic coating procedure allowed a large and constant electroosmotic flow whatever the pH of the buffer. A series of 19 BGEs with the same ionic strength was employed, covering a pH range from 1.4 to 11.3. Under these conditions, determined pKa values of various compounds ranging from 1.3 to 10.0 were in good agreement with the literature values. Therefore, short-end injection and dynamic coating procedure are of great interest for routine pKa value determination on a conventional instrument.

Recent Developments in the CE Separation of Enantiomers Using Single-Isomer Chiral Resolving Agents
Gy. Vigh, M.B. Busby, S. Lee, K. Nzeadibe, E. Tutu
Texas A&M University, College Station, TX, USA

In order to diversify the enantioselectivities of the available chiral resolving agents in CE, we have synthesized new, single-isomer sulfated alpha-cyclodextrin derivatives (hexakis(2,3-diacetyl-6-sulfo)cyclomaltohexaose, hexakis(2,3-dimethyl-6-sulfo)cyclomaltohexaose and hexakis(6-sulfo)cyclomaltohexaose), single-isomer sulfated beta-cyclodextrin derivatives (heptakis(2-methyl-3-acetyl-6-sulfo) cyclomaltoheptaose and heptakis(2-methyl-6-sulfo)cyclomaltoheptaose) and new, single-isomer, multiply-charged, quaternary ammonium beta-cyclodextrin derivatives (mono-6-deoxy-6-N,N,N’,N’,N’-pentamethyldiethylammonio-cyclomaltoheptaose and heptakis(6-deoxy-6-N-morpholinio)cyclomaltoheptaose) using sequentially-applied protecting group chemistry. Each intermediate and final product was thoroughly characterized by HPLC, indirect UV-detection capillary electrophoresis, 2D 1H and 13C NMR spectroscopy, and MALDI-TOF-MS and ESI-MS. When possible, single crystals of the pure products were grown to obtain the respective X-ray crystal structures. The new, single-isomer resolving agents were used for the CE separation of the enantiomers of acidic, basic, ampholytic and neutral analytes in aqueous low pH and high pH background electrolytes according to the guidance of the charged resolving agent migration model (CHARM model).

Recent Progress in Computerized Design of Robust MicroScale Separations
Dr. Imre Molnar
Molnar Institut für angewandte Chromatographie

Chemical, food and pharmaceutical products are today controlled to more than 80-90% with Reversed Phase Chromatography. The quality of the products is depending on the quality of the RP-methods. We need therefore robust and reliable RP-methods. The method optimization process in HPLC is a complex task and has many ways to achive the desired goal. Lloyd Snyder described several routes, which are today followed by the practicing chromatographer. The work of Heinz Engelhardt, studying the role of a wide scale of stationary phases helped to understand the complexity of retention process using new types of column packing materials. In industrial environment the first priority is adequate product quality. The "validation process" helps to establish reliable working conditions for the method. Later only minor changes will be accepted, which were clearly defined in the method description. However once a method is validated, its use in other laboratories can become often a problem. In some cases the results are not those, which are expected according to the method description. Such differences are found frequently between retention times, and more often in critical resolution values. In such cases the second laboratory has the problem to fix the method, to be able to produce the data, which have been expected. Sometimes it is possible to get the method working, if the right changes are done. Sometimes however this is not possible in a reasonably short period of time. There are several checks needed to find the source for such differing results. Columns and equipments are in most cases in order, as shown after tests with known mixtures, in a system suitability test (SST). Systematic investigation of the eluent parameter show, that small changes in the type of chemical bonding of the stationary phase, in eluent conditions, such as pH, temperature, form of the gradient, type of organic eluent, or in the buffer concentration, have a strong influence on the selectivity of the separation system. Peak tracking of multiple runs can be helpful to recognize peak position changes. The robustness of an HPLC method can be expressed in one and two-dimensional resolution maps as the ratio of the critical resolution at a working point to the critical resolution in an allowed distance from that point in a validated window of a working parameter. The resolution maps are helpful to define the critical parameters, which in turn allow to control the method more carefully. In this way, robust and transferrable methods are easier to be generated and communication between different groups using the same method is greatly facilitated. Practical examples of peak tracking and robustness studies will be presented.

Identification of a New Ligand for the Amyloidogenic Protein BETA-2 Microglobulin
C. Carazzone1, R. Colombo1, M. Quaglia13, S. Giorgetti23, V. Bellotti23, E. De Lorenzi1
1 Department of Pharmaceutical Chemistry, School of Pharmacy, University of Pavia, Italy. 2 Department of Biochemistry, School of Pharmacy, University of Pavia, Italy. 3 Biotechnology Laboratories, IRCCS, S.Matteo Hospital, Pavia, Italy.

The amyloidogenic potential of beta2-microglobulin (β2-m), a protein involved in dialysis-related amyloidosis (DRA), is due to the misfolding of the protein in pathological conditions. This occurs through the formation of folding intermediates and oligomers which lead to the generation of amyloid fibrils. A therapeutic approach could be the stabilization of the protein through the binding with a small molecule, in order to prevent the conformational changes responsible for the onset of the disease. Suramin is the only non-polypeptidic compound of pharmaceutical interest that we recently discovered to weakly bind β2-m. Part of a chemical library of 1000 compounds, including suramin analogues and substances of a diverse subset, has been thus screened in order to identify potential ligands of the protein. Given the affinity of suramin for β2-m, the drug has been used as reference standard for the evaluation of the binding properties of all the analysed compounds. As screening techniques, ultrafiltration and affinity capillary electrophoresis have been optimized. Our experiments led us to identify one molecule, called 573 and structurally classified as suramin analogue, able to bind β2-m. Both techniques have cross-confirmed the data obtained. Once the interaction between β2-m and 573 has been identified, studies have been carried out in order to characterize the binding affinity of the molecule for the protein and the results obtained have been compared with suramin data. Binding constants have been calculated through affinity capillary electrophoresis and surface plasmon resonance and the values obtained for both 573 and suramin are of the same order of magnitude, indicating that, albeit weak, the interaction takes place and can be quantitated. The potential of CE in separating conformational isoforms of proteins has been exploited to determine the interaction of the ligands with the native form of β2-m and for a folding intermediate as indipendent targets. The possibility of experimentally monitoring the interconversion of these two forms by CE allowed us for refolding kinetics studies for β2-m also in the presence of the newly identified ligand 573. Results indicate that 573, contrary to suramin, accelerates the refolding kinetics and this makes it a promising hit for further studies.

Chiral Separation of Four Fluoroquinolone Compounds using Capillary Electrophoresis
Willy R.G. Baeyens, Shanshan Zhou, Jin Ouyang
Department of Pharmaceutical Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium

Fluoroquinolone compounds including gatifloxacin, lomefloxacin, pazufloxacin, and ofloxacin (Fig.1) are most effective for the treatment of various bacterial infections, and they are generally marketed as racemates [1-2]. Differences in antibacterial activity of fluoroquinolone isomers are well-known [3]. Some reports discussed on the enantioseparation of fluoroquinolones by HPLC after the addition of chiral selectors to the mobile phase, using a chiral stationary phase, or by derivatisation of the analyte with chiral reagents [4-5]. In this communication a direct and relatively simple method is being described for the capillary electrophoretic chiral separation of fluoroquinolone enantiomers within about 12 min of migration time. Figure.1. Structure of the fluoroquinolones under investigation The four fluoroquinolones studied were well enantioseparated using a 50 cm (35 cm to detector)x50 mm I.D. fused-silica capillary and hydroxypropyl-β-cyclodextrin (HP-β-CD) as selector. A buffer consisting of 70 mM phosphate and 40 mM HP-β-CD at pH 3.90 was found highly efficient in the electrophoretic system for the separation of gatifloxacin, at pH 3.75 for lomefloxacin, at pH 4.90 for pazufloxacin and at pH 2.16 for ofloxacin (Fig. 2). To the best of our knowledge, this is the first report on the direct chiral separation of the enantiomers of the cited fluoroquinolones by capillary electrophoresis.

Retention behaviour and separation of flavonoid aglycones in mixed MEKC systems
C. Repollés, J.M. Herrero-Martínez, C. Rŕfols
Dept. of Analytical Chemistry, Universitat de Barcelona, 08028 Barcelona, Spain

Flavonoids are one of the largest groups of natural polyphenols widespread present in plants, that comprise an important part of our daily diet [1, 2]. The interest of these compounds lies in their wide pharmacological activities (e.g., antimicrobial, anti-allergic, anti-inflammatory, antiviral, anticarcinogenic, etc). They have structures based on 2-phenylbenzopyrone, and differ in their pattern of hydroxylation, methylation, and glycosilation, in the degree of unsaturation and in the type and position of sugar links [2]. In particular, we have based our study in several classes of flavonoid aglycones (without sugar component), among them flavanols, flavonols, flavones, flavanones and monohydroxyflavones, which are commonly present in food samples. The migration behaviour and separation of several flavonoid aglycones are investigated by micellar electrokinetic chromatography (MEKC) in the pH range 5.0-9.0 using different surfactants (sodium dodecyl sulfate, bile salts, and mixed surfactants systems). The influence of surfactant nature and buffer pH, as well as the choice of background electrolyte, on the separation and selectivity of analytes are examined. The effect of their structure (molecular size, number, position and acidity of free hydroxyl groups) and solute-micelle interaction, including hydrophobic, electrostatic and hydrogen bonding interactions are discussed in order to establish (elucidate) the selectivity changes observed (differences in their retention behaviour) in the studied MEKC systems. Also, the most suitable working conditions for separation of these compounds are applied to the analysis of these compounds in different samples. References 1. Robards, K., Antolovich, M., Analyst 1997, 122, 11R-34R. 2. Harborne, J.B. (Ed.), The Flavonoids: Advances in Research Since 1986, Chapman and Hall, London, 1994.

Development and optimization of a CE-MS/MS method for the determination of eight quinolones of veterinary use
F.J. Lara, A.M. García-Campańa, F. Alés-Barrero, J.M. Bosque-Sendra
Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Spain

Quinolones are synthetic antibiotics widely employed in both human and veterinary medicine for the treatment of pulmonary, urinary and digestive infections. The presence of quinolone residues in edible animal tissues could cause resistant human pathogens and hypersensitivity reactions. The European Union has set maximum residue limits (MRL) for these substances in foodstuffs of animal origin by means of the directive 2377/90/EEC. Therefore, techniques which provide unambiguous identification of quinolones are needed. In this sense, mass spectrometry coupled to CE can achieve the minimum number of identification points required for a reliable determination according to the 2002/657/EC European decision. We proposed a CE-ESI(+)-MS/MS(IT) method for the determination of danofloxacin, sarafloxacin, ciprofloxacin, marbofloxacin, enrofloxacin, difloxacin, oxolinic acid and flumequine at MRL levels. Electrophoretic separation was carried out taking into account compatibility with MS detection and a buffer consisting in 70 mM ammonium acetate at pH 9.1 was finally selected. Parameters affecting MS detection, such as nebulizer pressure, flow and temperature of the drying gas, and flow and composition of the sheath liquid, were optimized using experimental design to consider the possible interactions. In order to reduce the experimental work, a previous screening design 2^6-1 was carried out and it was concluded that the percentage of formic acid in the sheath liquid and the temperature of the drying gas were not significant in the studied range. Then, a Doehlert design was applied to obtain the optimum values for the significant factors in the response selected (signal to noise ratio of danofloxacin). Finally, and after optimizing the fragmentation and quantification of the selected quinolones, the method could be applied in food samples of animal origin using MRM mode. Acknowledgements The National Institute of Agricultural and Food Research and Technology (INIA, Ministerio de Agricultura, Pesca y Alimentación, Project Ref. CAL03-087-C2-1) supported this work. Francisco J. Lara thanks the Junta de Andalucía for a FPI grant.

Using HPLC and modern Voltammetric Techniques for Identification and separation of some Tea constituents.
N.Abo El-maali, D.Abd el-Hady
Assiut University, Assiut, Egypt

A comparison has been esabilshed for identification of some Tea's components using both voltammetry and HPLC. It is found that bioling tea has a bad effect on the constituent of some of its components.Both Voltammetry and HPLC have been successfuly applied to follow up such mechanism. Separattion and identification of such compounds was successfuly achieved and calibration plots have been constructed. Staistical parameters e.g. F-test and Q-test have been also calculated using MiniTab software.

Micro-Scale Self-Interaction Chromatography: A Bioseparation Process Design Tool
Tangir Ahamed, Lin Luo, Marcel Ottens, Gijs W.K. van Dedem, Luuk A.M. van der Wielen
Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands

Self-interaction chromatography (SIC) measures the interaction of immobilized protein molecules in the stationary phase with free protein molecules in the mobile phase. The average retention of a protein pulse represents the protein’s self-interaction, as it takes place in real solution. Higher retention than usual corresponds to attraction between stationary proteins and mobile proteins, and vice versa. Therefore, thermodynamic non-ideality of a protein solution can be well characterized by SIC methodology in term of potential of mean force, activity coefficient or osmotic second virial coefficient (B2). It was shown in previous study that SIC can measure B2 values efficiently with a comparatively minimum error limit [1]. The B2 along with protein solubility data is enough to predict and design protein crystallization or precipitation process [2]. Recent study shows that a number of downstream separation processes, for instance crystallization/precipitation, aqueous two-phase separation, size-exclusion chromatography, hydrophobic interaction chromatography, reversed-phase chromatography, can be described with these thermodynamic parameters and consequently be designed using SIC tool [3]. Therefore, B2 can be translated across the boundaries of different separation techniques using simple thermodynamic models. For example B2 can be interrelated to activity coefficient, solubility and reversed-phase chromatography distribution coefficient, which are important data for an integrated way of bioseparation process development. The presently existing experimental scale and throughput of SIC is an obstacle to allow extensive screening of protein’s thermodynamic data. Therefore, a micro-scale SIC methodology is proposed in this paper. Some preliminary experimentation suggests that the micro-scale SIC could mimic the presently existing SIC in term of their data reproducibility, whereas the experimental throughput increases by at least an order of magnitude. [1] Ahamed T., Ottens M., van Dedem G.W.K., van der Wielen L.A.M. J. Chromatogr. A 1089 (2005) 111-124. [2] George A., Chiang Y., Guo B., Arabshahi A., Cai Z., Wilson W.W. Methods Enzymol. 276 (1997) 100-110. [3] Ahamed T., Ottens M., Nfor B.K., van Dedem G.W.K., van der Wielen L.A.M. Fluid Phase Equilibr. Accepted.

Enantioseparation of nonsteroidal anti-inflammatory drugs using single-isomer amino cyclodextrin derivatives in nonaqueous capillary electrophoresis
M. Fillet1, I. Fradi1, M. Pedrini1, P. Chiap1, R. Iványi2, J. Crommen1, A-C. Servais1
1Department of Analytical Pharmaceutical Chemistry, Institute of Pharmacy, University of Ličge, CHU, B36, B-4000 Ličge 1, Belgium. 2Cyclolab Cyclodextrin R&D. Laboratory Ltd. P.O. Box 435, H-1525 Budapest, Hungary.

The enantiomeric separation of a series of acidic pharmaceuticals (mostly nonsteroidal anti-inflammatory drugs) has been investigated in nonaqueous capillary electrophoresis (NACE) systems using single-isomer amino
b-cyclodextrin (CD) derivatives. The first part of this study consisted in the selection of the basic experimental conditions to separate efficiently the enantiomers of acidic drugs. Several parameters, such as the nature of the ionic BGE components, were studied and a methanolic solution of ammonium acetate containing the cationic CD was selected as background electrolyte. A D-optimal design with 20 experimental points was then applied and the nature and concentration of the CD were found to have a significant effect on the enantiomeric resolution for all studied compounds. It also appeared that the influence of ammonium acetate concentration was related to the effect of the CD concentration for most of the compounds. On the other hand, Rs values were always higher with 6-monodeoxy-6-mono(3-hydroxy)propylamino-b-CD (PA-b-CD) compared to those obtained with 6-monodeoxy-6-mono(2-hydroxy)propylamino-b-CD (IPA-b-CD). However, the latter led to shorter migration times. Generic NACE conditions were then selected by means of the multivariate approach in order to obtain the highest Rs values in a minimum amount of time. Finally, dependence of separation selectivity, resolution as well as mobility difference on chiral selector concentration were discussed and binding constants with PA-b-CD were determined for the two enantiomers of one of the model compounds, suprofen in these NACE systems.

Development of Capillary Electrophoresis Method for the Determination of Related Impurities in Montelukast Sodium.
Yuliya Shakalisava, Fiona Regan
School of Chemical Sciences, Dublin City University, Glasnevin, Dublin 9. Ireland

Capillary electrophoresis (CE) offers the possibility of fast, cheap and reproducible separations for pharmaceutical preparations. Montelukast sodium is a specific cycteinyl leukotriene receptor antagonist. It was developed as a therapeutic agent for the treatment of bronchial asthma by Merck & Co. and for effective multi-aspect asthma control. Pharmaceutical impurities occur as part of processing of pharmaceutical products. An HPLC method by Radhakrishna et al. [1] for the determination of the related impurities in montelukast sodium is about 35 min long and requires big volumes of organic solvents as 80% of mobile phase is acetonitrile. For the first time this paper demonstrates the development of an alternative method for the separation of the related impurities in montelukast sodium by capillary electrophoresis. The separation is evaluated using several modes of CE: capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC) and cyclodextrin modified MEKC (CD-MEKC). The capillary electrophoresis method is highly effective and advantageous over the existing HPLC method. The CE separation is performed in 3 min, and combined with the rinsing steps for the capillary it is still 5 times shorter than HPLC method. The efficiency of the montelukast sodium peak is 250,000 theoretical plates, which is 70 times greater than the efficiency observed for the HPLC separation method. The % relative standard deviation of the migration time of the montelukast sodium is 0.14% (n=3), which proves it to be a highly reproducible method. 1. T. Radhakrishna, a. Narasaraju, M. Ramakrishna, A. Satyanarayana, J. Pharm. Biomed Anal. 00 (2003) 1- 10.

Stability studies of recombinant human Growth Hormone in pharmaceutical preparations by capillary zone electrophoresis with bilayer-coated capillaries
Javier Sastre Torańo, Jonatan R. Catai, Gerhardus J. de Jong, Govert W. Somsen
Dept. Biomedical Analysis, Utrecht University, Utrecht, The Netherlands

Pharmaceutical regulatory agencies, such as the European Pharmacopoeia, are starting to recognize capillary zone electrophoresis (CZE) as an effective means for the analysis of biopharmaceuticals [1,2]. CZE has a unique, charged-based separation mechanism which can be very useful for the study of protein degradation and glycoforms. Furthermore, CZE uses only minute amounts of sample and provides fast separations and high efficiency. However, interactions of proteins with the internal capillary wall may lead to band broadening, unstable electoosmotic flow (EOF) and poor migration-time reproducibility. This brings serious problems when monitoring the quality of biopharmaceutical preparations and comparing samples. An effective way to avoid these problems can be the coating of the capillary with polybrene (PB) and poly(vinyl sulfonate) (PVS), forming a bilayer of charged polymers. This double coating has shown excellent migration time reproducibility and high plate numbers for proteins [3]. Based on this work, we have developed an improved method for studying the stability of the recombinant human growth hormone (hGH) by CZE. Test samples of hGH were prepared by degradation with 3% hydrogen peroxide or by prolonged exposure to heat (40 °C). Aliquots were taken at appropriate time intervals and analyzed by CZE using PB-PVS coated capillaries and 400 mM TRIS phosphate (pH 8.5) as background electrolyte. Repeatable profiles with narrow and symmetrical peaks were obtained. CZE of hydrogen peroxide treated samples revealed 8 degradation products after 18h. Heated samples showed growing amounts of deamidated and dideamidated hGH in time. After 100h, deamidated hGH started to degrade further, resulting in a total of 10 peaks after 170h. The sum of the peak areas of the degradation products was in good correspondence with the decrease of the peak area of the mother compound hGH. These results show the potential of the system for quantitative degradation analysis in time. The applicability of the system is illustrated by the analysis of commercially hGH preparations, showing varying degree of degradation. Compared to the Pharmacopoeia method using bare fused silica capillaries [1], the resolution was improved, total analysis times were shorter (8.5 min vs. 24 min), plate numbers were higher (55000 vs. 300000) and migration time reproducibility was better (RSD=1.1% vs. RSD=8%). Moreover, additional degradation products were revealed demonstrating the feasibility of the system for biopharmaceutical quality control. [1] Monograph: Somatropin for injection, European Pharmacopoeia 5.3 2005, 3619-3621. [2] Monograph: Erythropoietin concentrated solution, European Pharmacopoeia 5.3 2005, 3494-3498. [3] Catai, J.R., Tervahauta, H.A., De Jong, G.J., Somsen, G.W., J. Chromatogr. A 2005; 1083, 185-192.

Analysis of Ce fotaxime and Cefoperazone Preparations by Capillary Electrophoresis
A.V. Shpak, A.I. Gremyakov, O.A. Shpigun
Moscow State University, Moscow, Russian Federation

Analytical interest to cephalosporines is maintained at a high level due to a wide range of antibiotics within this group and development of new cephalosporines. In this study, capillary electrophoresis was utilized for analysis of preparations of two cephalosporine antibiotics (cephotaxime and cephoperazone). The initial analysis was carried out to examine the applicability of either capillary zone electrophoresis or micellar electrokinetic chromatography, and the latter has been chosen for further optimization. Optimization criteria were the following: analysis time 30 min or less, high resolution of the main component and impurities and low LOD of impurities (0.05%). Optimal composition of background electrolyte was found to be the following: 5 mM borate, 140 mM SDS, with pH 9.2. Separation conditions were the following: 50 µm capillary, length 70(60) cm, +25 kV direct UV detection at 254 nm for cephoperazone and 235 nm for cephotaxime. The developed technique was applied for control of identity and purity of six commercial products of cephotaxime and cephoperazone. On the next stage of the study cross-validation of the developed method and HPLC was carried out to confirm accuracy of the CE technique. HPLC experiments were performed in accordance with USP 27. The results obtained by MEKC and HPLC for identity and purity of commercial preparations were in agreement. The developed technique was proven to be more effective compared to commonly used HPLC method in the number of theoretical plates, as well as in the number of impurities that could be separated within the anatysis. Low cost and simplicity of analysis by MEKC compared to HPLC make capillary electrophoresis an advantageous technique for use in pharmaceutical analysis.

Novel C18 reversed phase column for samll pharmaceutical compounds and small peptides
Bart Denoulet, Derek Chan, Reno Nguyen, Shawn Williams, Ning Mu
Grace Davison, Brandstraat 12, 9160 Lokeren, Belgium Grace Vydac, 17434 Mojave Street, Hesperia, CA 92345, USA

Over the last decade, there has been tremendous effort put into the development of silica reversed-phase (RP) columns that mainly focus on two areas : reducing metal content of silica materials and developing new bonding technologies to lower silanol activities. Although progress has been made in these areas, further improvement is still possible, especially in the area of monomeric C18 bonding. Using an ultrahigh purity silica and novel bonding chemistry, Grace Vydac has developed its new Denali line of monomeric C18 reversed phase columns and media. This product demonstrates excellent column efficiency, stability, lot-to-lot reproducibility and peak symmetry for basic and acidic compounds. Through using a combination of new technologies, undesirable silanol effects are reduced to a very low level, thus, at neutral pH symmetric peak shape of basic molecules, such as amitriptyline, was observed. At the same time, this material offers very low tailing for acidic organic compounds at controlled pH. It has been found that many other C18 RP columns showed much higher level of tailing for these acidic compounds under the same conditions. The Denali column also provides good resolution of a cytochrom-c tryptic digest and a mixture of small peptides containing basic amino acid residues, with better resolution in the hydrophilic region.

Determination of Phospholipids in Human Serum by Capillary Zone Electrophoresis with Indirect Ultraviolet Detection
Fei Gao12, Juan Dong1, Wei Li1, Tao Wang2, Huwei Liu1
1 College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China 2 Institute of Nephrol, First Hospital, Peking University, Beijing 100034, P.R. China

Phospholipids are not only essential structural constituents of biological membranes but also important substances in signal transduction pathways. Diacylglycerol and phosphatidylinositol, for example, are key players in cell proliferation, apoptosis, and intracellular membrane trafficking. In addition, phospholipids are important sources of arachidonic acid, which can be metabolized by cyclooxygenase or lipoxygenase pathways to produce biologically active prostaglandins or leukotrienes. Generally, the phospholipids are divided into several classes based on differences in their polar head groups, and their distributions are greatly different in different organs. In this work, a simple method for separation and quantitation of different anionic and zwitterionic phospholipid classes by capillary zone electrophoresis (CZE), using indirect UV detection with adenosine monophosphate (AMP) as background electrolyte and the UV-absorbing additive, was successfully developed. The separation conditions including pH of running buffer, concentration of AMP, organic solvent, applied voltage and capillary temperature were systematically optimized and 5 mmol L-1 adenosine monophosphate as BGE and UV-absorbing additive in methanol-water (9:1, v/v) under 25°C temperature and 30 kV applied voltage was selected. By using the proposed method, different phospholipids in human serum were successfully determined with satisfactory sensitivity, repeatability and recovery.

Applicability of Purine & Pyrimidine Compounds as Biomarkers for Diagnostics of Diseases
Yu. Tikhonov, R. Toguzov, R. Biktimerov, B. Lapin
State Medical University, Moscow, Russia

Diagnosis means complete knowledge. Naturally, each pathology is accompanied with specific metabolic alterations. So, it's possible to find a group of compounds which reflect the pathogenesis of a disease and analyze them using various analytical (especially chromatographic) procedures. On the other hand any disease involves a transformation of biochemical interactions at the level of the whole organism, and the final outcome of the pathological process is determined by these interactions. So, the problem is to find such compounds whose metabolic profile reflects systemic, i.e. interorganic and intertissue, interactions. In our view there is a class of compounds which can meet these requirements: namely, purine and pyrimidine derivatives which are involved in almost every area of metabolic pathways and alterations in nucleotide concentrations are associated with numerous pathological conditions. According our working hypothesis in the whole body there must be a dynamic system of purine/pyrimidine substrates cooperating between tissues and, accordingly, disorders of this cooperations generated by various pathological processes. To estimate the potential and prospects for the application of this hypothesis to diagnosis and prognosis of diseases in man we chose several ailments with a completely unlike etiology and pathogenesis and performed an HPLC and HPCE analysis of different patients' tissue: erythrocytes, lymphocytes, cerebrospinal fluid, stomach and intestinal mucosa etc. The obtained clinical evidence allows to regard the metabolic profiles and streams of free nucleotides and their derivatives as universal characteristics of pathological processes occuring in the organism.

Simultaneous detection of point mutations and large rearrangements by capillary electrophoresis
J. Weber, R. Looten, S. Miserere, D. Stoppa-Lyonnet, J.L. Viovy, C. Houdayer
Institut Curie, Paris, France

Recent studies showed that large genomic rearrangements (duplication or deletion of one or several exons) are an important cause of genetic diseases. Methods such as QMPSF, MLPA, real time PCRs, and MP/LC fit a routine use for these large deletions and duplications. They have to be performed additionally to a point mutation detection method (usually sequencing or DHPLC), which is labor intensive and expensive. We recently presented a method called Enhanced Mismatch Mutation Analysis (EMMA) based on heteroduplex analysis that offers great sensitivity for point mutation detection (1, 2). Separations are performed in multi-capillary electrophoresis. Poly(acrylamide-g-poly(dimethylacrylamide)) is used as a self coating sieving matrix. EMMA is able to detect point mutations in fragment ranging from 150 bp to 550 bp. It is thus possible to perform size multiplexing and analyze up to 5 fragments in the same run. We present here a way to simultaneously detect point mutations and large rearrangements using EMMA and semi-quantitative PCR. PCR multiplex fragments of different sizes are prepared by semi-quantitative PCR. One of the fragments is chosen as a reference for peak area normalization. A peak corresponding to a duplicated exon will have its normalized area 1.5 times higher than the control sample (3 copies of the exon instead of 2). A peak corresponding to a deleted exon will have its normalized area 2 times smaller than the control sample (1 copy of the exon instead of 2). EMMA was able to detect large duplication as well as deletion even in the presence of point mutations in certain fragments (see figure). It is thus a very promising method for genetic testing in diagnosis laboratories that offers high-throughput, ease of use and low cost. 1. J. Weber, R. Looten, C. Houdayer, D. Stoppa-Lyonnet, J.-L. Viovy, (submitted). 2. J. Weber et al., Anal Chem 76, 4839 (Aug 15, 2004).

Ion mobility spectrometry - a new method for the detection of lung cancer and airway infection in exhaled air? - First results of a pilot study.
J.I. Baumbach1, M. Westhoff2, P. Litterst2, L. Freitag2, V. Ruzsanyi1, S. Bader3, W. Urfer3
1 ISAS - Institute for Analytical Sciences, 44139 Dortmund, Germany 2 Lung Hospital Hemer, Theo-Funccius-Str. 1, 58675 Hemer, Germany 3 Department of Statistics of the University of Dortmund, Vogelpothsweg 87, 44227 Dortmund, Germany

Purpose: Lung cancer and airway infections gain increasing importance. Early diagnosis is desirable. We examined if volatile metabolites occurring in human exhaled air can be correlated directly to different kinds of diseases. Methods: An ion mobility spectrometer (IMS) coupled to a multi-capillary-column (MCC) was used to identify and quantify volatile metabolites occurring in human breath down to the ng/L- and pg/L-range of analytes within less than 500 s and without any pre-concentration. The IMS investigations are based on different drift times of swarms of ions of metabolites formed directly in air at ambient pressure. Results: During a pilot study data were obtained from 36 patients suffering with lung cancer and 54 healthy persons in a control group. A reduction from more than one million data points per IMS-chromatogram to 25 variables enabled a classification and differententiation of these two groups with an error of 1.3 %. A further study IMS-chromatograms were obtained from 30 patients with different airway infections (COPD-exacerbations, bronchiectasis, pneumonia). In comparison to healthy persons typical clusters of bacterial metabolites could be found. Conclusion: Ion mobility spectrometry seems to be a promising tool in the diagnostic approach to lung cancer and airway infections. These preliminary data need further confirmation by studies with greater populations.

High-throughput mutation screening for beta-thalassemia by single-nucleotide extension
Marina Cretich1, Marcella Chiari1, Silvia Galbiati2, Micol Macellari2, Maurizio Ferrari234, Laura Cremonesi2
1: Istituto di Chimica del Riconoscimento Molecolare (ICRM) – C.N.R.- Via Mario Bianco 9, 20131, Milano, Italy 2: Unit of Genomics for Diagnosis of Human Pathologies, IRCCS H San Raffaele, Milano, Italy. 3: Universitŕ Vita-Salute, H. San Raffaele, Milano, Italy 4: Diagnostica e Ricerca San Raffaele SpA, Milan, Italy.

Beta-thalassemia is a common monogenic disease caused by mutations in the human beta-globin gene (HBB). Point mutations in the HBB gene can be detected by several methods. These can be based on DNA hybridization alone (on microarrays or microelectronic chips), or on enzymatic nucleotide recognition followed by an electrophoretic separation. Due to the heterogeneous distribution of different defects in the HBB gene, the development of a flexible strategy that allows high-throughput detection of many different mutations is highly desirable. In this work we present a multicolour assay for some of the most frequent mutations of the HBB gene in the Mediterranean area by the use of the single-nucleotide extension (SNE) reaction. This assay is based on the extension, in the presence of fluorescence-labelled dideoxy nucleotides (ddNTP terminators) of an unlabeled oligonucleotide primer that binds to the complementary template immediately adjacent to the mutant nucleotide position. Since in the reaction there are no unlabeled dNTPs, a single labelled ddNTP is added to the 3’ end resulting in a fluorescent extended primer which is rapidly separated by capillary electrophoresis. Each individual ddNTP is assigned to a different fluorescent dye and therefore unambiguously assigns the genotype. Reaction conditions were developed for the analysis of CD 39 and IVSI.110 mutations and for the IVSI.1 and I.6. The reaction products were analysed by a 96 capillary DNA sequencer leading to a fast genotype detection. The possibility of using multiple injections further enhances the throughput of the mutation screening and facilitates automated genotyping for routine molecular diagnostics and large scale genetic studies.

Simultaneous determination of allantoin, hypoxanthine, xanthine and uric acid in serum/plasma by Capillary Electrophoresis
E. Caussé1, A. Pradelles1, B. Dirat1, A. Negre-salvayre1, R. Salvayre1, F. Couderc2
1. Labo. Biochimie/INSERM U466, CH Rangueil, 1av. Poulhes, TSA 50032, 31059 Toulouse cedex 9, France- 2. Eq.Chimie Analytique et Spectrométrie de Masse, Labo IMRCP, UMR 5623, Université P. Sabatier,31062 Toulouse Cedex, France.

Free radicals have been implicated in the development and progression of atherosclerosis. Allantoin is the catabolic end product of purines in mammals. The small amount of allantoin present in human serum results from free radical action on urate and may provide a stable marker of free radical activity in vivo. We propose a new fast capillary zone electrophoresis (CZE) method for the simultaneous determination of allantoin (All), uric acid (UA), hypoxanthin (HX) and xanthine (X) in human plasma. We estimated these products in human patients obtained from patients with chronic renal failure before hemodialysis (n=10), patients with chronic heart failure (n=10) and control (n=10). Deproteinized plasma samples were obtained by using millipore ultrafree 10 kD. These filtered serum or plasma were diluted 10 fold before direct injection in capillary electrophoresis with UV detection (195 nm, 28 KV, 33 µA). Separations were performed in 30 mM NaH2PO4 - 20 mM Na2B4O7 (pH 8.6) at 25 °C, in less than 13 min. The metabolites were detectable at concentrations of 0.23 - 0.63 µmol/L. The method was linear over the range 2 - 150 µmol/L for All, HX and X and 10 - 1000 µmol/L for UA (r > 0.99). The analytical performance of this method is satisfactory with intra-assay CV < 3.4 %, inter-assay CV < 5 % (HX and X < 7 %) and recovery (93 - 101 %) The CZE-UV method appears to be useful tool for studying changes of hypoxanthine, uric acid and allantoin levels in plasma samples. It seems that allantoin could be used as a possible indicator of free radical damage in vivo.

Separation of normal and modified nucleosides by MEKC
S.V. Sereda, A.V. Shpak, A.V. Pirogov, O.A. Shpigun
Moscow State University, Moscow, Russian Federation

In the study on the biochemical roles of urinary normal and modified nucleosides in carcinogenesis, as well as in the diagnosis of cancer at an earlier stage, simultaneous detection of diverse modified nucleosides in urine has become an important task. The main goal of this study was the development of approaches for finding the optimum conditions of capillary electrophoresis separation and detection of normal and modified nucleosides in urine. We use the hydrophobic coefficient, the distribution coefficient and the electrophoretic mobility of nucleosides as the basic parameters of optimization. The values of the hydrophobic coefficient and the distribution coefficient were calculated using software ACDLab
Ň. The values of electrophoretic mobility were determined experimentally using capillary zone electrophoresis under different values of pH and ionic strength of separation buffer. We determined the dependence of migration time, selectivity and efficiency of separation on the nature and concentration of the inorganic salts and the surfactant as pseudostationary phase in the separation buffer, pH of the buffer, the applied voltage, and the temperature of the thermostating system of the capillary and ionic strength of the sample matrix. Based on this dependence, we proposed an algorithm to search for optimal separation conditions of the model mixture of nucleosides. In addition, we compared main parameters of micellar electrokinetic chromatography (MEKC) separation of the nucleosides model mixture in 50 mkm and 75 mkm capillary of some different lengths. The experimental data confirmed the theoretical predictions used to search for the optimal conditions for separation of normal and modified nucleosides. In was found that the optimum conditions for MEKC separation was carried out with an uncoated fused-silica capillary (565 mm х 50 mkm I.D.) termostated at 20°C, using 25 mM borate-42.5 mM phosphate buffer (pH 6.7) containing 200 mM sodium dodecyl sulfate as the separation buffer under the applied voltage of 25 kV. Analysis under these conditions has allowed us to successfully carry out a separation of 16 nucleosides with a subsequent detection step in 15 minutes with efficiency close to 170000 theoretical plates per meter.

VEnture A : a high performance analytical column for monoclonal antibodies
Bart Denoulet, Jochen Saar, Gonda Van Essche
Grace Davison, Brandstraat 12, 9160 Lokeren, Belgium Grace Davison, In der Hollerhecke 1, 67545 Worms, Germany

Grace introduces the Venture A column : an affinity chromatography column utilizing ICE (Inert Coating Enhancement) surface passivation technology to eliminate non-specific binding on the silica surface. ICE technology enabled the Venture A columns to take advantage of silica gel’s rigid porous structure, providing greater productivity and capacity to users. Venture A columns use a recombinant protein A ligand for binding antibodies. The rigidity of the silica particles enables the columns to run under HPLC and FPLC modes with linear velocities from 150 up to 5000cm/h and its optimised structure results in capacities of 40 mg/ml for human polyclonal globulins.

Affinity capillary electrophoresis for bacterial toxins analysis
M.M. Fernandez(1), E. Baldini(2), M.C. De Marzi(1), E.L. Malchiodi(1), M.C. Vescina(2)
(1) Cátedra de Inmunología-IDEHU, CONICET, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina (2) Cátedra de Química Analítica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina

Protein-protein interactions are involved in many processes ranging from DNA replication, signal transduction, metabolism control and viral assembly. The characterization of molecular affinity combined with structural information about the interacting molecules is necessary to understand biological system mechanisms implicated in health and disease. Affinity capillary electrophoresis (ACE) is a powerful tool, where the combination of high resolution separation and functional molecular characterization, open the possibility to study molecular interactions. Superantigens (SAgs) are a class of immunostimulatory and disease-causing proteins of bacterial or viral origin with the ability to activate large fractions (5-20%) of the T cell population. Activation requires simultaneous interaction of the SAg with the variable (V) b domain of the T cell receptor (TCR) and with the major histocompatibility complex (MHC) class II molecules on the surface of an antigen-presenting cell. This activation leads to production of cytokines such as TNF-a, IFN-g and IL-2, which may result in acute toxic shock. The best characterized SAgs are produced by different strains of Staphylococcus aureus and Streptococcus pyogenes. These are implicated in a number of human diseases, including toxic shock syndrome, food poisoning, diabetes mellitus and multiple sclerosis and may be present in prosthesis and food. The aim of the present work was to analyze SAgs and T-cell receptors (TCRs) interaction and obtain a less expensive and fast method to measure analytes in different matrixes. For this purpose we have developed a two-dimensional ACE system where the first dimension is the separation of the SAg from other sample components via immobilized TCR specificity followed by separation and detection of the affinity analytes by capillary zone elctrophoresis (CZE) as a second dimension.

Determination of Mercaptopurine and its Four Metabolites by Large Volume Sample Stacking with Polarity Switching in Capillary Electrophoresis
Shou-Mei Wu1, Chun-Chi Wang1, Shyh-Shin Chiou2
1 Graduate Institute of Pharmaceutical Science, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan 2 Department of Pediatrics, Kaohsiung Medical University, Kaohsiung 807, Taiwan

This study describes approaches for stacking large volume of sample solutions containing a mixture of mercaptopurine monohydrate, 6-methylmercaptopurine, thioguanine, thioguanosine, and thioxanthine in capillary electrophoresis. After filling run buffer (50 mM borate buffer, pH 8.5), large sample volume was loaded by hydrodynamic injection (2.5 psi, 99.9 sec), followed by the removal of the large plug of sample matrix from the capillary using polarity switching (-15 kV). Monitoring the current and reversing the polarity when 95% of current recovered, the separation of anionic analytes was performed in a run buffer under 20 kV. Around 44-90 fold improvement of sensitivity for five analytes was achieved by large-volume stacking with polarity switching when compared with CE without stacking. This method was feasible for determination of the analytes spiked in plasma. Removing most of electrolytes from plasma is a key step for performing large-volume sample stacking. Solid-phase extraction was used for pretreatment of biological samples. To our knowledge, this study is one of few applications showing the possibilities of this stacking procedure to analyze biological samples by LVSSPS in CE.

Investigation of Physiology-active Peptides from fungal mycelium by Liquid chromatography/Mass spectrometry
O. Mikhailova1, B. Lapin2
1 “OAO Biomash”, Moscow, Russia 2 InterLab Inc.,Moscow, Russia

The crude extract from the fungal mycelium of Fusarium sambucinum Fuc. var. Ossicolum (Berk. ex Cart.) Bilai, strain of which was isolated from mycothallus of bracket fungus Polyporus squamosus Huds. ex Fr. in Bulgaria, was found to possess the neutropic activity. Preliminary investigation of this material by means of amino acid analysis, SDS-polyacrylamide gel electrophoresis and size-exclusion high-performance chromatography revealed near 5% compounds of peptide nature with molecular masses not exceed 4.5 kDa. The HPLC methods in size-exclusion, ion-exchange, normal and reversed-phase modes on Ultra Pac TSK G2000 SW, Aminex HP-72S, DEAE Si 100 Polyol, LiChrosorb NH2, LiChrosorb DIOL, LiChrosorb RP-18 and Si 100 Polyol RP-18 columns were applied for assay the constituents of mycelium crude extract. It was found that the most informative pattern of separation could be obtained by using reversed- phase column in acidic media. The optimization of chromatographic process was performed by studying the separation selectivity under different elution conditions. An on-line capillary-column HPLC-mass spectrometric procedure with an elecrospray atmospheric pressure ionization (ES-API) ion source was used for identification and characterization the peptide constituents from mycelium crude extract. The process of recognition the major peptide mycotoxins produced by species of the genus Fusarium and other microfungi was based on a comparative analysis of MS and MS-MS datasets with libraries containing chematoxonomic and molecular mass information. The peptides with linear chain structure were detected by means of an ion trap MS-MS system with ES-API ion source operated in the positive ion mode. Mascot search software and the protein sequence database National Center for Biotechnological Information (NCBInr), Version 20030215, containing 1338750 sequences were used for de novo peptide sequence interpretation of tandem mass spectra. The LC-ESI-MS-MS method revealed the bicyclic peptide phalloidin and some small peptides with linear chain structure in crude extract from the fungal mycelium.

Nano-LC/ESI Microfluidic Chip Based LC-MS Peptide Profiling
R. Ossola, H. Lee, L. Mueller, R. Aebersold
Swiss Federal Institute of Technology, Zuerich, Switzerland

A recently developed nano-LC/ESI microfluidic chip has brought high promises in quantitative proteomic researches. Its high reproducibility in retention times and peak intensities offer new opportunities to use LC-MS based peptide profiling as a fast and sensitive alternative to labeling based quantitative proteome analysis. Here we present a technology platform utilizing the LC/ESI chip as a crucial analytical tool for screening of biomarkers. The platform consists of automated isolation of formerly N-linked glycopeptides from human plasma or serum, high-throughput LC-MS analysis using the LC/ESI chip, and software identifying and selecting discriminatory peptides across multiple LC-MS runs. To demonstrate suitability of the LC/ESI chip for profiling of complex peptide mixtures, reproducibility of retention times and signal intensities of peptides, as well as chip-to-chip repeatability are presented using formerly N-glycosylated peptides prepared from human serum. Furthermore, used in-house developed software tools are described.

Chip-based Nanoelectrospray Ionization Tandem Mass Spectrometry for Determination of Ganglioside Pattern in Human Hippocampus
Alina D. Zamfir, Željka Vukelic, Jasna Peter-Katalinic
Institute for Medical Physics and Biophysics, Biomedical Analysis, University of Münster, Germany

In the last 2 years, carbohydrate research greatly benefited from the introduction of the microfluidic/mass spectrometry (MS) technology. Our laboratory pioneered the implementation of chip MS for carbohydrate analysis in general and glycoscreening/sequencing for biomedical and clinical research in particular. The advantages of the chip electrospray in terms of ionization efficiency, sensitivity, reproducibility, quality of data in combination to high mass accuracy and resolution of detection were systematically explored by us for several carbohydrate classes [1-3] extracted from human matrices in health and disease. Hippocampus is a part of the brain located inside the temporal lobe, highly contributing to learning and memory. It is one of the first regions of the brain affected by Alzheimer’s disease where disorientation, loss of memory and deterioration of the cognitive functions are the primary symptoms. Gangliosides are sialic acid containing glycosphingolipids particularly abundant in the central nervous system (CNS). Specific changes in their expression occur during brain development, maturation, and aging, and due to some neruological diseases, particularly in relation to neurodegeneration processes. Gangliosides were recently shown to have also a brain region specificity. Therefore, gangliosides represent an important class of biomarkers able to provide information upon various CNS processes and events. In the human brain, the specific developmental-, age- and disease-induced modifications in ganglioside expression have been demonstrated so far by thin-layer chromatography, immunochemical and immunohistochemical methods, which, however, showed limitation in detection sensitivity and providing structural data. In this study, fully automated chip-based nanoelectrospray quadrupole time-of-fight tandem mass spectrometry was employed and optimized for a systematic profiling of gangliosides followed by structural analysis of individual species in human fetal and adult hippocampus. 25 different glycoforms, exhibiting high degree of heterogeneity in both the oligosaccharide portion and the ceramide motif as well as biologically-relevant peripheral modifications of the oligosaccharide moiety and polysialylated structures not detectable before could be identified by our chipMS approach. The comparative chip-based nanoESI MS data revealed also a significant brain development-induced difference in ganglioside expression previously not evidenced by any other MS-based method. The unique feature of chip ESI tandem MS to provide structural data at elevated sensitivity offered an efficient sequencing and identification of several hippocampus-associated species. The obtained data indicate the benefits of ultra-sensitive and high performance microfluidics/mass spectrometry methods in determining the topo- and age-specific composition of brain gangliosides as well as in their detailed structural characterization. [1] A. Zamfir, S. Vakhrushev , A. Sterling, H. Niebel, M. Allen, J. Peter-Katalinic Anal. Chem.76, 2046, 2004 [2] A. Zamfir, N. Lion, Ž. Vukelic, L. Bindila, J. Rossier, H.Girault, J.Peter-Katalinic Lab. Chip, 5, 298, 2005 [3] A. Zamfir, Z. Vukelic, L. Bindila, R. Almeida, A. Sterling, M. Allen, J. Peter-Katalinic J. Am. Soc. Mass Spectrom. 15, 1649, 2004

Proteome Profiling by Multidimensional Protein Separation and Automated Lab-on-a-Chip Technology
R. Salowsky, M. Greiner, C. Buhlmann, P. Mrozinski, B. Wilson, H. Liu
Agilent Technologies, Deutschland GmbH, Waldbronn, Germany

Technologies and methodologies for proteomic studies often involve a combination of protein fractionation, identification, and quantitation by liquid chromatography tandem mass spectrometry (LC/MS/MS) and in many cases pattern comparison of mass spectra from a complex sample. A common goal of these efforts is the discovery and validation of biomarkers. While current mass spectrometry-based proteomic methods are highly developed and an important component for biomarker discovery, the time required for these methods makes them impractical for screening applications. In addition, current methods of pattern recognition, while capable of the speed required, suffer from a lack of reproducibility, tend to focus on high-abundance proteins, and fail to identify the actual biomarkers. We test here a concept to combine several novel separation approaches for protein fractionation with an automated Lab-on-a-Chip platform and nano LC/MS/MS based technologies to provide a rapid, quantitative proteome profiling method for the serum/plasma proteome. The Lab-on-a-Chip methodology affords high-throughput analysis combined with reproducible sizing and quantitation, making it a potentially powerful and enabling technology to rapidly compare the proteomes of multiple samples in a wide variety of applications.

Determination of D-Serine by Capillary Electrophoresis
D. Koval, J. Jirásková, K. Stríšovský, J. Konvalinka, V. Kašicka
Institute of Organic Chemistry & Biochemistry, Czech Academy of Sciences, Flemingovo nám. 2, 166 10 Prague 6, The Czech Republic

Identification of D-serine in the mammalian brain was surprising since up to recently it was thought that higher organisms used L-amino acids exclusively. Upon further investigation, it was found out that the distribution of D-serine throughout the brain matched that of the glutamate NMDA (N-methyl-D-aspartate) receptor. The NMDA receptor is a major class of receptor for glutamate, the main excitatory neurotransmitter in the central nervous system. The current hypothesis for D-serine’s role in the central nervous system is that D-serine coactivates the NMDA receptor through the “glycine” binding site. If true, this pathway represents a novel mechanism for intercellular communication in the brain. It would also offer a new pharmacological target for modifying NMDA receptor activity during stroke, epilepsy, or other periods of hyperactivity. In support of this hypothesis, serine racemase capable of synthesizing and removing D-serine have been identified in the mammalian brain. Measurement of D-serine concentration is not easy. D-Serine possesses neither any natural chromophore nor any other functional group, such as electroactive, which could be used for sensitive detection. The analysis is further complicated by the enantiomeric selectivity required to resolve D-serine from L-serine. A suitable approach for measurement of D-serine concentration involves formation of UV-absorbing or fluorogenic D-serine derivative and subsequent separation of D- and L-serine derivatives by a high-performance separation technique. Capillary zone electrophoresis (CZE) is well established separation technique for analysis of compounds of biochemical interest including amino acids. Despite the fact that CZE has been used for analysis of the amino acids since its early days, only a few successful CZE separations of serine enantiomers have been reported so far [1]. A modified CZE method was developed for direct determination of D-serine. Serine enantiomers are separated as derivatives formed during reaction with o phthaldialdehyde and 2-mercaptoethanol. The reaction is carried out directly in the separation capillary followed by subsequent CZE separation of the enantiomers in alkaline borate background electrolyte with 2-hydroxypropyl-g-cyclodextrin as a chiral selector. Analyses were performed in untreated fused silica capillaries of I.D./O.D 75/360 um and 30/40 cm effective/total length with UV-absorption detection at 230 nm, which enabled quantitative determination of D-serine at low micromolar level in 10 minutes of the analysis time. The assay was successfully applied to evaluation of serine racemase kinetics (KM) and characterization of serine racemase inhibitors (IC50). The work was supported by the Grant Agency of the Czech Republic, grants no. 203/05/2539, 203/04/0098 and by the Research Project Z40550506 of the Czech Academy of Sciences. References [1] O'Brien, K. B., Esguerra, M., Klug, C. T., Miller, R. F., Bowser, M. T., Electrophoresis, 24, (2003), 1227-1235.

MECC analysis of shikimate and shikimate-3-phosphate as biomarkers in plant materials treated with glyphosate
I.L. Petersen, H. Sřrensen, J.C. Sřrensen
Department of Natural Sciences, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark

Shikimate and shikimate-3-phosphate have a central position in the biosynthesis of aromatic amino acids, and they are of interest as biomarkers for the herbicidal effects of glyphosate (N-phosphonomethylglycine). These effects are considered to result from inhibition of the transformation of shikimate-3-phosphate into 5-enolpyruvyl-shikimate-3-phosphate (EPSP) by binding of glyphosate to EPSP-synthase (EC The possibilities for use of the shikimate and shikimate-3-phosphate as biomarkers for glyphosate effects have been investigated by a method developed for determination of their accumulation in plant materials. The micellar electrokinetic capillary chromatography (MECC) method developed for the analysis is based on a cholate-taurine system at pH 7.3 and with direct UV detection at 206 nm. This MECC method for shikimate analysis has a limit of detection (LOD) corresponding to 0.53 mM (~1.5 picomole). The limit of quantification (LOQ) corresponds to 2.7 mM, and the method has linearity for the quantification up to 150 mM with a correlation coefficient of 0.9942. The number of theoretical plates per meter (N/m) was found to be 250000 N/m for the optimised method. The method was successfully used to determination of the compounds in crude extracts from glyphosate treated plant materials. The concentration range determined for shikimate was from 4 to 270 µmole per g dry matter, and the identity of the analytes was confirmed by use of diode array detection and 1H-NMR spectroscopy.

MECC determination of oxidised DNA nucleosides resulting from induced DNA damage
J. C. Sřrensen, K. Ib, H. Sřrensen
Department of Natural Sciences, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark

An in vitro bioassay has been developed to determine oxidised DNA nucleosides for application in tests of potential anticarcinogenic compounds. Micellar electrokintic capillary chromatography (MECC) based on sodium cholate as surfactant have been developed for separation and quantification of DNA nucleosides and a biological marker of oxidative DNA damage, 8-oxo-7,8-dihydrodeoxyguanosine (8-oxo-dG). A diode array system was applied for peak identification and the wavelength 254 nm was used for analytical detection of the compounds considered. Optimisation of the MECC methods encompassed investigations of effects from temperature, voltage, surfactant concentration and buffer composition on separation efficiency, migration times, peak areas, resolution and number of theoretical plates per meter (N/m). Suitable resolution values (2.44-9.32) and up to 160,000 N/m were obtained. Linearity and repeatability were acceptable and detection limits was calculated to 8.56 pg. The developed system proved to be efficient in the detection and quantitation of 8-oxo-dG, resulting from the Fenton reaction. 8-oxo-dG induced damage on intact calf thymus DNA, which was determined directly by the developed MECC method following enzyme catalysed hydrolysis.

Analysis of Double-stranded DNA by Capillary Electrophoresis using Polymer Solutions Containing CTAB
Y.W. Lin, H.T. Chang
Department of Chemistry, National Taiwan University, Taipei, Taiwan

The impact of hexadecyltrimethylammonium bromide (CTAB) on the separation of ds-DNA by capillary electrophoresis in conjunction with laser-induced fluorescence (CE-LIF) is described. Prior to use, the capillaries were treated with 3 mM CTAB aqueous solution (pH 7) for 1 hr. The separation was conducted in the capillary filled with 0.75 % poly(ethylene oxide) (PEO) prepared in 100 mM TB buffer (pH 8.0) containing 25 µg/mL EtBr and 1 µM CTAB at 375 V/cm. In the presence of small amounts of CTAB (1.0 µM), the separation repeatability (RSD of the migration times < 3.0%), sensitivity, and resolution improved. As a result, lower concentrations of PEO solution can be used, leading to ease of filling the capillary with low-viscosity sieving matrixes. When DNA fragments migrate from aqueous solution to PEO solution, they slow down and are stacked as a result of sieving and increases in viscosity. This allows stacking of DNA samples that were injected at 1.0 kV for up to 2 min (about 38 nL). Having a 24-fold sensitivity improvement for the 603-bp fragment when compared to that injected at 1.0 kV for 10 s, the LOD for the DNA sample is 4.0 ng/mL. By using this approach, the separation of 38 nL of 0.1 µg/mL DNA markers V (pBR 322/Hae III digest) and VI (pBR 328/Bgl I digest & pBR 328/Hinf I digest) was completed in 9 min.

Comparison of Electromigration Properties of Stained Microoganisms, Cultured and from Body Fluids, in Capillary Electrophoresis
M. Horká1, F. Ružicka2, V. Holá2, J. Horký3, K. Šlais1
1 UIACH AV ČR Brno, Czech Republic, 2 MU Brno, Czech Republic, 3 SRS Olomouc, Czech Republic

The capillary electromigration techniques with UV/Vis and fluorometric detection were recently successfuly used for the on-line rapid separation of the bacteria and fungies dynamically modified by the non-ionogenic tensides. In future the capillary isoelectric focusing (CIEF) and the capillary zone electrophoresis (CZE) could be used for the study of the properties and identification of the microbial strains. In this contribution we suggest for the study and comparison of the properties of different microbial strains the use of the sensitive and selective UV/Vis and fluorescence detection of the bioanalytes, microorganisms, dynamically modified by the non-ionogenic tensides on the basis of lipophilic azo dyes, as the chromophore, or on the basis of pyrenebutanoate, as the fluorophore, respectively. Since the values of the isoelectric point of microbes are often in the range from 2 to 5 the segment of the selected simple ampholytes was necessary to inject into the fused silica capillary before the segment of modified bioanalytes and carrier ampholytes for the reproducible and efficient CIEF. For tracing of the pH gradient the low-molecular-weight pI markers were used here. The microorganisms, which are often the cause of common human or plant infections, were focused and separated by CIEF and CZE methods suggested. Isoelectric points, pI’s, of dynamically modified microbes from the body fluids and the other properties were compared with those obtained by IEF of the cultured microorganisms. This work was supported by the Grant Agency of the Academy of Sciences of the Czech Republic No. A4031302.

Hairpin Formation in Single-stranded DNA Oligomers
Earle Stellwagen, Qian Dong, Nancy C. Stellwagen
Department of Biochemistry, University of Iowa, Iowa City, IA U.S.A.

The free solution mobilities of a series of DNA oligonucleotides containing 26 residues, their complementary strands, and the corresponding double-stranded duplexes have been measured by capillary electrophoreis, using a common background electrolyte. While the mobilities of the duplexes were very similar, the mobilities of the single-stranded oligomers and their complements varied widely. To understand this behavior, we examined the thermal dependence of the mobilities of several oligonucleotides and their complements. If an oligonucleotide and its complement migrated relatively fast, both oligomers exhibited melting transitions within the range of 15-60ş C, accompanied by a significant decrease in mobility. Similar mobility decreases are observed for model oligomer hairpins upon increasing the temperature. Hence, the relatively fast mobilities observed for certain 26-nucleotide oligomers appear to be due to the formation of hairpins which can be unfolded by increasing the temperature. If a 26-bp oligonucleotide and its complement both migrated relatively slowly, neither oligomer exhibited a thermal transition between 15-60ş C, suggesting that these oligomers were in the random coil conformation at all temperatures. If an oligonucleotide and its complement exhibited one fast and one slow mobility, the one with the fast mobility exhibited a thermal transition consistent with hairpin formation. Comparison of the sequences of the various oligomers with predictions of the thermal stability of various hairpin structures provides a rationale for the variable mobilities observed for the different oligonucleotides.

Extremely Fast, Long-Read DNA Sequencing in a Microfluidic Chip Using a Dynamic Wall Coating: 550 bases in 5.5 Minutes
C.P. Fredlake, D.G. Hert, B.E. Root, R.E. Forster, A.E. Barron
Northwestern University, Evanston, IL, United States

The recent recommendation for a Human Cancer Genome Project calls for the determination of all genetic variations of every major type of cancer by sequencing the entire genomes of approximately 15,000 cancer samples. To achieve this ambitious goal, the current sequencing costs must be reduced by two orders of magnitude to about $100,000 per genome. Although alternative sequencing technologies are being developed, electrophoresis-based Sanger sequencing is the only method which can achieve individual reads long enough for assembly of a human-sized genome. Microchip electrophoresis holds the key to cost reduction. Compared to current capillary electrophoresis systems, microchannel systems deliver faster sequencing times, require lower reagent volumes, and can integrate all aspects of sequencing from sample preparation and cleanup to separation in one device. Further cost savings can be realized by using a dynamic polymer coating for these microchannels rather than applying time-consuming and complicated covalent coating procedures. Here, we report sequencing up to 550 bases in just 5.5 minutes using poly(N,N-dimethylacrylamide) as a separation matrix with an acrylamide-based polymer wall coating, using a microchannel with a 7.5-cm effective separation length. This surprising result demonstrates reduction of sequencing time by two-thirds compared to any previously published microchip sequencing result, while maintaining comparable read lengths. This increased speed is attributed to the choice of sequencing conditions including channel length, polymer matrix attributes and temperature. Analysis of polymer radius of gyration at different temperatures as well as videomicroscopy suggests that the migrating DNA may disrupt the entangled polymer network. While unoriented reptation is the dominant separation mechanism, this proposed network disruption leads to faster mobilities while at the same time providing size based separation as the DNA becomes entangled with free polymer chains. Through further study and optimization of this system, longer read lengths will be achieved and thus advance the development of the next generation of sequencing technology.

New Microchips and Analytical Methods Designed with the Aid of Computer Simulation and Application to the High-sensitive Analysis of DNA Fragments and Proteins
T. Hirokawa1, Y. Takayama1, A. Arai2, T. Nishine2
1 Applied Chemistry, Department of Chemistry and Chemical Engineering, Graduate School of Engineering, Hiroshima University 2 Life Science Laboratory, Analytical & Measuring Instruments Division, SHIMADZU CO.

A major problem facing microchip electrophoresis (MCE) is its low concentration sensitivity due to inherent geometrical restriction. A possible approach to address this issue is developing on-chip preconcentration methods, which is more convenient and costs less than depending on advanced detection systems such as LIF. With the aid of computer simulation (CFD-ACE+, CFDRC, AL, USA), we have developed new microchips with a preconcentration channel suitable for electrokinetic injection with transient isotachophoresis (tITP) preconcentration (with the name of electrokinetic supercharging [EKS] for such a procedure). The microchip was applied for the analysis of DNA fragments (50 bp step ladders) and SDS-proteins. Although the EKS method previously developed by us[1] was difficult to apply to a cross-geometry microchip, a new analytical procedure was developed using the EKS technique with floating injection. This enabled more rapid and convenient analysis of a low-level concentration of DNA sample using MCE-2010 (Shimadzu) with a linear imaging UV detector. The proposed method improved the detection sensitivity of the biopolymers 40-fold in comparison with the conventional pinched injection method. The detailed results will be discussed in the presentation. [1] Z-Q. Xu, T. Nishine, A. Arai, T. Hirokawa, ELECTROPHORESIS, 25, 3875-3881,2004.

Increasing Resolution of Capillary LC Reversed-phase Analysis of Antibodies and other Proteins of Biotechnical Importance
R.L. Chien, D. Rakestraw, D.E. Hughes
Eksigent Technologies (R.L. Chien and D. Rakestraw); Chromatographic Excellence (D.E. Hughes)

Liquid chromatographic (LC) separation of peptides and small proteins has been successfully performed for at least two decades while the reversed-phase (RP) separation of large biomolecules, such as antibodies, fusion proteins, and enzymes has experienced more modest development. Peptides act chromatographically much like small drug molecules and that empirical base has an extensive literature for the analyst to employ. The advent of biotechnology and the need for analytical techniques to examine protein-based therapeutic agents has resulted in the RP of large proteins being studied more carefully. The standard RP method is capable of determining the protein concentration and a retention time characteristic to the main species of interest. In the most favorable cases, the relative abundance of the various glycoforms and impurities can also be determined. For these reasons, the RP gradient determination of protein molecules has become a standard assay as part of most biologic drug species examination protocols. The RP separation of large protein molecules by conventional LC often requires very long (>60 minutes) analysis times to achieve reasonable peak shape and isoform resolution. The mechanism for the attachment, residence time, and release of large protein molecules in RP chromatography requires the mobile and stationary phases to be nearly in kinetic equilibrium. Conventional LC conditions required for analysis therefore often require very slow flow rates to allow analyte/stationary phase interaction. The chromatographic peak profiles are nonetheless often lacking in efficiency and selectivity. The very precise gradient control of capillary LC, rapid re-equilibration, and the use of multiple-slope gradients allow a dramatic reduction in analysis time and increased isoform/impurity resolution when compared with conventional LC. Several separations of antibody and other protein molecules employing multiple-slope gradients will be presented to demonstrate the total analysis time and resolution advantages capillary LC offers when compared with conventional-bore LC.

Determination of dendrigraft polylysines diffusion coefficients by Taylor diffusion analysis using capillary electrophoresis apparatus
E. Souaďd, H. Cottet, A. Papillaud, H. Collet, J.P. Biron, A. Commeyras
Equipe « Dynamique des Systčmes Biomoléculaires Complexes », Organisation Moléculaire, Evolution et Matériaux Fluorés, UMR CNRS 5073, Université de Montpellier 2, Place Eugčne Bataillon, 34095 Montpellier cedex 5, France.

This work focuses on the physico-chemical characterization of dendritic polypeptides, namely, dendrigraft poly-L-lysines (DGPL) that were synthesized by ring opening polymerization of amino acid N-carboxyanhydride. Diffusion coefficients (D) and hydrodynamic radii (Rh) of five DGPL generations were determined by Taylor Diffusion Analysis (TDA) using a capillary electrophoresis apparatus. TDA is an absolute, simple and rapid method for determining D (or Rh) values. It is based on the dispersion of a solute plug in a solvent flow under dispersive velocity profile (laminar Poiseuille flow). The band dispersion is directly related to the molecular diffusion that redistributes the molecules over the cross section of the tube. TDA is applicable on (macro)molecules of virtually any molar mass. Despite the advantages of this method, TDA has not been extensively used. Up to our knowledge, this is the first experimental work on the use of TDA for the characterization of dendrimer-like structures. Experimental Rh values obtained by TDA for the five generations of DGPL were compared to those derived from dynamic light scattering and size exclusion chromatography coupled to a triple detection (refraction index, viscosity, and static light scattering). Important differences were obtained, especially for the highest generations, due to the inherent contribution of aggregates to the light scattering intensity. For that reason, TDA was found to be the most appropriate technique for determining D values. The influence of the pH and of the addition of organic solvent on the DGPL Rh was studied. Regarding to their physico-chemical behaviour, the experimental results confirm that DGPL are very similar to trifunctional dendrimers (linear variation of the hydrodynamic radius, exponential growth of the molar mass, high branching density, maximum of the intrinsic viscosity or of the free volume fraction for generation 4).

Macromolecular coatings of silica capillaries for tuning EOF and improving the separation efficiency for basic proteins in Capillary Electrophoresis
N.T. Tran*, A. Pallandre*, J. Zhang*, J. Weber**, M. Villain*, J.L. Viovy**, M. Taverna*
*Faculté de pharmacie, Université Paris-Sud, JE Protéines et Nanotechnologies en Sciences Séparatives, 5, rue J.B. Clément, 92 290 Châtenay-Malabry, France **Institut Curie, UMR 168, 11 Rue P.M. Curie, 75005 Paris, France

The control and modification of surface state is a major challenge in bioseparation sciences, and in particular in electrokinetic separation methods, due to the importance of electroosmosis (EOF) . Moreover, in proteins analyses, efficiency and method reproducibility are strongly dependant on the prevention of protein adsorption on the capillary wall . This topic has gained recently a renewed interest, associated with the development of “lab-on-chips” systems that extend the range of materials in which separation channels are fabricated. We precisely present in this communication, various routes to dynamically coat capillaries with macromolecular layers. Several families of polymers have been studied. Two protein neutral repellant block copolymers based on a poly(acrylamide) backbone and poly(N,N-dimethylacrylamide) grafts were synthesized in our laboratory and were recently evaluated . They were compared to the cationic coating, hexadimetrine bromide (polybren), and commercial ones, the EOTrol LR and UltraTrol HR acrylamide/N-substituted acrylamide copolymers from Target Discovery, Inc. Macromolecular coatings were obtained by dynamic treatment of silica capillaries, and various procedures were tested to optimize the stability and homogeneity of the coatings. The performance, homogeneity and stability of these coatings were characterized by EOF measurements as well as by microscopy based techniques. Their performance, in preventing protein adsorption, were evaluated from the peak efficiencies and shapes obtained for strongly basic proteins and from reproducibility studies. Finally, these coatings were applied to the separation of RNAse B glycoforms.

H.Y. Zhang*, J.Y. Zhang**, I. Le Potier**, M. Taverna**
* college of chemistry and environmental science, hebei university, Boading 071002, China **JE nanotechnologies and proteins separation science, Université Paris sud, faculté de pharmacie, 5 rue JB Clément, 92296 Chatenay-Malabry cedex, France

“In-capillary” derivatization is the most efficient and straightforward approach to enhance the detection sensitivity of peptides and proteins through combination of fluorescence labeling and laser-induced detection. Compared to conventional procedures, pre- or post-capillary derivatization, in-capillary derivatization has a remarkable merit due to a small reaction chamber volume which results in a minimum sample consumption, handling and dilution. In the present work, 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was selected as a derivatization reagent to label peptides, considering that it can react with primary and secondary amines. Since its first introduction in 1981 by Watanabe and Imai as a labeling reagent, there have been many reports on amino acids, peptides and proteins analyses after NBD-F derivatization by both HPLC and CE. To our best knowledge, no systematical work has reported yet the use of this reagent for the “on line” derivatization of amines. In this study, we focused on how to obtain a strong, reliable and repeatable fluorescent signal for each peptide or amino acid derivatized with NBD-F. As NBD-F can easily get hydrolysed, we have developed a strategy to introduce the sample and the reagent into the capillary while avoiding the contact between the NBD-F solution and the BGE. Sodium borate was selected as BGE due to its ability to generate a stable EOF and in order to perform the derivatization in alkaline conditions. We investigated the influence of various factors such as pH value, plug lengths or molar concentrations reagent to sample ratios, addition of organic solvent to BGE, on the intensity of fluorescence of amino acids and then of peptides. The validation of the in-capillary derivatization with NBD-F was also studied in terms of linearity, reproducibility and sensitivity. The method was finally applied to the analysis of amyloid peptides mixture. References: 1.W. J. M. Underberg, J. C. M. Waterval, Electrophoresis, 23(2002) 3922 2. K. Imai, Y. Watanabe, Anal. Chim. Acta 130 (1981) 377. 3. S.Uchiyama, Biomed. Chromatogr. 15 (2001) 295 4. I. Le Potier, G. Franck, C. Smadja, S. Varlet, M. Taverna, J. Chromatogr. A 1046 (2004) 271

Are Aptamers able to Specifically recognize Endogenous Enzyme in Single Cells?
J. Jitkova, M. Berezovski, S.N. Krylov
York University, Toronto, Canada

Farnesyltransferase (FTase) is a cytosolic enzyme that catalyzes the first step in post-translational modifications (transfer of farnesyl group) of a number of cellular proteins including Ras, lamin B, prelamin A and others. FTase has attracted attention for its role in the processing of Ras proteins whose oncogenic mutations have been found in 30% of human cancers, and inhibition of FTase shown to prevent tumor growth. In addition, recent findings suggest that FTase is a potential marker of tumourigenicity. Western blotting and transfer of radioactive farnesyl group are the techniques that are commonly used for determination of FTase level and activity in cell population. In this study we apply capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection for aptamer-based determination of FTase concentration in single cells. Single-stranded DNA aptamer to human FTase has been selected by recently introduced NECEEM method. The fluorescein-labelled aptamer has 36 oligobases and Kd=0.5 nM. It has been shown that the presence of both substrates and inhibitors doesn’t influence FTase-aptamer interaction. Four types of cells have been chosen for analysis. These are murine (NIH3T3 and B-16), monkey (COS-7) and human (MCF-7) ones. The level of FTase was determined by Western blotting, and its concentration was similar for all cells. The cells were destroyed with a lysing agent, mixed with FTase aptamer and subjected to CE-LIF analysis after 10-min incubation. The pattern for human and monkey cells was different from that of mouse cells. The specific peaks that can be ascribed to the aptamer binding to FTase were observed only for COS-7 and MCF-7 cells. The absence of these peaks for mouse cells proves that the aptamer can distinguish FTase of different species. To confirm aptamer binding to endogenous FTase in cells, co-injection experiments have been done, and no additional peaks appeared. Cells treated with a specific FTase inhibitor (FTI-276) have been subjected to the same analysis and it has been shown that the aptamer-mediated method allows measuring the total concentration of endogenous FTase. The average concentration of FTase per cell has been calculated using the calibration curve for human recombinant FTase and the estimated data were in consistent with data obtained by Western blotting. Moreover, this method allows quantitative determination of FTase concentration of in 5-cell suspension. Thus, the reported aptamer-based method of determination of FTase concentration by CE-LIF is quantitative, sensitive and fast. Also it opens the door for measurement of enzyme content in individual cells.

Multi-residue analysis of glucocorticosteroids in sports doping and veterinary control using high resolution UPLC/TOFMS
M.E. Touber1, J.A. van Rhijn1, C. Georgakopoulos2, M.W.F. Nielen1
1) RIKILT-Institute of Food Safety, Wageningen, The Netherlands; 2) Doping Control Laboratory of Athens, OAKA, Athens, Greece.

The recent introduction of short columns packed with 1.7 micrometer materials offers unprecedented chromatographic resolution and speed in bioanalysis. Hyphenation with a fast MS technique such as electrospray ionisation TOFMS is essential in order to exploit fully the separation efficiency and the increase in signal-to-noise. The first part of the presentation is about multi-target analysis of corticosteroids in urine. More than 20 corticosteroids were separated in 4 minutes, accurate masses were determined, element compositions calculated and spectra were searched against a spectrum library. In the second part of this presentation focus is on the discovery of unknown (illegal) bioactive steroids. We developed an LC/bioassay-UPLC/QTOFMS identification system in which a bioactivity chromatogram (“biogram”) is generated with in parallel a set of narrow LC fractions which can be injected into the UPLC/QTOFMS for subsequent identification using full-scan accurate mass measurement. Thus identification and confirmation efforts are focused on the relevant bioactive peaks in the chromatogram only. The element compositions obtained are used to interrogate electronic chemical databases rather than spectrum libraries in order to find out whether the unknown has been ever commercialised or studied. If not, structure elucidation by UPLC/QTOFMS/MS and comparison with a synthesized reference compound will disclose the identity of the designer steroid. The validity of this approach will be highlighted using THG in urine as a model case. Acknowledgement This project is financially supported by the World Anti-Doping Agency and the Dutch Ministry of Agriculture, Nature and Food Quality. References M.W.F. Nielen, C.T. Elliott, S.A. Boyd, D. Courtheyn, M.L. Essers, H. Hooijerink, E.O. van Bennekom and R.E.M. Fuchs, Rapid Commun. Mass Spectrom., 17 (2003) 1633-1641. M.W.F. Nielen, E.O. van Bennekom, H.H. Heskamp, T.F.H. Bovee, L.A.P. Hoogenboom, Anal. Chem. 76 (2004), 6600. M.W.F. Nielen, T.F.H. Bovee, P. Rutgers, J.A. van Rhijn, L.A.P. Hoogenboom, Anal. Chem. 77 (2005) submitted.

Nonaqueous Capillary Electrophoresis of a-Helical Polypeptides
A. Psurek1, C. Neusuess2, T. Degenkolb3, G. K. E. Scriba1
1 School of Pharmacy, University of Jena, Philosophenweg 14, 07743 Jena, Germany 2 Bruker Daltonik GmbH, Leipzig, Fahrenheitstr. 4, 28359 Bremen, Germany 3 University of Giessen, Interdisciplinary Research Center, 35392 Giessen, Germany.

The capillary electrophoresis separation of a-helical polypeptides containing of 14 - 31 amino acid residues has been investigated using aqueous and nonaqueous background electrolytes. Generally, higher separation selectivities were observed in nonaqueous electrolytes. This may be explained by a change in the secondary structure when changing from water to organic solvents. Circular dichroism spectra revealed a significant increase in helical structure in methanol-based buffers compared to aqueous buffers. The advantages of nonaqueous capillary electrophoresis (NACE) for the analysis of a-helical polypeptides were utilized for the characterization of the microheterogeneous alamethicin F30 by NACE coupled to electrospray ion trap mass spectrometry (ESI-IT-MS) and electrospray time-of-flight mass spectrometry (ESI-TOF-MS). Tandem ESI-IT-MS was used for elucidation of the amino acid sequence based on the fragmentation pattern of selected parent ions. Eleven amino acid sequences were identified, characterized by the exchange of Ala to Aib in position 6, Gln to Glu in positions 7 or 19 as well as the loss of the C-terminal amino alcohol. In addition, two truncated pyroglutamyl peptaibols were found which have not been described for peptaibols from fungal sources before. Additional time of flight mass spectrometry measurements were performed in order to resolve the ammonium adducts from comigrating compounds (i.e. Aib-Ala exchange) and to confirm the amino acid composition of the individual components. Mass accuracy well below 5 ppm was observed using the major component ALM F30 as mass calibrant.

Transferrin Glycoforms Characterization by CE-UV and CE-ESI-MS applied to Congenital Disorders of Glycosylation Diagnosis
E. Balaguer, F. Benavente, J. Barbosa, V. Sanz-Nebot
University of Barcelona, Barcelona, Spain

Glycosylation is the most common posttranslational modification of proteins. The type and amount of glycoforms for a certain glycoprotein may change as a consequence of pathological processes. Patients with Congenital Disorders of Glycosylation (CDG) present hypoglycosylation of several plasmatic glycoproteins. This is the case of human transferrin (Tf), a 79600 Da glycoprotein which 85% is in the tetrasialo form. Carbohydrate deficient transferrin (CDT) containing less than three sialic acids is a well known marker for CDG and chronic alcoholism [1]. Most of the capillary electrophoresis (CE) approaches for Tf glycoform separation were based in the use of non-volatile buffers and additives not compatible with MS detection [2]. In the present study, a new CE separation method based on a PB-DS permanent coating is developed [3]. Reasonable good separation is obtained in a volatile background electrolyte based on ammonium acetate without any other modifier. The separation method is applied to serum samples from healthy and CDG patients, previously treated with an Albumin Depletion Kit. The suitability of the method for CDT detection and CDG type identification is demonstrated. In this work, a CE-ESI-MS approach for intact transferrin is carried out. The main parameters for CE-ESI-MS coupling, as sheath liquid composition, are optimized for standard Tf [4]. The method is applied to different serum samples, obtaining the separation of the different proteins present in serum and partial separation of Tf glycoforms. Different mass spectra are obtained from each glycoform which are deconvoluted separately, obtaining different molecular masses for Tf glycoforms from healthy and CDG sera. 1.G. Keir, B.G. Winchester, P. Clayton. Ann. Clin. Biochem. 36 (1999) 20-36. 2.C. Lanz, M. Kuhn, F. Bortolotti, F. Tagliaro, W. Thormann, J. Chromatogr. A, 970 (2002) 43-57. 3.H. Katayama, Y. Ishihama, N. Asakawa, Anal. Chem. 70 (1998) 2254-2260. 4.V. Sanz-Nebot, F. Benavente, E. Balaguer, J. Barbosa. Electrophoresis, 24 (2003) 883

Identification and Quantitation of trans-Ketoconazole as Impurity in cis-Ketoconazole by Capillary Electrophoresis-Mass Spectrometry
M. Castro-Puyana1, C. García-Ruiz1, A. Cifuentes2, A.L. Crego1, M.L. Marina1
1 Department of Analytical Chemistry, Faculty of Chemistry, University of Alcalá,Alcalá de Henares (Madrid), Spain. 2 Department of Food Analysis, Institute of Industrial Fermentations (CSIC),Madrid, Spain.

Impurities are defined as any component of a new drug product that is not the drug substance or an excipient. Their detection, identification, and quantitation are important aspects in the development of new drug substances and products. The ICH guidelines on impurities define certain thresholds for the content of impurities above which they should be identified and/or quantified. In fact, an accurate analytical profile of a drug substance must fulfill the requirements of Regulatory Agencies with respect to toxicity and safety aspects from the initial stage of the development of a potential drug to the quality control of a marketed pharmaceutical product. In this work, the main impurity of cis-ketoconazole has been detected by CE with UV detection, and identified and quantified by electrophoresis-electrospray-mass spectrometry (CE-ESI-MS). An ammonium formate buffer was employed to separate cis-ketoconazole from its main impurity, trans-ketoconazole, for CE-ESI-MS identification and quantitation. The optimization of ESI-MS parameters was carried out to obtain the best sensitivity. The optimized conditions enabled the identification of the impurity of cis-ketoconazole as trans-ketoconazole. Quantitation of this impurity was achieved in different samples: cis-ketoconazole standard and different pharmaceutical formulations. A comparative study on the quantitative capabilities of CE-UV and CE-MS analysis of ketoconazole was carried out. In all cases, percentages higher than 2.0 % were determined for the impurity. According to ICH guidelines, these values require the identification and quantitation of any impurity in drug substances and products.

Combined Injection and Fractionation Capabilities of an Autosampler for Automated Off-line Multidimensional LC Methods in Proteomics
Guillaume Tremintin, Bas Dolman, Evert-Jan Sneekes, Remco Swart
LC Packings, Abberdaan 114, 1046 AA Amsterdam, The Netherlands

The high sample complexity in proteomics requires the use of multidimensional separation techniques, such as gel-electrophoresis or liquid chromatography. The development of multidimensional LC methods involves optimization of many experimental parameters. Two main issues that must be considered are the way columns are coupled, on-line or off-line, and whether to perform separations at the peptide or protein level, i.e. the bottom-up or top-down approach. Off-line multidimensional LC techniques have several advantages over on-line approaches: i) more efficient separations, ii) higher flexibility with respect to column dimensions and mobile phase selection, iii) easier method development and trouble-shooting. Here we report on the fractionation capabilities of an autosampler. The instrument allows for sample injection and automated fraction collection at flow rates from 5-250 µl/min for capillary and microLC applications. The performance of the instrument is discussed for injection reproducibility, carry-over and recovery of sample. Multidimensional LC applications for the separation of peptides and proteins will be discussed as well as an instrument set-up for fully automated off-line 2D-LC of peptides including ESI or MALDI MS/MS detection.

Towards Oligosaccharide "Fishing" by On-line Frontal Analysis Continuous Capillary Electrophoresis-electrospray Mass Spectrometry (FACCE-ESI-MS)
A. Varenne, S. Fermas, T. Le Saux, F. Gonnet, P. Gareil, R. Daniel
Laboratoire Analyse et Environnement, CNRS UMR 8587, Université d’Evry-Val d’Essonne and Laboratoire d’Electrochimie et Chimie Analytique, CNRS UMR 7575, ENSCP

The interactions of polysaccharides with proteins represent a major issue in glycobiology, since resulting protein/carbohydrate complexes are involved in fundamental biological processes. The increasing interest in these non-covalent interactions is accompanied by a demand for viable, accurate and high throughput methodologies for their characterization. The structural identification of a targeted carbohydrate ligand remains a challenging task because of the huge structural complexity of oligosaccharides and of the molecular complexity of their mixtures. In order to overcome these difficulties, a new analytical strategy based on the hyphenation of capillary electrophoresis (CE) to mass spectrometry (MS) has been implemented in this work. The application of this strategy to the prototypic anti-thrombin/heparin pentasaccharide complex is presented. Frontal analysis continuous capillary electrophoresis (FACCE) consists in electrokinetically and continuously injecting targeted protein/ligand mixtures, leading to the formation of two migration fronts, the first one corresponding to the free protein and the second to the protein/ligand complex in equilibrium with the free protein. The hyphenation of CE to MS allowed on-line structural characterization. The ability of this method to identify either the protein or the complex in positive ionization mode and in non-denaturing electrospray conditions is shown. In addition, as ESI-MS has been widely recognized as a powerful and highly sensitive analytical method for the characterization of oligosaccharides, experiments have been carried out to perform on-line detection of the sulfated pentasaccharide ligand involved in the complex by mass fragmentation in negative ionization mode.

Detection and Characterization of Chemical Warfare Agent Degradation Products by Capillary Electrophoresis Coupled to Mass Spectrometry
M. Lagarrigue1, A. Bossée1, A. Begos1, A. Varenne2, J.C. Tabet3, P. Gareil2, B. Bellier1
1 Centre d’Etudes du Bouchet, Vert-le-Petit, France 2 Laboratoire d’Electrochimie et Chimie Analytique, Paris, France 3 Laboratoire de Synthčse, Structure et Fonction des Molécules Bioactives, Université Pierre et Marie Curie,Paris, France

In the last few years, capillary electrophoresis coupled to mass spectrometry (CE-MS) has gained a wide interest1,2. This technique represents a promising alternative to liquid chromatography-mass spectrometry LC-MS) especially for the analysis of charged or polar compounds in the presence of complex matrices containing surfactants. CE-MS combines separation principles which are complementary to those of chromatography, miniaturization implementation affording high separation efficiency and low sample consumption, and powerful detection capabilities with respect to sensitivity, identification and structural characterization. The aim of this work was to evaluate the performances of CE-MS coupling for the detection and identification of the degradation products of chemical warfare agents (phosphonic acids, phosphonates…) present in soil extracts or samples containing surfactants using a coaxial sheath liquid interface and an electrospray ionization source coupled to an ion trap mass spectrometer. Preliminary CE experiments were realized with a standard mixture containing five alkylphosphonic acids (diacids) in pure water, using indirect UV detection and separation electrolytes of varying pH composed of sorbate anion as chromophore in water-methanol media (60/40, v/v). A baseline separation of isomeric propyl and isopropylphosphonic acids was obtained at pH 8.3, which was attributed to different acidity constants of these compounds. Prior to CE-MS experiments, the nebulization conditions (drying and nebulizing gas) and ion trapping parameters were adjusted by direct sample infusion in order to maximize the abundance of phosphonic acid quasi-molecular ions on the MS spectra. The influence of the nature and concentration of the volatile separation buffer (ammonium acetate or carbonate) on resolution and sensitivity were studied and the sheath liquid composition was optimized. Thus, a series of 15 alkylphosphonic acids (diacids) and alkyl alkylphosphonic acids (monoacids/monoesters) in pure water were identified in 15 min in negative ionization mode with detection limits of so far 5-50 ppm. Similar results were already obtained in soil extracts of different origins. Even in cases where the resolution between the isomeric alkyl alkylphosphonic acids was limited, the investigation of their specific fragmentations in source or by MS/MS (isolation followed by fragmentation by resonant excitation in ion trap) allowed each isomer to be identified. Various approaches (preconcentration, detection) are presently being considered to improve overall method sensitivity. [1] P.Schmitt-kopplin, M.Frommberger, Electrophoresis 24, 2003, 3837-3867 [2] A. von Brocke, G. Nicholson, E. Bayer, Electrophoresis 22, 2001, 1251-1266

Microchip Electrophoresis-Mass Spectrometry for the Analysis of Peptides and Proteins
Takahide Kameda1, Fumihiko Kitagawa1, Akihiro Arai2, Koichi Suzuki2, Shin Nakamura2, Koji Otsuka1
(1) Department of Material Chemistry, Graduate School of Engineering, Kyoto University; (2) Shimadzu Corporation

Application of a microchip electrophoresis-mass spectrometry (MCE-MS) system employing an electrospray ionization (ESI) interface with a spray tip and a liquid junction was investigated for the analysis of peptides and proteins. To improve the MS sensitivity, reproducibility, and separation efficiency, the successive multiple ionic polymer layer (SMIL) coating was applied to the microchip and/or the spray tip. To evaluate the fundamental analytical performances in MCE-ESI-MS, two peptides, Pro-Leu-Gly amide and [Val4] angiotensin, were used as test analytes, and successful separation was achieved with the resolution of 1.3 within 70 s using the SMIL coated microchip. In the MCE-ESI-MS analysis of proteins, effect of the property of the SMIL coating on the separation performance was investigated. When a SMIL coated microchip with a bare spray tip was employed in the analysis of cytochrome c, a broader peak was observed, while with the SMIL coated spray tip a slightly sharper peak was obtained. By using the SMIL coated microchip and spray tip, cytochrome c and myoglobin were separated with the resolution of 1.1 within 80 s. The molecular weight of cytochrome c determined from the MS spectrum was accorded within a 0.5% difference from the literature value. Therefore, faster and efficient separation, and selective detection of peptides and proteins were achieved by the present MCE-ESI-MS system. To achieve further improvement of the MS detectability for proteins, an application of on-line sample preconcentration by stacking was investigated. As a typical example, the 10-fold sensitivity enhancement as well as improved resolution was obtained when a sample solution containing cytochrome c and myoglobin, of which the conductivity was lower than of the running buffer, was injected as a long plug.

Coupling of non-aqueous electrokinetic chromatography to electrospray ionization mass spectrometry (NAEKC-ESI-MS) for drug analysis
R. Mol, G.J. de Jong, G.W. Somsen
Department of Biomedical Analysis, Utrecht University, P.O. Box 80082, 3508 TB Utrecht, The Netherlands

Non-aqueous capillary electrophoresis (NACE) represents an attractive separation technique exhibiting different and/or enhanced selectivities in comparison to aqueous CE. Furthermore, NACE allows analysis of compounds that are poorly soluble in water. By adding pseudo-stationary phases, such as (charged) cyclodextrins (CDs) and ion-pairing agents to the NA background electrolyte (BGE), favorable separation systems with both chiral and non-chiral selectivity can be obtained. The non-aqueous electrokinetic chromatographic (NAEKC) systems, therefore, seem very promising for drug analysis, including impurity profiling. In order to achieve sensitive and structure-elucidative detection, coupling of NAEKC to electrospray ionization mass spectrometry (ESI-MS) seems very attractive. However, NAEKC-MS may not be straightforward due to the presence of the non-volatile phases in the BGE. In this study, NAEKC-ESI-MS using the anionic CDs heptakis(2,3-di-O-methyl-6-O-sulfo)-β-cyclodextrin (HDMS-β-CD) and heptakis(2,3-di-O-acetyl-6-O-sulfo)-β-cyclodextrin (HDAS-β-CD) was investigated. The NACE system is combined with an ion-trap MS equipped with an ESI source using a sheath-flow interface. Interface parameters like sheath liquid composition and nebulizing gas pressure were optimized. The effects of the CDs on the ionization efficiency of model drugs, was studied under infusion (no voltage) and CE (high voltage) conditions. It appeared that suppression of the analyte signals by the CDs was significantly less when a voltage was applied. The role of the counter ions (sodium) of the CDs in ion suppression was also studied. Full NAEKC-MS runs of (racemic) salbutamol, and mebeverine with related (chiral) compounds will be shown, indicating the chiral/achiral selectivity of the system. Satisfactory sensitivities (LOD of ca. 10 ng/ml injected) were obtained which is sufficient for a wide range of analytical queries like impurity profiling and bioanalysis. The enantio-selective analysis of salbutamol in urine using NAEKC-ESI-MS after SPE is demonstrated showing good repeatability (<7%) and linearity (R2>0.996) for both enantiomers.

Evaluation of ESI and APPI for CZE-MS of pharmaceuticals using non-volatile buffers
P. Hommerson, A.M. Khan, G.J. de Jong, G.W. Somsen
Utrecht University, Utrecht, The Netherlands

Capillary zone electrophoresis (CZE) is a highly efficient separation technique that is well suited to the separation of complex mixtures of ionizable solutes. In order to further extend its applicability, coupling to mass spectrometry (MS) is essential to allow sensitive detection and molecular structure characterization. Until now, the coupling of CZE with MS has predominantly been performed through electrospay ionization (ESI). Although CZE-ESI-MS has become an accepted analytical approach, the ESI of analytes is negatively affected by other ionic species such as (non-volatile) buffer salts. Conventional CE background electrolytes like phosphate and borate buffers are therefore known to cause ionisation suppression, and thus lead to reduced or even complete loss of analyte signals. Advances in bench top MS interface design saw the introduction of atmospheric pressure photoionization (APPI), a novel MS ionization source for liquid chromatography [1]. Recently, in our laboratory the feasibility of CZE-APPI-MS has been demonstrated [2]. The aim of the present study was to draw a quantitative comparison between the performance of ESI and APPI for CZE-MS, with emphasis on analyte characteristics and BGE effects. A sheath flow interface was used to combine CZE to an ion-trap mass spectrometer equipped with an ESI or APPI source. As analyte responses in CZE-MS are dependent on both intrinsic ESI/APPI efficiency and band broadening effects, the responses of test compounds and influences of BGEs were investigated in two ways. Firstly, the MS responses of various pharmaceutical compounds and influences of volatile and non-volatile BGEs were measured by infusion of test solutions in the respective BGEs. Secondly, signal-to-noise ratios of tested compounds were determined under actual CZE-MS conditions. An overview of analyte responses, obtained electropherograms and signal-to-noise ratios (S/Ns) is presented. The influence of BGE on ESI and APPI signals is evaluated and discussed in relation to the molecular structures of the test compounds. [1] D.B. Robb, T.R. Covey, A.P. Bruins, Anal. Chem. 72 (2000) 3653. [2] R. Mol, G.J. de Jong, G.W. Somsen, Electrophoresis 26 (2005) 146.

Hyphenation of CE with APPI/MS for pharmaceutical applications
Davy Guillarme, Julie Schappler, Josiane Prat, Serge Rudaz, Jean-Luc Veuthey
Laboratoire de Chimie Analytique Pharmaceutique

The use of capillary electrophoresis coupled with mass spectrometry (CE-MS) has evolved as a useful tool to analyze charged species with extremely small sample quantities. While CE confers high resolving power and speed of separation, MS provides high selectivity and sensitivity. The electrospray ionization (ESI) interface is currently the method of choice for CE-MS because of its sensitivity, versatility and ease of implementation. Nevertheless, ESI also possesses some inherent drawbacks: whereas non-volatile buffers like phosphate or borate provide better peak shapes and thus higher separation efficiency, only volatile buffers can be used. Moreover, ESI could provide large signal suppression with complex matrices. Finally, partial filling techniques have to be implemented for chiral separations and MEKC analysis to avoid problems of background noise and source contamination. An alternative to ESI can be the new atmospheric pressure photoionization (APPI) source which has been recently introduced. This ionization source is similar to APCI but the corona needle has been replaced by a krypton discharge lamp. Molecules are either ionized by absorption of photons or via a charge transfer mechanism using a dopant (acetone, toluene…). Like in APCI, the sample is vaporized before the ionization process is initiated and non-volatile salts can be eliminated during this step. Because of the gas ionization process, APPI is also adapted for non polar compounds ionization. However, many parameters have to be optimized to fully exploit the potential of APPI hyphenated with CE, such as the sheath liquid nature, its composition and flow rate, the dopant nature and composition, the drying gas flow rate and temperature, the nebulizing gas pressure, the vaporizer temperature and the capillary voltage. In order to optimize both principal parameters and interactions, experimental design procedures were used. In the optimized conditions, limits of detection were determined in CE-APPI/MS using a mixture of pharmaceutical compounds and compared with conventional CE-ESI/MS.

CE-MS of non-denatured proteins using bilayer-coated capillaries
Jonatan R. Catai, Javier Sastre Torańo, Gerhardus J. de Jong, Govert W. Somsen
Utrecht University

Capillary electrophoresis (CE) is an attractive tool for purity and stability studies of proteins as it provides fast and efficient separations and requires only minute amounts of sample. Changes in protein charge and shape are reflected in the electrophoretic mobility and, moreover, CE can be carried out using quasi-physiological conditions. The combination of CE with mass spectrometry (MS) is even more powerful as it offers both separation and characterization of proteins and their degradation products. However, CE of proteins using bare fused-silica capillaries may suffer from analyte-wall interactions leading to band broadening, unstable electroosmotic flow (EOF) and poor migration time reproducibilities. To overcome these problems, various capillary coating procedures have been proposed but they are often laborious and lack stability. Recently, we have suggested a simple, fast and reproducible coating method based on a bilayer of charged polymers and showed its usefulness for the analysis of proteins and peptides [1,2]. In this paper, the feasibility of this coating for the CE-MS analysis of (pharmaceutical) proteins was investigated. CE was coupled to an ion-trap mass spectrometer via an electrospray ionization (ESI) sheath-flow interface. Capillaries were coated with a bilayer of polybrene (PB) and poly(vinyl sulfonate) (PVS). The coating showed full compatibility with MS detection, and yielded a significant and constant EOF independent of the pH. The influence of several volatile background electrolytes (BGE) (pH 7.0-8.5) on plate number, migration time reproducibility, and MS signal intensity was examined for the tested proteins. Very stable protein separations (migration-time RSDs < 1%) and satisfactory plate numbers (ca. 100,000) were obtained with CE-MS using a BGE of 75 mM ammonium formate (pH 8.5). The applicability of the CE-MS method using bilayer coatings will be demonstrated by the stability monitoring of the biopharmaceutical human growth hormone (hGH) revealing e.g. deamidation and oxidation products. Furthermore, the results of the CE-MS analysis of several (expired) hGH preparations of commercial sources will be discussed. [1] Catai, J. R., Somsen, G. W., de Jong, G. J., Electrophoresis 2004, 25, 817-824. [2] Catai, J. R., Tervahauta, H. A., de Jong, G. J., Somsen, G. W., J. Chromatogr. A 2005, 1083, 185-192.

Monolithic silica supported electrospray interface for LC/MS
Shota Miyazaki1, Masanori Motokawa1, Kazushi Hayakawa1, Atsushi Sato1, Hideyuki Uzu1, Kazuki Nakanishi2
1 GL Sciences Inc., Saitama, JAPAN 2 Graduate School of Science, Kyoto University, Kyoto, JAPAN

The monolithic material has been widely investigating for its unique separation properties in the fields of analytical sciences. In recent years, nano- or micro-scaled monolithic separation media was commercialized and came into use in the bioanalysis. Especially, the monolithic separation media which is used in micro fluidic device coupled to mass spectrometer is powerful analytical tool for obtaining higher efficiency and sensitivity in microscale bioseparations. Hence, the capillary monolithic column with electrospray emitter (MonoSpray) was developed for electrospray ionization mass spectrometry (ESI-MS). This electrospray emitter was fabricated from silica monolithic capillary column so that monolithic silica was packed in fused silica capillary tube. When the MonoSpray was applied to the LC/ESI-MS, this electrospray tip which has 0.1 mm i.d. produced stable electrospray at the wide flow rate range (300 - 5000 nl/min). In addition, the peak with at the half height was narrower than the use of unpacked electrospray needle in the analysis of tryptic digest of protein. This result showed that the effect of band broadening was easily decreasing by means of forming a monolithic material in the nano electrospray emitter. Therefore, the MonoSpray has advantage over unpacked electrospray tip in nano-LC/ ESI MS. The MonoSpray C18 which is inner silica monolithic material was modified the C18 phase, which has both functions of analytical column and nano electrospray emitter. Thus, the use of MonoSpray C18 is able to separating the multicomponent sample without analytical column and it is able to introducing directly mass spectrometry without connecting other electrospray tip. When it was compared to general method of nano LC / ESI-MS analysis, the equal or better result was obtained for using MonoSpray C18. While the performance of MonoSpray C18 was compared with nanospray emitter filled with octadecyl silica particle, the separation efficiency of MonoSpray C18 was better than particle packed emitter. Indeed, the tryptic digest of protein (10 femto mol) could be separated using MonoSpray C18 having 0.1mm i.d. at the flow rate 300 nl/min in nano-LC / ESI-MS. The experimental results suggest that newly developed silica monolithic emitter utilizing for electrospray emitter achieved higher resolution and sensitivity in nano-LC / ESI MS analysis.

Chip-Based Ultra-Miniaturized Anchors for High Sensitivity in MALDI-MS
J. Sjödahl1, O. Öberg2, J. Roeraade1
1 Royal Institute of Technology, Dept. of Analytical Chemistry, SE-100 44 Stockholm, Sweden 2 Acreo AB, Electrum 236, SE-164 40 Kista, Sweden

Ultra-miniaturized anchors have been fabricated and employed for sample preparation and subsequent MALDI-MS analysis of peptides. The anchors are defined by the top surface of round pillar structures, which were fabricated by means of deep reactive ion etching, from doped silicon of low resistivity in diameters down to 5 µm. With anchors of this size, sample volumes in the femtoliter range can be deposited. A precision robotic setup was employed to transfer sample/matrix solutions onto the anchors, which were covered by a liquid lid of fluorocarbon during sample deposition. The liquid lid eliminates evaporation and enables a transfer of known amounts of analyte. The ultra-miniaturized anchors confine the sample within an area, significantly smaller than the cross-section of the ionizing laser beam. Thus, the whole sample is utilized for analysis. Furthermore, the small sample volumes allow minute amounts of analyte to be handled at high concentration, while the surface area, which the sample molecules are exposed to, is reduced. In this way, a considerable improvement in sensitivity can be obtained. Using a 30 µm anchor, we were able to detect 70 zeptomole of angiotensin I with a signal-to-noise ratio of 27.

Electrospray ionization generated from a gap with adjustable width
P. Ek, J. Sjödahl, J. Roeraade
Royal Institute of Technology, Dept of Analytical Chemistry, SE-100 44 Stockholm, Sweden

An electrospray nozzle with variable orifice size has been developed. The electrospray is generated at the end of an open gap with adjustable width formed between the edges of two triangular tips. The tips were fabricated from a plastic film. To improve imbibition and sample confinement, the edges of the tips forming the gap were hydrophilized. The gap width was adjusted in-situ between 1 and 20 um during the mass spectrometry experiments. Using angiotensin I as analyte, the signal to noise ratio increased as the gap width was decreased. Moreover, a shift towards higher charge states was observed. A 0.1 uM sample of angiotensin I yielded a signal to noise ratio of 11:1 when a gap width of l µm and a flow rate of 100 nL/min were employed. An important feature of the presented design is that it allows optimization of the electrospray in terms of sample consumption and sensitivity during operation. Also, nozzle clogging can be easily managed by simply widening the gap.

Amino Acids Screening in the nM Range in Body fluids by CZE-ESI TOF MS
Christian Neusuess, Matthias Pelzing
Bruker Daltonik GmbH

Amino Acids are the base for life and thus of importance in various life science related applications like nutrition science, sequencing, metabolomics or as early disease markers (Phenylketonuria). LC-based methods require the derivatization or ion pairing for the separation of the amino acids. Unlike these techniques there exist a couple of studies on the determination of underivatized amino acids by capillary zone electrophoresis-mass spectrometry (CZE-MS), however lacking appropriate concentration sensitivity. Here we present an improved method for the determination of amino acids and related compounds by CZE - ESI-TOF MS, enabling the sensitive and fast determination of amino acids profiles directly in various body liquids without derivatization. Capillary zone electrophoresis - electrospray time-of-flight mass spectrometry was used, coupling the fused silica capillary via a coaxial sheath-liquid interface to an orthogonal accelerated TOF MS. Separation was performed in a formic acid based electrolyte in uncoated fused silica capillaries (50um ID). The background electrolyte was optimized applying an amino acid standard to achieve best separation. Finally, all 20 natural amino acids could be separated, including baseline resolution of leucine and isoleucine. The concentration sensitivity is improved by on-line pH-mediated stacking. The method allows injecting more than 10% of the capillary. Thus, most amino acids can be determined down to 50 nM at an S/N ratio of 10-100. The calibration curve was linear over up to 4 orders of magnitude, enabling quantification on the level of 50nM to 250µM, without dilution or adaptation of the injection amount. Results for amino acid screening in various body fluids will be presented. The method has been applied to the analysis of urine, cerebrospinal liquid or blood without any sample preparation beside dilution. Beyond the standard amino acids, unknown compounds could be identified by their elemental composition based on the accurate mass of the ESI-TOF MS. Thus, the approach is an excellent tool for screening of known and rare amino acids without derivatization in body fluids.

Intact glycoform characterization of erythropoetin-a and erythropoetin-β by CZE-ESI TOF MS
Christian Neusuess, Elvira Balaguer
Bruker Daltonik GmbH, University of Barcelona

Glycosylation of recombinant human erythropoietin (rhEPO) is a post-translational process which depends on the type of cell in which rhEPO is synthesized, but also on the cell culture conditions and the final purification steps. These glycosylation modifications alter the biological activity, solubility and lifetime of rhEPO in blood. Thus, a rapid and simple method for the elucidation of the carbohydrate microheterogeneity of rhEPO is needed in order to evaluate a certain manufacturing process or assure the quality of the final product. Based on a recently developed method, the accurate mass determination of the intact glycoforms from two types of commercial rhEPO (epoetin a and epoetin β) by capillary electrophoresis-electrospray-time of flight-mass spectrometry is presented. The sample treatment consists in a fast and simple preconcentration step of the ready-to-use drug achieved by a centrifugal filter device. Characterization of the carbohydrate composition of each single glycoform is performed, in agreement with the results in glycan and glycopeptide analysis reported by other authors. The main differences between the carbohydrate structures of both epoetins are shown: the existence of two additional basic sialic acid isoforms for epoetin β and the higher degree of acetylation for epoetin a. The agreement of the main glycoforms for both epoetins is shown by molecular mass agreement. The high accuracy and reproducibility of the mass measurements with a standard deviation below 1 Da is proved by repeated analysis of European Pharmacopoeia rhEPO. Thus, in combination with a novel method for glycan analysis by CZE-MS an attribution of the overall composition can be given.

An Integrated Microfluidic Sampling/Electrophoretic Analysis System for In Vivo Chemical Monintoring
N.A. Cellar, R.T. Kennedy
University of Michigan, Ann Arbor, USA

A microfluidic chip with a pump for continuous sampling and an integrated flow-gated injector for capillary electrophoresis - laser induced fluorescence (CE-LIF) is described. The tunable elasticity of poly(dimethylsiloxane) (PDMS) in combination with multi-layer soft lithography allows for the creation of microfluidic valves and pumps that enable sample independent fluidic control. This technology is harnessed to combine a pneumatically-actuated peristaltic pump capable of generating flow rates between 20 and 200 nL/min with a microfluidic, flow-gated injector on a single chip. Because the device is intended for sampling and monitoring of neurotransmitters in the brain of living animals, the chip must be capable of driving many fluids. The microfluidic pump is designed with four channels: one pushes artificial extracellular fluid (aECF) into the brain, a second pulls sample, a third combines the resulting sample stream with o-phthaldialdehyde (OPA) for on-line derivatization, and the fourth provides separation buffer to the flow-gated injector. A pneumatic valve embedded in the chip is employed to divert separation buffer to make electrokinetic injections with the microfluidic flow-gate, and detection is performed off-column by LIF in a sheath-flow cuvette. The device allows for separation of eight amine neurotransmitters with separation efficiencies in excess of 400,000 plates, sub-100 nM detection limits, and 20 s temporal resolution. Stable measurements for over three hours have been achieved allowing for monitoring of dynamic changes caused upon the application of external stimuli such as perfusion of high potassium concentration aECF.

Study of Modulation of Signal Transmission in Reconstructed Neuronal Networks with Microfluidic Devices
Y. Mourzina, A. Steffen, S. Böcker-Meffert, B. Hoffmann, A. Offenhäusser
Research Centre Juelich, juelich, Germany

Biochemical modulation of synaptic communication with agonists and antagonists of membrane receptors is based on the activation of multiple pathways and accompanied by changes in protein expression. We combine biochemical and electrophysiological approaches to study modulation of neuronal activity in cultured cortical neurons by selective agonists/antagonists of metabotropic glutamate receptors (mGluR1, 5). To apply methods of expression proteomics for neuronal circuits, miniaturization of bioanalytical tools, in particular, capillary electrophoresis is desirable. The results of our work in two directions will be presented: 1) development of analytical techniques for proteomic analysis of the expression level of glutamate receptors in cell culture of rat cortical neurons, and 2) on-chip electrophoresis of amino acids and peptides and development of on-chip CE-MS interface with further aim to analyze membrane receptors in cultured neuronal cells. These results will be important for modulation of signal transmission in reconstructed neuronal networks in biomedical studies and bioelectronic devices.

D.R. Knapp, J. Liu, K.W. Ro, R. Nayak
Medical University of South Carolina Charleston, SC, USA

As part of our U.S. National Heart, Lung and Blood Institute-sponsored Cardiovascular Proteomics Center efforts to develop improved technologies for proteomic analysis, we are working on inexpensive, disposable microfluidic devices for sample preparation prior to mass spectrometric (MS) analysis. As the initial step toward more complex devices, we are developing arrays of capillary reversed phase HPLC columns in cyclic olefin copolymer (COC) chips that interface to MS via either matrix assisted laser desorption ionization (MALDI) or electrospray ionization (ESI). The microfluidic chips are prepared by hot embossing in COC wafers using photo-lithographically formed masters of SU-8 photoresist on silicon wafers or electroplated nickel masters. Cover wafers are attached using solvent-enhanced thermal pressure bonding utilizing a new vapor phase solvent enhancement method or by conventional heated press methods. The microfluidic channel walls are functionalized to facilitate bonding of the monolithic columns using photoinitiated graft polymerization via a photomask. Acrylate-based porous monolithic chromatography columns are then prepared in the channels using photopolymerization via a photomask. Samples and mobile phase are applied via an external pumping system. The MALDI interfacing utilizes simultaneous electrostatic transfer of fractions from multiple columns onto MALDI plates with pre-applied matrix. ESI interfacing utilizes planar carbon electrode connections to the channels formed by embossing traces of a carbon ink, which was formulated to resist the solvents used in column preparation. To confine the ESI Taylor cone to the diameter of the column exit (to maximize sensitivity and avoid postcolumn mixing), the edges of the chips are coated with a fluorocarbon layer using radio frequency chemical vapor deposition. A second approach to ESI interfacing uses carbon fiber ESI emitters. These reversed phase column arrays with MS interface comprise one module of the microfluidic devices for proteomic analysis.

Rapid Characterization of Sieving Gels for DNA Electrophoresis Using Microfluidic Devices
R.C. Lo, V.M. Ugaz
Department of Chemical Engineering, Texas A&M University, Texas, USA

Many research groups have made great efforts to optimize separation performance and maximize throughput of current gel electrophoresis technology. The ultimate goal is to quantitatively determine achievable separation resolution from direct measurements of parameters associated with the fundamental physics of DNA migration in the sieving gel matrix (mobility, diffusion, and dispersion coefficients) instead of by the traditional trial-and-error process. Unfortunately, acquisition of all the data needed for detailed gel characterization using conventional DNA sequencing instruments is very tedious and time-consuming, and thus has hindered advances in electrophoresis technology and development of improved theoretical models to describe electrophoretic DNA migration phenomena. We seek to address this issue by developing an automated microfabricated gel electrophoresis system capable of performing rapid and systematic characterization of mobility, diffusion, and dispersion behavior of double- and single- stranded DNA fragments (70-1000 bases). Experiments are carried out in 6, 9, and 12 %T photopolymerized crosslinked polyacrylamide gels at electric fields ranging from 15 to 40 V/cm, and the collected data are used to estimate mean gel pore size and to predict separation resolution under a variety of run conditions. These results are then compared with the predicted mean gel pore size of several models and with experimentally measured separation resolution. We also compare these results with those from a conventional slab gel DNA sequencer under the same run conditions. This automated microfluidic system allows the time required to collect a complete set of mobility, diffusion, and dispersion data to be reduced from several months to 2 hours. Using this platform, it is possible to quickly and consistently screen potential materials for DNA gel electrophoresis in order to improve separation performance, advance understanding of the physics of DNA electrophoresis, develop and refine theoretical models, and describe and predict the observed behavior.

Isotachophoresis for Integrated Microfluidic Analysis of Nucleic Acids
L. Chen, J. Prest, P. Fielden, N. Goddard, A. Manz, P. Day
Institute for Analytical Sciences, Dortmund, Germany School of Chemical Engineering and Analytical Science, The University of Manchester, Manchester, UK CIGMR, The University of Manchester, Manchester, UK

The analysis of DNA and RNA is central to functional genomics and gene assay development. However despite the highly heterogeneous composition of most tissue samples, currently most bioassays do not distinguish from which cell subtypes the assayed gene has been derived. The interfacing of the processes leading from gathering a biological sample through to data generation and analysis would gain greatly from standardisation and particularly by implementing integrated microfluidics, or more aptly, quantitative micro total analytical systems (mTAS). The biological problem to be addressed relates to the quantification of the leukemic cancer cell sub-population in a whole blood sample. The chosen route to resolving this important issue is to reduce a sample down to its cellular constituents and then undergo homogeneous analysis encompassing from sample selection, sample preparation, nucleic acid manipulations through to the detection of reporter moieties. Isotachophoresis (ITP) has been used widely to separate and pre-concentrate inorganic and organic analytes including those from biological samples. ITP advantageously features separation parameters that are controlled by electrolyte composition and high sample load capacity, and makes ITP a promising tool to improve the inherent limiting ability of mTAS to detect extremely low-concentration species. We are developing a microfluidic chip format employing ITP to concurrently purify and pre-concentrate nucleic acids from crude cell lysates prior to the introduction of a particular amount of nucleic acid template into subsequent steps of the bioassay analyses, including PCR and isothermal gene amplification. Primary studies have been carried out on standard DNA and RNA fragment separations using ITP microchips, as well as mixtures of DNA and proteins, and yeast cell lysates. We report our results and link the ITP studies to different PCR miniaturized reactors for in-vitro gene amplification.

A Microfluidic Chip Coupled to Microdialysis for in vivo Monitoring of Primary Amine Neurotransmitters by Capillary Electrophoresis
Z.D. Sandlin, R.T. Kennedy
University of Michigan, Ann Arbor, The United States of America

The ability to monitor neurotransmitters in vivo in the extracellular space of the brain can facilitate the elucidation of normal and pathological nervous system functions. Previously we have developed an on-line microdialysis based capillary electrophoresis (CE) system that utilizes laser induced fluorescence (LIF) with a sheath-flow cuvette detector capable of measuring in vivo basal and stimulated levels of primary amine neurotransmitters with high temporal and spatial resolution. However, the operational complexity of the system is a hindrance to the dissemination of the CE-LIF methodology. In response, we have developed microfluidic devices to replace the mixing tee, reaction chamber, flow-gate interface, and separation capillary of the CE-LIF system. In vitro testing of the initial microfluidic design resulted in detection limits for amino acids of ~200 nM, relative standard deviation of peak heights of 2%, and separations within 95 s with up to 30 200 theoretical plates. A second device design that allowed fields of 1436 V/cm to be applied while preserving the reaction time allowed separations within 20 s with up to 156 000 theoretical plates with on-line derivatization. For in vivo monitoring, fluidic devices were coupled to microdialysis to monitor glutamate concentrations in the striatum of an anesthetized rat during infusion of the glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC). A 272% increase in glutamate basal levels was observed after a 30 min infusion of 400 uM PDC. These results prove the feasibility of using a microfabricated fluidic system coupled to sampling probes for chemical monitoring of complex media such as mammalian brain. In the future, the microfluidic chip could be readily expandable to an array format to allow for multiple analyses from a single run.

Flexible microfluidic device for investigating coupled mass and charge transport in systems with charge-selective interfaces by quantitative confocal laser scanning microscopy
S. Ehlert, U. Tallarek
Otto-von-Guericke-Universität, Magdeburg, Germany

We have designed a flexible microfluidic device with external hydraulic flow and electrical field control, allowing the quantitative study of coupled mass and charge transport in systems of immobilized single adsorbent particles and discrete, as well as random arrangements of selected particles. For this purpose a flexible microfluidic device has been constructed that can be fixed and operated under a conventional microscope. Confocal laser scanning microscopy (CLSM) can reveal the influence of applied electrical field strength, mobile phase viscosity and ionic strength, particle size and surface charge density, as well as the resulting hydrodynamics in dilute and dense multiparticle systems on the local transport and distribution of co-ionic and counter-ionic analytes. A particular characteristic of these systems is that they contain interfaces which separate quasi-electroneutral fluid in the interparticle macropore space from fluid entrained in the charge-selective intraparticle mesopore space. This charge-selectivity originates in a mesopore-scale electrical double layer interaction (or overlap) causing co-ion exclusion and counter-ion enrichment at electrochemical equilibrium. When an electrical field is applied concentration polarization (CP) is induced in the bulk electrolyte solution as a result of coupled mass and charge transport normal to the charge-selective interfaces. CP depends on applied field and mobile phase ionic strengths, as well as the local (electro)hydrodynamics and it sensitively influences intraparticle transport and the distribution of charged analytes relevant to electrochromatography and electrical field-assisted processes [1,2]. [1] Tallarek, U., Leinweber, F. C., Nischang, I., Electrophoresis 2005, 26, 391-404. [2] Leinweber, F. C., Pfafferodt, M., Seidel-Morgenstern, A., Tallarek, U., Anal. Chem. 2005, 77, 5839-5850.

Application of micro multiphase laminar flow for extraction and detection of carbamate pesticides derivatives in microchip
A. Smirnova, A. Hibara, T. Kitamori
The University of Tokyo, Tokyo, Japan

Newly designed microchip with modified complex-shape microchannels was developed for efficient solvent extraction. As an important application of this system, pesticides determination is proposed. Determination of carbamate pesticides such as carbaryl, carbofuran, propoxur and bendiocarb with thermal lens microscope detection was demonstrated. The pesticide was hydrolyzed in an alkaline medium to 1-naphthol, was coupled with p-nitrobenzenediazonium fluoborate reagent, and, then, was extracted to n-butanol as a colored azo dye. A new microchip was used for mixing, reaction, extraction, and detection. A thermal lens microscope was used for the detection and ultra trace determination of the colored product. Optimum conditions for aqueous phase and organic phase flow rates inside the microchannels were identified. The calibration line showed good linearity for concentrations of 5 × 10-7 - 5 × 10-6 M and a detection limit of 7 × 10-8 M was obtained that at least two orders less than LODs for conventional spectrophotometric methods. Analysis time for such concentrations level can be estimated 15 min for microchip techniques comparing to 1 hour for bulk scale pre-concentration and separation by conventional methods. In order to distinguish target pesticides, potentials of separation methods such as HPLC and MEKC with thermal lens microscope detection were utilized for combination with the present method.

Quantification of Fluorescently Labeled Thiols by Microchip Electrophoresis
T. Revermann, S. Götz, U. Karst
University of Twente, Enschede, The Netherlands

Thiols play a major role in biological processes and it is therefore of importance to determine thiol functionalities in proteins and peptides. In particular, the determination of cysteine, homocysteine and glutathione in biological fluids and tissues is of importance. Other applications include environmental analysis and the quantification of thiols in depilatory cream and cold wave suspensions. The derivatization of thiol functionality with ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F) stabilizes the reactive analyte. Furthermore SBD-F is advantageous for electrophoretic separations due to the good solubility of the derivatives in water and the incorporated charge within the sulfonate functionality. As SBD-F is tailored for the derivatization and analysis of thiols, it shows quick reaction and significant fluorescence only for thiol derivatives and not for the respective amine analogues. The emission wavelength of thiol derivatives is 530 nm if excited at 380 nm. These thiol derivatives are separated on glass microchips. Samples are introduced through a double tee into the separation channel. A self-assembled fluorescence microscope based setup is used for detection. This instrument allows freely selecting the point of detection on the microchip and has the feature of wavelength resolved detection. The advantages of microchip separation are low sample and reagent consumption and short analysis times. With the developed microchip method, thiols were analyzed quantitatively with limits of detection of 2*10-6 mol/L and three decades of linear range. A commercial CE instrument was used as a reference.

Polystyrene-Divinylbenzene Monolithic Filled 50-100 µm i.d. Electrospray Needles for nanoLC-MS Analysis
Bjorn de Haan1, Vanessa Crenn1, Lennard Dekker2, Peter C. Burgers2, Th.M. Luider2, Remco Swart1
1 LC Packings - A Dionex Company, Abberdaan 114, 1046 AA Amsterdam, The Netherlands 2 Department of Neurology and Center for Biomics, Erasmus MC, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands

Polystyrene-divinylbenzene (PS-DVB) monolithic filled electrospray needles with internal diameters of 50-100 µm i.d. have been prepared for reversed phase nanoLC-MS analysis. Integration of the monolithic LC column and electrospray needle provides a dead-volume free and highly efficient separation system for peptides and proteins. The chromatographic performance of the monolithic filled electrospray needles is demonstrated by separation of tryptic peptides and intact proteins in gradient LC mode. Peak widths at half height of 2 s. and peak capacities up to 100 in less than 15 minutes were obtained. The monolithic columns showed reproducible and stable chromatographic performance in LC-MS over a wide flow rate range of 0.2-2.5 µl/min. NanoLC ESI-MS/MS analysis of several tryptic digests in less than 10 min yielding high sequence coverage demonstrated the potential of the monolithic filled electrospray needles for proteomics applications. We are now applying this technology to microdissected tissue from glioblastoma on small numbers of cells (200-1000) that are measured in FT-ICR mass spectromery.

Capillary-Assembled Microchip (CAs-CHIP): Reagent-Release Capillary towards Multiple Chemical Sensing
T. Henares, M. Takaishi, N. Yoshida, H. Hisamoto, S.Terabe
University of Hyogo, Ako-Gun, Japan

Simultaneous chemical sensing of various biological molecules from cell lysate is an indispensable endeavor in the microfluidic research. Capillary-assembled microchip(1) (CAs-CHIP) offers a potential solution. CAs-CHIP is fabricated by embedding various chemically functionalized square capillaries onto a PDMS lattice channel having same channel dimensions as outer dimensions of the square capillaries. The entire chemical sensing occurred inside the capillary. Thus, development of a simple, selective and sensitive capillary is of great importance. Here, we developed the chemically functionalized capillary by non-covalently immobilizing various reagents on the four corners of the square capillary in which the fluorescent reagent releases and reacts with specific analyte when a solution is introduced via capillary force. We have named this capillary as “ reagent-release capillary”. As a proof-of-concept, we used different reagents such as (a) fluorescein, for pH-sensing, (b) benzoyl-arginine-4-methyl-coumaryl-7-amide (Bz-Arg-MCA), for trypsin activity sensing, and (c) fluorescamine, for total protein sensing. In all cases, the reagent-release capillaries demonstrated excellent fluorescence response to specific analyte. Also, preliminary application of multiple chemical sensing using pH-sensing capillary as a model reagent-release capillary integrated onto the CAs-CHIP was successfully implemented. (1)Hisamoto, H.; Nakashima, Y.; Kitamura, C.; Funano, S.; Yasuoka, M.; Morishima, K.; Kikutani, Y.; Kitamori, T.; Terabe, S. Anal. Chem. 2004, 76, 3222-3228.

Development of Electrically Driven Separations on PDMS Microsystems.
Nathalie Delaunay-Bertoncini, Maximilien Blas, Jean-Louis Rocca
Laboratoire des Sciences Analytiques, UMR 5180 CNRS - Université Claude Bernard Lyon I, Bât CPE, 43 boulevard du 11 novembre 1918, 69622 Villeurbanne Cedex, France

Miniaturization of analytical instruments has attracted a wide interest in analytical chemistry over the past decade because of the advantages of reduced reagent consumption, better analytical performance, and shorter analysis time. The widespread interest in this field has resulted in efforts to develop chips. The use of plastic materials such as poly(dimethylsiloxane) (PDMS) may be a simple alternative process to expensive and time-consuming glass and silicon microfabrication and thus has been preferred in this study. We also decided to focus on chips involving electrically driven flows. Indeed, the application of voltage across the terminals of microchannels is much easier to achieve than the application of a pressure difference and the resulting flat profile allows to minimize dispersive effects. Injection is a key step to control in order to have efficient and sensitive analysis. Thus, we considered all the procedures described in the literature and we also proposed new ones to do an exhaustive and comparative study. Injected amount, reproducibility, and analytes discrimination have been determined for both cross and double-T design and different injecting modes (classical, gated, pinched, pinched with counter-flow, dynamic). The experimental results have been successfully correlated with numerical simulations. This allowed to draw innovant conclusions on the degree and the domain of interest of each injection mode and injection geometry. Then, we studied the separation step. Highly efficient and fast zone electrophoresis simultaneous separations of fluorescent labelled amino acids and catecholamines, which are neurotransmitters, have been carried out with the chips. However, a lack of selectivity has been observed. The introduction of interactions between analytes and a stationary phase was thus necessary to improve the separation. One approach consisted in using micelles and gave improved selectivity. A second option was to synthesize directly in the channels an organic monolithic stationary phase. Unfortunately, it was observed that polymerization of acrylate-based monoliths in PDMS channels was highly challenging because of the PDMS absorption abilities. Thus, an original approach has been evaluated, which consists in assembling pieces of classical capillary, filled with the desired monolithic stationary phase, and pieces of PDMS chips for injecting and detecting the target analytes.

Harnessing the Power of the NanoBioProcessor Engine for Genomics and Biodefense.
Stevan Jovanovich, Rich Belcinski, Iuliu Blaga, Allen Boronkay, Norman Burns, William Gurske, Roger McIntosh, Margaret Roy, Barney Saunders, Robert Stack, Jacklyn Taal, Mattias Vangbo
Microchip Biotechnologies Inc

Microfluidics and nanofluidics offers potential advantages in performance, reagent consumption, and size. However, the potential has seldom been realized and the impact of miniaturization technologies remains largely a future vision. In this presentation, we describe a new microfluidic and nanofluidic platform, the NanoBioProcessor TM, which integrates novel valves, pumps, and routers on-chip with simple control of their operation using full-scale pneumatics. The NanoBioProcessor engine is being applied to develop biodefense and genomic instruments. For biodefense, the NanoBioProcessor is being developed as a biosensor for both pathogens and toxins with bead-based bioagent capture followed by on-chip sample processing and detection. For genomics, the NanoBioProcessor is a central part of a major collaborative effort to develop a Microbead-based INtegrated DNA Sequencer (MINDS) as a next-generation completely integrated platform to achieve a $100,000 mammalian genome. The overall strategies, designs, and initial results will be presented to illustrate how the NanoBioProcessor harnesses the power of microfluidics to create a widely applicable sample preparation and platform.

Chip-based Magnetic Separation Technique for Simplification of Peptide Mapping.
L. Korecká12, J Krenková13, N. Minc2, J.L. Viovy2, Z. Bílková1
1 University of Pardubice, Pardubice, Czech Republic 2 Institut Curie, Paris Cedex 05, France 3 Institute of Analytical Chemistry, Brno, Czech Republic

Technique of peptide mapping consists of number of time consuming steps such as protein unfolding and digestion, peptide separation and identification. Moreover low concentration of specific peptides, undesirable sample dilution during protein unfolding and/or digestion and even a contamination of peptide mixture are possible limitations of this technique. Due to the excellent characteristics the magnetic particles were used for preparation of the enzyme and affinity microreactor for highly efficient protein fragmentation and affinity isolation of specific peptides in relation to application of prepared reactors to the channel of magnetically active microfluidic device. Miniaturization of analytical systems for several applications (fragmentation, purification, separation, detection) is now subject of interest. Utilization of chip-based magnetic separation for specific enzymatic fragmentation with following isolation and purification of specific peptides in one step is presented in this entry. Proteolytic enzyme trypsin was selected for specific fragmentation of selected proteins. Anhydrotrypsin, catalytically inactive form of trypsin, which enables highly specific trapping of peptides which correspond to the products generated by the action of various trypsin-like proteases and binds only specific peptides containing arginine and lysine residues at their C-termini, was selected as an affinity ligand. Both proteins were immobilized on carriers with magnetic nature and with special parameters (size, porosity, hydrophobicity). High purity and preconcentration of peptides isolated on immobilized anhydrotrypsin enables significant facilitation of the protein fingerprint identification even for protein in submicromolar concentration. Acknowledgements: This work was supported by grants MSM0021627502, Czech Science Foundation GACR 203/05/0241 and GACR 203/05/2106. The development of microfluidic device was supported by French Government ACI NMNC and EU Project “Allergy-Card” (contract 012793).

Dielectrophoretic Immobilization of Mammalian Neural Cells in Microfluidic Devices
S.P. Forry, D.R. Reyes, M. Racic, M. Gaitan, L.E. Locascio
National Institute of Standards and Technology

Much attention has been focused on the use of microfluidics to improve cell culture and manipulation through the development of biomimetic microenvironments.(1) These systems would allow continuous nutrient delivery to as well as waste removal from the culture area, transient and/or localized chemical treatments, and parallel cultures on a single device of individual cells or small cell colonies. However, to take full advantage of microfluidic systems as culture sizes decrease toward single cell assays, it becomes important to accurately control cell position and surface attachment within the microfluidic network. We have designed and fabricated electrode arrays for generating AC dielectrophoresis (DEP) forces within the microfluidic device geometry. Neural cells suspended from bulk cultures into isotonic sugar solutions have been individually immobilized by positive DEP as they moved through microfluidic devices under hydrodynamic flow. Approximately 85% of cells passing the DEP electrodes were immobilized, correlating well with the fraction of viable cells under conventional cell culture conditions. Interesting cell array geometries were generated as cells were immobilized one at a time (e.g. a linear arrangement perpendicular to solution flow - see Figure). Prior surface treatment with polyelectrolyte multilayers (PEMs) at the site of DEP immobilization prevented disruption of the cellular arrays after the DEP electrodes were deenergized. While PEMs encourage surface attachment of various mammalian neuronal cell lines,(2) the process was dramatically accelerated from several hours in bulk cultures to just seconds with DEP trapping. The immobilized cells remained adherent for >24 hours even when the flow rate was varied, or when various chemicals were introduced. Treatment with Trypan blue, an indicator of cell viability, demonstrated that the immobilized cells are not damaged by the DEP forces even after prolonged exposure. Laminar flow past the cell array trapped with DEP allowed localized delivery of chemicals. For example, when a dilute toxin was administered only to specific cells within the immobilized array, the treated cells rapidly died and detached from the surface while untreated cells remained unaffected. This demonstrates the utility of the current methodology for toxicity or drug efficacy investigations with in situ controls. 1. Beebe, D.;Folch, A. Lab on a Chip 2005, 5, 10-11. 2. Forry, S. P.;Reyes, D. R.;Gaitan, M.;Locascio, L. E. Langmuir, In Preparation.

Use of Self-assembled Magnetic Beads for On-chip Protein Analysis
Z. Bílková1, M. Slováková12, L. Korecká12, N. Minc2, J.-L Viovy2
1University of Pardubice, Department of Biological and Biochemical Sciences,Štrossova 239, CZ-530 03 Pardubice, Czech Republic 2 Laboratory of Physical Chemistry, Institute Curie (UMR CNRS/IC 168), Rue d´Ulm, Paris, Cedex 05, France

There is a continuous push to miniaturize and integrate as many laboratory instrumentation pieces as possible in the quest for a “lab-on-chip”. Three of the most important advantages of using microfluidic system of reduced dimension for analytical applications are known to be: 1) using minute quantities of sample and reagents (micro-,nanoliters), 2) relatively fast reaction times when molecular diffusin lengths are of the order of the microchannel dimension, 3) a large surface-to-volume ratio offering an intrinsic compatibility between the use of a microfluidic system and surface-based assays (1). Parallel to the boom of microfluidic systems, nanomaterials and nanoparticles with paramagnetic properties have become a hot topic in recent research. Functional nano- and microparticles offer a large specific surface for ligand binding. The concept of using immobilized ligands has extended beyond just chromatographic applications. Using carriers with magnetic property overcomes many of the problems associated with the use of liquid gel slurries in high-throughput and standard laboratory applications. Magnetic particles provide universal system with additional convenience, consistency, stability, ease of handling and exceptional flexibility compared to standard chromatography resins. Verpoorte (2003) (2) and Gijs (2004) (1) recently reviewed new technology based on the integration of magnetic functionalized carriers within microfluidic systems. Microanalytical system with packed plug of bio-active beads profit from a larger surface-to-volume ratio, faster analysis time due to favorable reaction kinetics on the microscale format. Furthermore, integration of several assay functions on a single chip leads to assay automation and elimination of operator involvement as a variable. An objective of this presentation is to perform new microfluidic analytical system using superparamagnetic nanoparticles as a self-organizing matrix. The versatility of new microfluidic device enables integration of different reaction steps, e.g. peptide mapping technique (3-5)
], epitope extraction technique (6), and immunospecific detection of pathological proteins (7). Acknowledgements: the authors wish to acknowledge Czech Science Foundation GA 203/05/0241 and GA 203/05/2106, Ministry of Education (MSMT 0021627502) for financial support of research program. The development of microfluidic device was supported by EU Project Project "Allergy-Card" (012793). 1. Gijs M.A.M.: Microfluidic Nanofluid 2004, 1, 22-40. 2. Verpoorte E., Lab chip 2003, 3(4), 60-68. 3. Korecká l., et al., J. of Chromatography B 2005, 808, 15-24. 4. Slováková M., et al., Lab-on-chip 2005, in press. 5. Bílková Z., et al., Electrophoresis 2005, special issue - Miniaturization, submitted. 6. Bílková Z., et al., EJMS 2005, submitted. 7. Bílková Z. et al., Proteomics 2005, 5, 639 - 647.

Automated On-chip Electrophoresis Enables Fast and Efficient Monitoring of Protein Expression and Refolding Screen - an Example of Applied Microscale Bioseparation
R. Salowsky, C. Buhlmann, M. Greiner, T. Wulff, Paul Hawtin1
Agilent Technologies Deutschland GmbH, Waldbronn, Germany 1 Department of Global Sciences and Information, AstraZeneca, Cheshire, United Kingdom

In the areas of target validation and high throughput screening, the need for pure and correctly folded target protein is of primary importance. Furthermore, the growing use of structural biology to support medicinal chemistry efforts makes similar demands on a viable protein supply. To this end, a growing number of research organizations are employing a multiple parallel protein supply paradigm. In such approach proteins are produced, purified, and processed in titer plate format for subsequent experiments, the need for high throughput, parallel protein analytical systems is also increasing. Through the use of high throughput expression and purification systems, investigators are able to produce multiple protein targets under a variety of conditions. The automated Lab-on-a-Chip Platform used in this study addresses these new needs by providing parallel protein analysis in a reusable microfluidic format. Due to the microscale of the chip only a few hundred Pico liters sample are loaded from 96 well plates onto the chip. Sizing and quantitation can be performed in an unattended fashion on twelve titer plates in a single “job”. Data for the utility of this platform for protein expression, protein purification, and protein refolding optimization experiments is presented. A new results flagging software feature affords rapid, high-level review of analyses with all data from the platform database. This permits straightforward optimization of protein expression conditions, protein purification protocols, and protein refolding screens.

Development of a Portable, Ultra Low-cost Plastic Microfluidic System for Multiple Simultaneous DNA Separations
W.N. Vreeland, N.Y. Morgan, J. Kakareka, J. Shah, C.W. Kan, L.E. Locascio, P.D. Smith, T. Pohida, M. Gaitan
National Institute of Standards & Technology, Gaithersburg, Maryland, USA National Institutes of Health, Bethesda, Maryland, USA

We have developed a portable microfluidic system with auxiliary optics, pneumatics, and software to facilitate DNA electrophoretic separations. This system requires only 10 minutes of electrophoresis to separate DNA fragments up to 400 bases in length. The microfluidic devices are fabricated from common commercial plastics, thus are inherently of low material cost, enabling them to be single-use, disposable devices. To facilitate high efficiency separations, the plastic channel surface is passivated with a polymeric coating to ameliorate non-specific interactions of the DNA with the channel surface. Coatings that are well developed for passivation of glass and silica channels are less ideal in plastic systems; hence, we have developed new chemical surface treatment procedures to effectively passivate plastic channel surfaces with polymeric coatings. Specifically, this is accomplished by oxidizing the plastic surface with oxidizing acids or UV-ozone treatment to create a higher concentration of hydrogen-bonding species on the surface. This allows for better adsorption of hydrophilic polymers to the channel surface and effective reduction in analyte-wall interactions. For fluorescent optical detection methods, plastic devices have relatively high levels of autofluorescence, when compared to traditional glass and silica systems that can decrease detection sensitivity. To address this challenge, we have developed a novel, spatially selective detection system. A free-space laser is spread into a collimated line with two cylindrical lenses and then focused into 8 individual spots by an array of spherical plano-convex lenses to serve as light for fluorescent excitation. At each excitation spot a ball lens and optical fiber is positioned underneath a microfluidic channel. Spatial selectivity is achieved by using a high refractive index ball lens and a much smaller optical fiber positioned so that the fiber collects focused light from the channel, while rejecting most of the plastic autofluorescence. The other ends of the fibers are then positioned in an array a directed to the entrance of a spectrograph for spectral separation and the fluorescent spectra of the channels recorded using a cooled CCD camera. This system allows for limits of detection of approximately 10 pM of fluorescein in a plastic microchannel. Furthermore, in contrast to scanning confocal systems, this ball lens and fiber system can be used with channels at an arbitrary spacing has no moving parts and uses low-cost optical components. The complete system fits into a 60 cm x 60 cm x 30 cm enclosure mounted on a small cart and is powered by a standard domestic electrical outlet. This allows for a portable device that does not require special laboratory environment. To date we have demonstrated our device in hotel meeting rooms after transportation in a standard size automobile.

Electrochromatography in Microchip:Performant Tool for Efficient Preconcentration Combined with Fast Separation
S. Descroix, V. Augustin, G. Proczek, P. Gareil, M.C. Hennion
Lab. Environment and Analytical Chemistry, UMR CNRS 7121, ESPCI, 11 rue Vauquelin, 75231 Paris cedex 05, France Lab. Electrochemistry and Analytical Chemistry, UMR CNRS 7575, ENSCP, 11 rue Pierre et Marie Curie, 75231 Paris cedex 05, France

Since few years, the analytical needs have evolved towards rapidity, high throuhgput, efficacity and sensitivity. In this context, microsystems for total analysis (µ-TAS) have been designed to integrate the whole analytical chain: sample pretreatment, compounds of interest separation, detection and quantification. Before this whole integration could be achieved, the strategy we adopted was to fisrt optimize each analytical system in capillary format, then to transpose it in microdevice format. This work presents the results of in situ synthesis of monolithic stationnary phases for CEC. CEC is considered as a promising analytical technique because it combines the efficiency of zone electrophoresis and the selectivity of liquid chromatography. First, the in situ synthesis conditions were optimized in capillary columns then carried out in glass microdevices. An homogenous acrylate based monolithic stationnary phase has been successfully photopolymerized in a chosen zone of the microsystem. Then, separations have been first performed in capillary electrochromatography in order to validate the analytical system performances. Environmental compounds (pesticides, pollutants) as well as biological ones (proteins, peptides) have been succesfully separated within short analysis time (less than 10 minutes) and with high efficiencies (ranging generally from 150 000 to 300 000). Concerning, the channel electrochromatography, the separation performances were first studied trough the analysis of model compounds of environmental interest. Five polycyclic aromatic hydrocarbons (PAH) were separated with satisfying resolution within less tan three minutes with efficiencies ranging from 100 000 to 200 000 plates per meter. And this sepration has been achieved with an effective length of 1.5 cm. We are currently confirming the separation potentiality of CEC in glass microsystem containing a monolithic stationnary phase for proteins and petides separation. Finally, one of the main drawbacks of microsystem analysis is their lack of sensitivity due to the very low volume injected. These difficulties shoud be overcome by achieving a proper concentration step of the compounds of interest before the separation step. Studies were fisrt conducted on model environmental compounds and biomolecules in capillary format. Preconcentration factor higher than 10 000 were obtained while maintaining at the separation level satisfying resolution and efficiencies. The preconcentration procedure was then adapted to microsystems device leading to promising results for PAH preconcentration and separation. Indeed, the on-line preconcentration allows a sensitivity enhancement factor about 500 for a preconcentration step of one minute without loss of separation performances. We are currently confirming the separation and preconcentration potential of CEC in glass microsystem containing a monolithic stationnary phase for protein and petide analysis.

Miniaturized Fluorescence Detection Chip for Capillary Electrophoresis Immunoassay of Atrazine
S.K. Kim, J.Y. Kang, Y.H. Kim, J.A. Min, K.S. Shin, E.K. Yang, T.S. Kim
Micro system Research Center, Korea Institute of Science and Technology

Fluorescence detection in microfluidic system is a sensitive method for chemical analysis. However, fluorescence microscope is too big to be portable for on-site analysis. This paper reports a miniaturized fluorescence detection system in microfluidic chip for CEIA (capillary electrophoresis immunoassay) of atrazine. It consists of a microfluidic chip, a silicon based PIN photodiode and an interference optical filter. For higher sensitivity of fluorescence detection, the finger type PIN photodiode was designed which uses the side depletion region of p+n junction. Its low parasitic resistance enhanced the sensitivity by 60% compared with conventional rectangular type. The interference filter was directly deposited on the photodiode substrate to block the background noise from excitation light. The microfluidic channel was bonded with the photodiode using the interlayer of polydimethlysiloxiane (PDMS) as shown in Figure 1. The fabricated detection chip could detect the concentration of a few-nM with TMR (tetramethyl rhodamine) dye with laser light (Figure 2). Miniaturized detection system analyzed atrazine using CE competitive immunoassay. As a preliminary test, a mixture of 100nM atrazine and 100nM anti-atrazine was pinched and separated in 20mm long microfluidic channel. The Ab-Ag* and Ag* were separated and two fluorescence peaks were detected by miniaturized sensor. External PMT (Photo Multiplier Tube) also recorded the fluorescence signal of microfluidic channel to confirm the reliability of the photodiode sensor. The graph in Figure 3 shows that both sensors detected separation of complex and antigen, and that two lines of results agreed quite well. These results support the feasibility of incorporated on-chip fluorescence detector for quantitative determination of CRP by CE immunoassay. [1] K. B. Mogensen, H. Klank, and J. P. Kutter, “Recent developments in detection for microfluidic systems”, Electrophoresis, vol. 25, pp. 3498-3512, 2004. [2] J. R. Webster, M. A. Burns, D. T. Burke, C. H. Mastrangelo, “Monolithic Capillary Electrophoresis Device with Integrated Fluorescence Detector”, Anal. Chem., vol. 73, pp. 1622-1666, 2001

Designing Microchip for High Performance Electrophoretic Analysis
Kenji Sueyoshi1, Hidenori Nagai2, Shin-ichi Wakida2, Junji Nishii2, Fumihiko Kitagawa1, Koji Otsuka1
(1) Department of Material Chemistry, Graduate School of Engineering, Kyoto University; (2) National Institute of Advanced Industrial Science and Technology, AIST

In microchip electrophoresis, the concentration sensitivity is generally insufficient due to a short optical pass length. To improve the detectability, we designed microchips with single a cross- and T-form channel geometry, which was applied to stacking using reverse migrating micelles and a water plug (SRW) in micellar electrokinetic chromatography on microchip (MCMEKC). Under an optimal condition, a baseline separation of three rhodamine derivatives was successfully attained with the 65-fold increase in the detectability by SRW.MCMEKC compared with a conventional MCMEKC. It has been noted that both resolution of two rhodamine dyes and the efficiency of the preconcentration increased with increasing the injection time of water from 0.5 to 3.0 s. To achieve further high performance sample injection, we designed microchips with a new channel geometry, which enable both two sample solutions to be introduced into the separation channel with desired volumes by a gated injection technique. In this study, the developed microchips were applied to MCMEKC using a partial filling (PF) technique. In the PF technique, to suppress the increase in a background noise due to the presence of a pseudostationary phase (PSP), e.g., ionic surfactant micelles in MCMEKC with mass spectrometric detection, in a background solution (BGS), the separation channel is partially filled with a PSP while the rest of the channel including detection point is filled with the BGS without a PSP. As a result, both the micellar solution and sample were successfully injected into the separation channel as a long and short plug, respectively, and a baseline separation of rhodamine derivatives was achieved by MCMEKC in the micellar zone injected with the PF technique. We will demonstrate that a combination of the PF technique with on-line sample preconcentration to attain a high performance analysis on the microchip.

Pressure driven flow control system for nanofluidic channels
E. Tamaki, A. Hibara, T. Tsukahara, H. B. Kim, T. Kitamori
University of Tokyo, Kanagawa Akademy of science and Technology, Japan Science and Technology Agency

We developed pressure-driven flow control method for nanochannel by using back-pressure regulating and achieved chemical reaction in nanospace. Integration of flow chemical processing has been an important investigation. In researches on microchip chemistry, short diffusion length and high specific interfacial area were utilized. Further integration of chemical systems will enable us to utilize these characteristics more effectively. From the basic chemistry viewpoint, it should be investigated how the channel size affects behaviors of molecules in a nanospace. We have investigated liquid properties in nanochannels by NMR and time-resolved fluorescent spectroscopy and the results indicated higher viscosity and higher proton mobility. We expected such property change will affect chemical process in nanospace. We chose pressure-driven flow as flow control system rather than electro-osmotic flow because various solvent should be used for general chemical process. In this report, pressure-driven flow control method in the nanochannels was evaluated and chemical reaction in the nanochannels as a single unit of nanofluidic chemical process was demonstrated. HPLC pumps were used for controlling liquid flow in the microchannels. A Nanochannel was connected to two microchannels. One microchannel was connected to a back-pressure regulator in downstream and controlled to a specific pressure. Another microchannel was not connected to a back-pressure regulator. As a result, pressure difference between two microchannels was controlled by back pressure regulator and applying pressure to nanochannel could be determined. Liquid flow in nanochannel is linear to pressure. By utilizing the flow control system, two different solutions can be simultaneously introduced in Y-shaped nanochannel (390 nm width, 240 nm depth). Figure (a) shows the scheme of pressure driven flow in Y-shaped nanochannel. In order to demonstrate mixing in nanochannel, two different fluorescent solutions were introduced into Y-shaped nanochannel using flow control method for nanochannel. Rhodamine B 10-4 M aqueous solution and Fluorescein 10-4 M aqueous solution are used as fluorescent probe. Furthermore, chemical reaction in nanochannel was performed. Tokyo Green (TG), which is a derivative of Fluorescein molecule, 10-5 M solution was used. Fluorescence intensity of TG depends on pH condition. Reaction scheme and results of reaction in nanospace was shown in figure (b). TG in acid buffer, which did not generate fluorescent, was mixed with alkali buffer in Y-shaped nanochannel. When the two solutions meet at the meeting point of nanochannel, TG was protonated and generates fluorescence. However the mixed solution in nanochannel except for the meeting point was not fluorescent. We speculated that the change of fluorescent molecule condition was affected by size-effect of nanochannel. We will investigate details of size-effect by trying various pattern of nanochannel.

H. Mühlberger, A.E. Guber, W. Hoffmann
Forschungszentrum Karlsruhe, Institute for Microstructure Technology, Germany

This paper details an initial approach towards polyether ether keton (PEEK) based lab-on-a-chip systems by introducing PEEK capillary electrophoresis (CE) chips combined with contactless conductivity detection (CCD). Difficulties in PEEK processing which have been limiting until now have been overcome by a new plasma enhanced thermal bonding process. A significant decrease in electroosmotic flow (EOF) was found using PEEK. Initial CE experiments demonstrated the analytical suitability of these new PEEK chips. Although PEEK shows outstanding material features for applications in analytical chemistry, such as extremely high chemical resistance and mechanical stability, high temperature resistance, very low adsorption and absorption etc., no adaptation of microchannel structures to the lab-on-a-chip world have been found until today, mainly due to difficulties in processing. Furthermore, PEEK microparts are opaque preventing the use of established optical detection methods. But, CCD utilizing capacitive high frequency electrical signal transfer can be used for broad non-specific detection of any charged species in CE where the intrinsic property of ion mobility is essential for the separation process itself [1]. It does not need sample labelling, and because external detection electrodes outside the microchannel are used in CCD, any sample contamination and electrode degradation are prevented. Therefore, advancing PEEK chip processing technology and combining these chips with CCD might be a substantial contribution to lab-on-a-chip development. To find optimal conditions for PEEK bonding - which is one of the most critical fabrication steps - several pretreatments with respect to resulting bonding strength were optimized. Nitrogen plasma proved to be superior compared to other plasma or UV pretreatments. Bonding was so strong that fractures predominated in the bulk material. Additionally, the wetting contact angle was reduced by N plasma treatment. Using this technique, we were able to bond standard cross shaped microchannel CE structures, prepared by hot embossing, with a cover foil. Additional Au electrodes for CCD were sputter deposited on the channel cover. Although a small increase in the surface charge caused by the plasma pretreatment was observed, a significant decrease of electroosmotic flow (EOF) was found using PEEK compared to other conventional polymer materials such as poly (methyl methacrylate) (PMMA), polypropylene (PP), polycarbonate (PC), polystyrene (PS) and cycloolefine copolymer (COC). These results may be attributed to specific inert surface properties of PEEK. In some initial separation experiments amino acids could be separated, furthermore sacharide could be analyzed in diverse beverages. [1] W. Hoffmann, H. Mühlberger, B. Gaš, A. E. Guber, T. van de Goor, K.Witt, A. Gerlach, N. Gottschlich, "Polymer Capillary Electrophoresis Chips - Evaluation and Optimization by Model Aided Contactless Conductivity Detection", ECS Meeting, Honolulu, USA, May 2004.

An isoelectric focusing chip with microstructured superhydrophilic open channels for coupling with MALDI-MS detection
W. Hattori, H. Someya, M. Fujita, T. Sano, H. Kawaura
Proteomics Research Center, Fundamental and Environmental Res. Labs., NEC Corp., Tsukuba, Japan

To enable the automation of a high-speed two-dimensional mapping system for protein analysis, we have now fabricated a sealed chamber and an isoelectric focusing chip with microstructured superhydrophilic open channels. We have been developing the system that is based on the off-line coupling of capillary isoelectric focusing on a chip with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The system we reported at MSB 2005 uses the chip in which the channels were covered with removable resin tape during the isoelectric focusing [1]. With this system, the whole process comprises from seven steps: 1) separate the sample in the channel by isoelectric focusing, 2) freeze the separated sample, 3) remove the tape covering the channels, 4) freeze-dry the frozen sample, 5) apply matrices, 6) perform MALDI-MS analysis along each channel, and 7) map sample proteins two-dimensionally with their isoelectric point (pI) and molecular weight. Since the tape is removed manually, the system cannot be automated. Then, we fabricated a chip with open (uncovered) channels. This 21-mm-square fused-silica chip has four straight 12-mm-long, 400-micron-wide and 10-micron-deep separation channels (Fig. a). Generally, it is difficult to fill a shallow channel across the whole length with liquid solution because it forms droplets on the surface with the finite hydrophilic property. Therefore, we introduced micro-pillars (Fig. b) in the channel and made it superhydrophilic by increasing the surface area. This property prevents the sample solution from forming droplets and makes it spread out evenly in the channel. To prevent the solution in the separation channel from drying up during the isoelectric focusing, we also developed a sealed chamber into which the chip was placed. This chamber controls the chip temperature and the humidity and atmosphere around the chip, thereby enabling proper focusing, freezing, and drying. Using the chip and the chamber, we demonstrated continuous processing between isoelectric focusing and freeze-drying. During the processing, we used a photon counter built in a fluorescent microscope to measure the patterns of separated fluorescent pI markers at three stages: immediately after focusing, freezing, and drying. Four marker peaks with pIs of 5.1, 5.5, 7.2, and 7.6 were observed (Fig. c). Each peak was in the same position at all three stages. This shows that the sample movement during the processing was sufficiently suppressed. In consequence, we successfully automated the continuous processing between isoelectric focusing and freeze-drying, and simplified the operation of the high-speed two-dimensional mapping system. A part of this work was supported by NEDO. [1] L19-L6-W in MSB 2005, M. Fujita, et al., “High-throughput and high-resolution two-dimensional mapping of pI and m/z using microchip and MALDI-TOFMS.”

Magnetic reactors, highly studied tools for proteolytic cleavage in a microchannel.
M. Slováková, N. Minc, J.L. Viovy, A. Le Nel, C. Smadja, Z. Bílková
University of Pardubice, Pardubice, Czech Republic Institute Curie, Paris, France Université de Paris XI, Chatenay Malabry Cedex, France

Peptide mapping of recombinant and therapeutic proteins is one of the most relevant tools for the quality control. It is also a key element for the identification of proteins in proteomics. We present examples of bioanalysis based on the self-organization of superparamagnetic beads under a magnetic field in microfluidic device fabricated in polydimethylsiloxane (PDMS) by soft lithography and rapid prototyping. We use here a magnet arrangement leading to field lines parallel to the flow. This reduces flow resistance and increases digestion efficiency. This arrangement retains the advantages of self assembled magnetic bead arrays: when contaminated, the plug can be easily replaced by fresh beads. Proteolytic enzymes were immobilized by covalent grafting method on magnetic beads. Aspects of immobilization method, grafting efficiency, orientation of the enzyme molecule in microspace of the chip channel and enzyme activities were examined by reaction with synthetic substrates and model proteins. A plug of trypsin-functionalized magnetic particles was immobilized between two magnets in a microchannel for flow-through protein digestion with off-chip analysis. Kinetics studies of the hydrolysis of a model peptide BApNA show a 100 fold increase in digestion speed obtained by the microsystem when compared to a batch system. High performance and reproducible recombinant human growth hormone digestion was confirmed by analysing the digest products in both CE and MALDI-TOF-MS. Similar sequence covering (of about 35%) are obtained from MS analysis of products after 10 minutes on-chip and 4h with soluble trypsin in batch. Proteinase K (PK) was covalently bound to magnetic nanoparticles functionalised with carboxylic group. The PK magnetic beads were self-assembled into the channel of the microchip by a strong magnetic gradient produced by permanent magnets. Activity and reaction kinetics of the immobilized enzyme were determined by monitoring the hydrolysis of succinyl-ala3 -p-nitroaniline as a low-molecular synthetic substrate, and showed a 100-fold increase in digestion speed. Finally, the device was applied to the “online” digestion of cell lysate and brain homogenate of transgenic mice over-expressing prion protein (non-pathogenic form), a protein that exhibit a shift of resistance to PK in its pathological form. This device represents an inexpensive way of fabricating a multi open-tubular-like column with an appropriate pore size for proteins. Since the grafting of proteins is becoming a well-developed field, we emphasize that this device could be used in any kind of chromatographic application. Acknowledgement: This work was supported in part by the Czech Ministry of Education (MSMT 0021627502) and part by Czech Science Foundation GA203/05/0241. The development of microfluidic device was supported by E.C. project “Microproteomics” (QLG2-CT-2001-01903).

Microfluidics-Based Nano-biosensing of PCR Products - Hybridization Induced Fluorescence Quenching
Shu-Hui Chen, Yu-Ting Li
Department of Chemistry National Cheng Kung University

Colloidal gold nanoparticles were used to develop a simple microfluidics-based bioassay that is able to recognize and detect specific DNA sequences via conformational change-induced fluorescence quenching. In this method, a self-assembled monolayer of gold nanoparticles was fabricated on the channel wall of a microfluidic chip and DNA probes were bonded to the monolayer via thiol groups at one end and a fluorophore dye was attached to the other end of the probe. The created construct is spontaneously assembled into a constrained arch-like conformation on the particle surface and under which, the fluorescence of fluorophores is quenched by gold nanoparticles. Hybridization of target DNAs results in a conformational change of the construct and then restores the fluorescence, which serves as a sensing method for the target genes. The nanocomposite constructed on the glass surface was characterized by UV absorbance measurement and the quenching efficieny for different fluorophores was evaluated by Stern-Volmer studies. The applicability of proposed assay was first demonstrated by the use of a pair of synthesized complementary and non-complementary DNA sequences. The method was further applied for the detection the PCR product of danguevirus with the use of enterovirus as the negative control and results indicate that the assay is specific for the target gene. Moreover, using this approach, dehybridization, hybridization, and detection of the target genes can be performed in-situ on the same microfluidic channel. Thus, this method could be regarded as one-pot reaction and it holds great promises for clinical diagnostics.

Protein depletion and desalting with microchip CE.
L.H.H. Silvertand1, E. Machtejevas2, R. Hendriks3, K.K. Unger2, W.P. van Bennekom1, G.J. de Jong1
1 Utrecht University, Utrecht, The Netherlands 2 Johannes Gutenberg Universitat, Mainz, Germany 3 Merck KGaA, Darmstadt, Germany

One of the problems in the separation of complex biological samples with liquid chromatography and capillary electrophoresis, is the presence of large amounts of salts, proteins, peptides, and other biological compounds. With the microchip CE system described, a part of the sample can be injected into a second separation channel after preconcentration or preseparation. Both columns can be run in different capillary electrophoresis modes with this column coupling microchip device (The Merck IonChipTM), for example isotachophoresis-zone electrophoresis (ITP-CZE) or zone electrophoresis-zone electrophoresis (CZE-CZE). Sample preparation, e.g. desalting or protein depletion, is the first step and is followed by an electrophoretic separation step. The sample used is a mixture of five peptides spiked with Cytochrome C and Human Serum Albumin. A 1 µL sample volume is injected onto the first capillary, which can be used for preconcentration (ITP mode) and/or separation (CZE mode). When the sample reaches a first conductivity detector (CD1), a time-lag can be programmed, which directs part of the sample into the second separation channel, via a T-split implemented on the chip. The rest of the sample is injected into a waste channel. When the depletion or selection of certain compounds is completed, the compounds are separated in the second separation channel and detected with a second conductivity detector. Results of desalting a mixture of proteins with ITP-CZE and depletion of proteins from a mixture of peptides and proteins, using CZE-CZE are presented. Furthermore we attempted to couple the IonChipTM to an ESI-iontrap mass spectrometer in two different ways. The direct coupling was unsuccessful due to software problems. In the second coupling method, the compounds are first trapped on a C18 monolithic column and also separated by a C18 monolithic separation column and subsequently directed to the MS. First results of transferring a peptide successfully from the IonChipTM to the MS are shown. Depletion and subsequent mobilization of the compounds to the MS has not yet been successful.

EOF-driven Hydrodynamic Filtration for Continuous Particle Sorting in Microfluidic Devices
T. Kawamata1, M. Yamada2, M. Yasuda1, M. Seki1
1 Department of Chemical Engineering, Graduate School of Engineering, Osaka Prefecture University, Japan 2 Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Japan

A method for continuous particle separation and concentration using electroosmotic flow (EOF) has been developed. In our previous study, we have proposed a method for continuous particle concentration and separation in microfluidic devices utilizing a laminar flow profile, named hydrodynamic filtration [1]. In this study, we have tried to apply EOF to the presented hydrodynamic filtration. By using EOF, the flow rates distributed into branch channels can be accurately and easily tuned. Therefore, it is expected that the separation efficiency can be improved. Microdevices were fabricated using usual rapid prototyping and replica molding methods, and were made of PDMS and glass substrate. These microdevices have two inlets and three outlets. The microchannel was designed regarding the channel structure as an electric circuit. The main channel width was 15 micrometer, and the channel depth was 5 micrometer. As model particles, fluorescent polystyrene beads with diameters of 2.1 or 3.0 micrometer were used. These particles were suspended in 15 mM ammonium hydrogencarbonate aqueous solution.
 After introducing solutions with and without particles into the microchannel, electrodes were inserted into inlets and outlets, and then electroosmotic flow was generated by applying voltage to inlets and outlets. As a result, 2.1 and 3.0 micrometer particles were precisely separated and concentrated. This method is advantageous in the sense that particles can be sorted continuously without using any kinds of syringe pumps. [Reference] [1] “Hydrodynamic Filtration for On-chip Particle Concentration and Classification Utilizing Microfluidics,” M. Yamada, and M. Seki, Lab chip, 5, 1233-1239 (2005)

Dynamic surface modification of PMMA microchip and its applicability to the analysis of biogenic amines and amino acids
N. Naruishi, Y. Tanaka, T. Higashi, S. Wakida
Human Stress Signal Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Osaka, Japan

Generation and control of electroosmotic flow (EOF) are critical for resolution, speed, and reproducibility in microchip CE separations. Dynamic coating is a simple and rapid surface modification technique that has been used widely in manipulation of EOF and suppression of analyte adsorption. For this purpose, surface-active compounds like polymers or surfactants are added to the run buffer or are applied within a preconditioning step prior to analysis. One of the most popular materials for preparation of microchips is poly(methyl methacrylate) (PMMA), nevertheless well-established techniques from classical CE could be only applied for the surface modification of glass substrates. In this work we have investigated some novel coating agents, blocking agents for ELISA such as BlockAce from Dainippon Sumitomo Pharma and ULTRABLOCK from Serotec, for the surface modification of a PMMA microchip. The electroosmotic mobility was determined by measuring a current monitoring using two different concentrations of HEPES-Na buffer. For an untreated PMMA microchannel, the electroosmotic mobility was 1.2×10-4 cm2/V
sec in the buffer at pH 7.4. It shows a lower magnitude of EOF than glass or fused silica at an equivalent pH. Dynamic coating with BlockAce increased the electroosmotic mobility from 1.2×10-4 to 3.1×10- cm2/Vsec. Even if the channels were flushed with the buffer prior to each EOF measurement, the highly stable coating was maintained during five successive measurements at least. The increase in EOF can be useful in significantly reducing analysis times. On the contrary, dynamic coating with ULTRABLOCK reduced the electroosmotic mobility from 1.2×10-4 to 6.4×10-5 cm2/Vsec, whereby the technique is useful for the analysis of negatively charged compounds. We have applied these dynamic coating techniques to the analysis of biogenic amines and amino acids labeled with 4-fluoro-7-nitrobenzofurazan (NBD-F). The NBD-labeled amines and basic amino acids could be analyzed using a BlockAce coating-chip. Negative charged analytes, such as NBD-labeled acidic and neutral amino acids, could be successfully analyzed towards the cathode under the reduced EOF mode using a ULTRABLOCK coating-chip. These coating techniques have expanded the applicability of microchip CE.

DNA separation via dielectrophoresis on a microfluidic chip
J. Regtmeier, T.T. Duong, D. Anselmetti, A. Ros
Experimental Biophysics & Applied Nanoscience, Bielefeld University, Germany

DNA separation by length is a very important analysis tool in molecular biology, medicine and biochemistry. However, the separation of long DNA molecules remains a major challenge. Here, we present a microfluidic chip for the separation of long DNA molecules without the need for gels or sieving matrices. The design consists of a cross injector and a separation channel, structured with posts. These constrictions focus the electric field and create an array of locations, which act as dielectrophoretic traps for DNA molecules, upon application of an AC electric field. DNA is trapped or retarded in the post array via a careful combination of AC- and DC-voltages. The detection of fluorescently labeled DNA molecules was either achieved by recording an electropherogram at a specific detection position in the microchannel or by scanning the post array. Preliminary recorded electropherograms demonstrated the length dependent DNA migration behavior. Furthermore, adjusting AC-conditions to complete trapping of DNA molecules in the post array, revealed that fragments of varying length (larger than 7 kbp), can be trapped at different positions along the channel, when analyzed by fluorescence scanning. By this method, the complete analysis time could be reduced to 400sec in a 4 mm long channel. Currently, we are extending these studies to advanced geometries and AC conditions in order to enhance the separation efficiency and to determine the applicable size range for dielectrophoretic genomic DNA separation and purification.

Capillary-Based Microfluidic Instrument for Rapid Clinical Assessment
M.C. Peoples1, T.M. Phillips2, H.T. Karnes1
1Department of Pharmaceutics, VCU School of Pharmacy and 2Ultramicro Analytical Immunochemistry Resource, NIH Bethesda, MD

A prototype microfluidic system is presented for rapid assessment of clinical samples. Immunoaffinity chromatography is proposed as a means for separating analytes of interest from biological samples. The instrument is capable of injecting sub-microliter samples and detecting labeled antigen by laser-induced fluorescence. The laboratory-constructed device is assembled from an array of components including two syringe pumps, a nano-mixer, a micro-injector, and a separation capillary packed with antibody coated glass beads. Two prototype columns currently in use are made from PEEKSIL with a 200 µm inner diameter and lengths of either 25 or 50 mm. For the immunoaffinity matrix, antibody would be attached to glass beads via a streptavidin-biotin linkage. Thus, initial columns have been packed with plain silica beads to test the system. Optimization of the device has been achieved by measuring flow accuracy with respect to column length and particle size. Syringe size and pressure effects have also been used to characterize the capability of the pumps. Inaccuracy of flow rate has been linked to syringe failure due to exceeded pressure ratings. Reducing the syringe size increases the pressure tolerance of both the pumps and syringes. Additionally, pressure is lowered by reducing column length or increasing particle size. An in-house program written with LabVIEW software controls the syringe pumps to perform gradient elution and collects the LIF signal as a chromatogram. A twenty-step input allows for timed changes of flow rate for each of the two pumps. The software program is triggered to collect the detector signal at a particular time for a desired duration. Chromatographic peaks are selected and outputs of height, area, and signal to noise are generated.

Comparison of analysis parameters affecting on on-line performed ITP-ZE separation on PMMA microchips and silica capillaries
Heli Sirén, Nina Virkkala, Elina Välinen, Ari Hokkanen, Ingmar Stuns

In biological fluids drugs and metabolites have very low concentration levels. Isotachophoresis (ITP) which is made in a capillary or a microchip channel is well suited for these kinds of applications due to its high separation efficiency, large sample loading capacity and ease of interfacing other techniques, e.g. zone electrophoresis (ZE). In the past, microchips which are made of polymethylmethacrylate (PMMA) polymer have been used for both separation and determination of inorganic ions and drugs and metabolites successfully. Determinations were made with combining ITP and ZE, where the ITP step was used to concentrate the analyte zones. On-line monitoring was made by the conductivity detector placed in the concentration channel and the LIF detector (
lex 488 nm, lem 520 nm) in the separation channel. In this study, PMMA chips were used for separation of amines. The method, used, was a combination of ITP and ZE. The identification was made by a 2D conductivity detector, which was self-constructed. The method development was done in a silica capillary (50 mm i.d.) by using the capillary electrophoresis (CE) with UV detection. The electrolyte solution pair was either 10 mM MES/Na (leading electrolyte, LE) or 5 mM glutamic acid (terminating electrolyte, TE) or 10 mM MES/Na (LE) and 5 mM acetic acid (TE). The method was transferred into a microchip channel, but it was further optimized due to the structural differences concerning the patterns on the chip. The detection of 90 ng of three analytes was obtained with the chip method. However, in CE the LOD values of the analytes were at pg-level. The parameters influencing on the separation were the lengths of the ITP and sample zones.

Continued development of on-Chip Gel Electrophoresis - New Series II Agilent 2100 Bioanalyzer Kits
Marc Valer, Susanne Glueck, Meike Kuschel, Martin Greiner, Tobias Preckel
Agilent Technologies

In the past fifteen years there have been significant advances in the automation of gel electrophoresis. First, the introduction of capillary electrophoresis instruments allowed researchers to move from slab gels to a more reproducible although slightly more finicky technology. Then, several years ago the advent of microfluidics promised to revolutionize the field of biomolecule analysis. Agilent Technologies introduced the first commercially available Lab-on-a-Chip system in 1999. In the meantime, the application range has been extended to automated analysis of RNA, DNA, cells and protein samples and the product family comprises 10 different chip kits. Agilent Technologies has now upgraded all of the kits and assays to improve sensitivity, robustness and precision of the measurements. In particular, in the field of protein analysis Lab-on-a-Chip technology provides significant advantages over traditional slab gel analysis. In comparison to its predecessor it provides significant improvements in sizing range and accuracy and quantitation accuracy and reproducibility though a more stable and pure internal standard. Here we present experimental data obtained with the new 2100 bioanalyzer protein kit and detailed information on how the size calibration of the assay has been done in order to align sizing information to that of traditional SDS-PAGE.

Microchip-based liquid-liquid extraction for gas-chromatography analysis of amphetamine-type stimulants in urine
Hajime Miyaguchib12, Manabu Tokeshi2, Yoshikuni Kikutani2, Akihide Hibara23, Hiroyuki Inoue1, Takehiko Kitamori23
1National Research Institute of Police Science, Kashiwa, Chiba, Japan 2Micro Chemistry Group, Kanagawa Academy of Science and Technology, Kawasaki, Kanagawa, Japan 3Department of Applied Chemistry, The University of Tokyo, Tokyo, Japan

Methamphetamine (MA) abuse accounts for the most of drug offenses in Japan. In consequence, urine tests for MA have been routinely performed at any local police laboratory, and the results are considered the most reliable evidences of MA abuse. For the rapid, laborsaving and small-scale sample preparation of urinary drug analysis, we have investigated microchip-based liquid-liquid (LL) extraction for gas chromatography (GC) analysis of amphetamine-type stimulants in urine. A glass microchip which has the shallow and deep microchannels having separate inlet and outlet holes and asymmetric LL contact region was fabricated by two-step photolithographic wet-etching process and thermal bonding method (Fig.1). The capillarity restricted modification (CARM) method [Anal. Chem., 77, 943 (2005)] was employed for patterning the surface of microchannels with perfluorooctylsilane in order to stabilize organic/aqueous interface. Using syringe pumps, an organic solvent and a diluted urine sample containing aqueous ammonia and EDTA were simultaneously introduced into the modified and bare microchannels, respectively. LL extraction was carried out through the organic/aqueous interface in the LL contact region. The organic phase was separated at the branch of the microchannels, and the organic phase was pooled into a glass vial. The extract was injected into a capillary GC equipped with a flame ionization detector. When using a water-insoluble solvent for extraction, no drying process is required before injection into GC. Then, n-hexane and 1-chlorobutane (BuCl) that have lower water-solubility (<=0.5 g water/L) were examined for the extraction solvents instead of common extraction solvents for urinary drug analysis such as chloroform and ethyl acetate. In order to realize effective LL extraction and phase separation on the microchip, it is necessary to maintain a long organic/aqueous interface even at low flow rates. The organic/aqueous interface inside microchannels can be stabilized by using the patterned hydrophobic modification by the CARM method. The controlled interface at the branch of the microchannels automatically contributed complete phase-separation, especially using BuCl as the extraction solvent. The main reason for the stability may be attributed to the wettability between the fluoroalkylated surface and the solvent. When comparing BuCl with hexane, BuCl showed not only the better stability of the interface but also the sufficient extraction recoveries of amphetamines the stimulants. The recoveries of MA and MPN (methoxyphenamine
reached to ca. 90%, and that of MA reached to ca. 80% when employing at the lower flow rate of BuCl (BuCl, 5 ul/min; the aqueous phase, 25 ul/min). Under this condition, it takes about six minutes to prepare a GC sample. The validation and applicability studies using the fortified and real urines obtained from the three subjects who were administered MPN are now under investigation.

Engineering of polymer based HPLC-Chip devices and instrumentation
Karsten Kraiczek, Hans-Georg Weissgerber, Patrick Kaltenbach, Georges Gauthier
Agilent Technologies R&D and Marketing GmbH & Co.KG, Germany

In recent years, nano LC-MS has become established as the state-of-the-art analytical platform for high sensitivity identification of proteins and peptides from proteomics samples. Although this technology provides very high sensitivity it is not considered an easy-to-use and robust analytical technology. Fragile nano-spray needles have to be properly assembled with the nano-LC column in order to get the maximum efficiency and lot of connectors are being used to hydraulically connect the individual components. A polymer based µ-fluidic HPLC-Chip platform integrates, for example, sample preparation, a separation column, complex hydraulic connections and an electrospray device and provides much greater ease of use, reliability and faster set up time relative to current column nano-spray based protein ID solutions. The fabrication method of HPLC-Chip devices is fairly simple and basically suited for both rapid prototyping and large scale production. UV laser ablation in combination with vacuum lamination of polyimide films is used to create multilayer polymer based µ-fluidic devices with excellent chemical resistance to solvents and bio-friendly environment for proteins and peptides. Open micro channels are packed with reversed phase and/or ion exchange materials to create high performance HPLC columns. Non-straight analytical columns ranging from 2cm up to 50cm length have been realized in a chip format smaller than a credit card. Patterned noble metals are applied by thin film deposition on the polymer film surfaces to create electrical contacts for electrospray biasing. A chip handler, a major part of the HPLC-Chip/MS instrumentation, automatically loads and positions the chip tip to the MS inlet. The chip is identified by an integrated RF-Tag and all hydraulic connections are made by the system according to chip and application type. A high pressure, ultra low dead volume concentric rotary valve is being used for highly integrated applications.

Development of New Non-aqueous Capillary Electrophoresis Method with chromatographic mechanism for separation of non-charged substances
B. Lapin1, O. Mikhailova2
1 InterLab Inc.,Moscow, Russia 2 “OAO Biomash”, Moscow, Russia

During last year there had been published a large number of results obtained by non-aqueous capillary electrophoresis. Most of scientific papers describe work with very small electric current levels. There is small amount of indications of micelles occurrence in pure organic media. We attempt to find such organic phase composition that is capable of providing best results with a relative large electrical current. It was shown by us that using of CTAB as a current supporting agent can help meet our application needs. We use the short capillary with effective length of 85 mm. Such length selection enable us possibility to perform tens of analysis without needs to replace the run buffer (with no obvious depletion of charge carrier). In attempting to improve peak shapes we have chosen to use small amount of non-ionogenic surfactant addition (in our case it was Brij 35). This approach has provided us reversing of osmotic flow direction and a very significant improving of peaks shaping. Finally, the selected organic phase comprised about 70 - 80% acetonitrile, 10% methanol; 10 to 20% Brij-35; 0.082 mM CTAB. We have not found any signs of micelles occurrence, but the observed elution order was reminding us that being very typical for micellar electrokinetic capillary chromatography. Close observation of the electropherograms has revealed some peaks belonging to CTAB or Brij-35 displacements. Therefore, we are able to name this report as having relation to chromatographic separation mechanism. We do not use any pseudostationary phases. Moreover, we have found the developed organic phase as being rather universal and can be used for indirect substances detection as well. We will show results for determination of following substances: antitubercular drugs (rimfampicin, pyrazinamide, isoniazid and ethambutol), fat-soluble vitamins, water, and water contaminants (such as amines, ethylene glycol and some cations).

Chemiluminescence Detection in Capillary Electrophoresis (CE-CL) for the Determination of Phenothiazines in Pharmaceuticals and Biological Fluids
F.J. Lara, A.M. García-Campańa, J.M. Bosque-Sendra, F. Alés-Barrero
Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Spain

In this communication, a home-made CE-CL device is used for the determination of two phenothiazines: promazine (PMH) and promethazine (PTH). Different CL reactions were tested, selecting the enhancement produced by the analytes on the CL emission of luminol with potassium ferricyanide as analytical detection. Electrophoretic separation was carried out using a buffer of 20 mM tetraborate at pH 8.5 with 1.25 mM luminol and the CL reagent was a solution 0.27 mM K3[Fe(CN)6] with 0.01 N NaOH. Traditionally, the optimization of the CL reagent concentration is mainly carried out following a step-by-step approach requiring a large number of experiments and without taking into account possible interactions between the different factors or quadratic effects. We propose for the first time, the use of experimental design in CE-CL for optimization purposes by means of Doehlert designs with three central points. Signal to noise ratio was selected as the most suitable response and it was demonstrated that no similar conclusions were obtained when peak height is used. Injection by gravity was applied in pharmaceutical formulation achieving LOD of 80 ng/ml for PMH and 335 ng/ml for PTH. Electrokinetic injection was used for human urine after a solid phase extraction method giving a LOD for PMH of 1 ng/ml (PTH used as internal standard). The proposed method was validated in terms of accuracy, linearity, repeatability, intermediate precision and robustness. Relative standard deviations lower than 12% were achieved in all cases. Finally, it was applied to the determination of PTH in pharmaceuticals formulations and to the determination of PMH in human urine with recoveries higher than 85 %. Acknowledgements The authors thank the Fondo de Investigación Sanitaria (Instituto Carlos III, Ministry of Health) (Project PI021369) and EU Funds (FEDER) for financial support. Francisco J. Lara thanks the Junta de Andalucía for a FPI grant.

High-Performance Two-Dimensional Gel Electrophoresis Using a Low-Concentration Isoelectric Focusing Gel Supported by a String
T. Odake, T. Saheki, H. Hotta, K. Tsunoda
Gunma University, Kiryu, Japan

Two-dimensional gel electrophoresis (2-DE) is a powerful separation tool in proteome analysis, because proteins can be separated according to their isoelectric point and size by isoelectric focusing (IEF) in the first dimensional electrophoresis and by SDS-PAGE in the second dimensional electrophoresis at a time. However, the conventional 2-DE using the self-made (agarose) IEF gel has some limitations; total analysis time is as long as almost one week, handling the fragile IEF (tube) gel is difficult, and reproducibility is not sufficient due to fluctuation of pH gradient during IEF. The immobilized pH gradient strip gel (typically 4%T) commercially available as the IEF gel can solve the handling problem and provide stable pH gradient to some degree, but proteins of high-molecular-weight above 100 kDa are difficult to be analyzed because of the relatively small pore. We proposed as a new IEF gel a low concentration polyacrylamide gel supported by a string. By using a multifilament string as an IEF gel support, the total acrylamide concentration of the gel could be reduced to 2%T with sufficient mechanical strength, and the gel was easily handled by picking up with tweezers. The low-concentration gel was expected to have large pore, providing fast IEF and effective protein transport. By using the 2%T polyacrylamide gel, proteins were focused at least twice faster and were observed more clearly, especially in the high-molecular-weight region, on the silver-stained 2D map than by using the 4%T gel, and high-molecular-weight proteins were observed no less clearly on the 2D map than by using the agarose IEF gel. One or two days was enough to carry out all analytical operations of 2-DE. It could be concluded that this system using the string-supported low-concentration IEF gel could be expected as a fast and facilitated 2-DE system applicable to high-molecular-weight proteins.

Mass assembly technology for large arrays of capillaries
C.R. Forest, D. Buckley, I. Collier, I.W. Hunter
Massachusetts Institute of Technology

Manual assembly of capillary arrays comprising hundreds of capillaries for electrophoresis instrumentation is laborious and costly, and becomes impractical for ultra-high throughput arrays of thousands of capillaries. A semi-automated capillary array assembling machine has been created to facilitate this task, reducing assembly time by two-thirds as compared to manual assembly. In this device, a linear stage is automatically indexed to successive positions in a two-dimensional array. At each position, a cartridge containing 10-30 capillaries is repeatably attached to the stage using kinematic couplings. The key feature of this design is the steel cartridge that contains a row of evenly spaced trapezoidal troughs accurately fabricated by microelectrode discharge machining (microEDM) to
<5 µm process variation. Each trough non-destructively pinches a capillary upon application of a preload force, thus constraining it for insertion into the array. The cartridge is easily filled with capillaries by rolling a bundle of them across the troughs. This array assembly technology eliminates time-consuming handling and meets assembly alignment tolerances, enabling array manufacture with dramatically reduced time and cost.>

Capillary zone electrophoresis method for the determination of inorganic anions in honey
Silvia Suárez-Luque, Inés Mato, José F. Huidobro, Jesús Simal-Lozano
Facultad de Farmacia. Departamento de Química Analítica, Nutrición y Bromatología. Área de Nutrición y Bromatología. Universidad de Santiago. 15782 SANTIAGO DE COMPOSTELA (Galicia). SPAIN

A capillary zone electrophoresis method for the determination of inorganic anions in honey samples was developed for the first time. The mineral content of honey samples could give an indication of environmental pollution and herewith also an indication of the geographical origin of honey [1]. The complete separation of chloride, nitrate, sulphate and phosphate can be achieved in 12 min with a simple electrolyte composed by 2 mM potassium dichromate as the carrier buffer and background absorbance provider and 0.05 mM tetraethylenepentamine (TEPA) as flow modifier (pH 4.00). Injection was performed hydrostatically by elevating the sample at 10 cm for 10 s. The running voltage was +27 kV at 25 şC. Indirect UV-absorption detection was achieved at 254 nm. The detection limit was in the range between 0.06 and 1.02 mg/l and the quantification limits ranged from 1.12 to 2.66 mg/l. The calibration graphs were linear in the concentration range from the quantification limit till at least 250 mg/l for chloride, 25 mg/l for nitrate, 75 mg/l for sulphate and 50 mg/l for phosphate. The repeatability, intraday and interday analysis were <= 1.00 % and <= 1.35 % for migration time and <= 1.54 % and <= 3.26 % for peak area. The developed method has been applied to honey samples with only a simple dilution and filtration treatment of the sample. This method is precise and accurate. Results obtained by this method are similar to those obtained by other authors with reference methods. [1] E. Anklam, Food Chem. 4 (1998) 549.

Capillary zone electrophoretic analysis of metal cations in honey samples and their relationships with colour
Inés Mato, Silvia Suárez-Luque, José F. Huidobro, Jesús Simal-Lozano
Facultad de Farmacia. Departamento de Química Analítica, Nutrición y Bromatología. Área de Nutrición y Bromatología. Universidad de Santiago. 15782 SANTIAGO DE COMPOSTELA (Galicia). SPAIN

A capillary electrophoresis system for the simultaneous determination of cations in honey samples has been developed for the first time [1]. The complete separation and quantification of K+, Ca2+, Na+, Mg2+, Mn2+, Ni2+ and Li+, which represent more than 99% of the total content of cations in honey, can be achieved in 4 minutes with only dilution and filtration of the honey sample. Electrolyte solution was composed by 10 mM imidazole as the carrier buffer and background absorbance provider and 1 M acetic acid as the complexing agent (pH 3.60). Injection was performed hydrostatically by elevating the sample at 10 cm for 30 s. The running voltage was +25 kV at 25 şC. Indirect UV-absorption detection was achieved at 185 nm. Under the optimum conditions the detection limits ranged from 0.02 to 48.2 mg/kg and the quantification limits have ranged from 0.41 to 48.7 mg/kg. Precision data in honey samples analysed have shown repeatability and reproducibility RSD (%) lower than 2.84% and 6.62%, respectively. Recoveries of cations in honey samples analysed have ranged from 88.5 to 101.8%. Twenty-five honey samples were classified according to their colour determined by the method of Brice et al. [2] and they were analysed by the proposed method. Results showed a great variability in the quantitative composition of cations in honeys, which could have any relation with the colour of the honeys because the content of K+, Na+, Mg2+ and Li+ cations were greater in dark honeys and Ni2+ was only detected in dark samples. [1] S. Suárez-Luque, I. Mato, J.F. Huidobro, J. Simal-Lozano, J. Chromatogr. A, 1083 (2005) 193. [2] B.A. Brice, A. Turner, J.W. White Jr., J. Assoc. Agric. Chem. 39(4) (1956) 919.

A Nanoscale HPLC System Capable of 10,000 psi Operation for High Efficiency Separations Utilizing Sub 2 Micron Particles
J.W. Finch, H. Liu, M.D. Stapels, J.P. Murphy, K. Fadgen, G. Gerhardt, S. Ciavarini, D. DellaRovere, C.C. Benevides, A. Wallace, J.C. Gebler
Waters Corporation, Milford, Massachusetts, United States of America

Increased MS sensitivity achieved using nanoscale capillary chromatography offers significant advantages for analysis of complex proteomic samples, where variability in dynamic range exists. Significant improvements in LC/MS separation efficiency can be realized by packing nanocolumns (i.d.'s of 75 µm or less) with bridged-ethyl hybrid (BEH) stationary phase particles < 2 microns at longer lengths (up to 30 cm). Due to the greater density of particles per length, these columns generate significantly higher backpressures than columns packed with conventional 3 to 5 micron particles. There are significant challenges in designing a nanoscale LC system which is robust and can operate routinely at pressures in excess of 10,000 psi. Furthermore, highly reproducible chromatography and MS response is desired for quantitative proteomics. A direct-flow high pressure gradient mixing system is most suitable for meeting these requirements. Here we describe and present results for a direct-flow nanoscale UPLC (Ultra Performance Liquid Chromatography) system. The system is uniquely optimized to deliver highly reproducible gradients while operating at 10,000 psi or higher. All consumables and fittings are designed to preserve chromatographic performance while providing ease of use and robustness. Data will be presented demonstrating that peak capacities in excess of 700 can be achieved with a 75 µm x 30 cm BEH column for a gradient separation over 540 minutes. In addition, the nanoscale UPLC capabilities are combined with an on-line 2D method, where the first dimension utilizes a capillary-scale strong cation exchange (SCX) column (salt steps injected from the autosampler), with UPLC reversed-phase (trap and analytical nanocolumn) as the 2nd dimension.

Integration of IEF and SDS-PAGE into a Miniaturized Device for Two Dimensional (2-DE) Electrophoretic Separation of Proteins
Z. Demianova1, M. Shimmo1, E. Pöysä1, S. Franssila2, M. Baumann1
1 Protein Chemistry Unit, Institute of Biomedicine, PO BOX 63, University of Helsinki, 00014 Finland 2 Microelectronics centre, PO BOX 3500, Helsinki University of Technology, 02015 Finland

Two-dimensional gel electrophoresis (2-DE) is a biochemical method, which separates proteins by two independent parameters according to their net charge and mass. Although it is one of the most powerful protein isolation methods, it is currently mostly used in proteomic research. If faster and automated, it would probably be accepted by far more users than now and even applied for diagnostic purposes. In the present work we described a miniaturized form of such a 2-DE instrument. Our 2-DE miniaturized device is based on the classical 2-DE technology including polyacrylamide (PAA) matrixes. The device consists of a small IEF unit (IEF gel size: 2 x 24 x 1mm; (width x length x depth)) and a SDS-PAGE unit (SDS-PAGE gel size: 25 x 37 x 0, 5 mm) both unified to be run in one single analysis. IEF was carried out using a pH gradient from 3 to 10 made by carrier ampholytes in a 4% PAA gel. The second dimension gel was usually a 15% Laemmli gel. The performance of the instrument was tested by a set of naturally colored standard proteins. With approximately 1-2 micrograms of the total protein amount, focusing was completed in ~50 minutes. IEF focused proteins were then electophoretically transferred to the SDS-PAGE and were separated according their molecular weights in ~20 minutes (2-DE pattern is shown in the figure). To mimic an authentic proteome study, we excised protein spots from Coomassie/silver stained 2-DE gels, digested them with trypsin and analyzed the released peptides by MALDI-TOF-MS. Our further experiments show that the device is also able to focus and separate samples obtained from cytoplasmatic extract of mammalian cells after sample pretreatment.

Determination of the Herbicide Metribuzin and its Conversion Products in Soils by Micellar Electrokinetic Chromatography
J. F. Huertas-Pérez1, M. del Olmo Iruela1, A. M. García-Campańa1, A. González-Casado1, A. Sánchez-Navarro2
1 Departament of Analytical Chemistry, Faculty of Sciences, University of Granada, Spain. 2 Departament of Agriculture Chemistry, Geology and Edafology, Faculty of Chemistry, University of Murcia, Spain

Metribuzin (M) is a selective systemic herbicide used for pre- and post- emergence control of many grasses and broad-leaved weeds in soya beans, potatoes, tomatoes, sugar cane, alfalfa, asparagus, maize and cereals at 0.07-1.05 kg a.i./ha. Metribuzin belongs to the group of triazinone herbicides, it is highly water-soluble (1.05 g/L) and adsorption in low-organic sandy soils is rather weak, sorption coefficients vary from 0.56 in a very sandy loam to 31.7 in a soil containing 60% organic matter. The decomposition of metribuzin in the environment is due to microbiological and chemical processes. Only deaminometribuzin (DA), diketometribuzin (DK), deaminodiketometribuzin (DADK) and two glycoside conjugates are known as metabolites. We propose a micellar electrokinetic chromatography (MEKC) method for the determination of M and its three main conversion products with UV-diode array detection, being an alternative to HPLC method due to its low cost, short separation times, high efficiency and no need of high volumes of organic solvents. The applicability of off-column SPE procedure for the enrichment of the analytes prior to MECK analysis, using LiChrolut EN cartridges, has been carried out to achieve satisfactory detection limits and efficient clean-up. A deep study of the different steps involved in the proposed method was carried out in order to obtain the best conditions for both treatment of sample and CE separation. Under the optimal conditions the method presents a detection limit of 21.25 ng mL-1 for M, 33.87 ng mL-1 for DA, 27.91 ng mL-1 for DK and 26.97 ng mL-1 for DADK and a linear range of 50 to 500 ng mL-1. The method has been successfully applied to the determination of M and its by-product in soil samples. The National Institute of Agricultural and Food Research and Technology (INIA, Ministerio de Agricultura, Pesca y Alimentación, Project ref. CAL03-087-C2-1) supported this work.

Analysis of Amino Acids in Human Plasma by Capillary Electrophoresis with Miniaturized Laser-induced Fluorescence Detection Cell
V. Kostal, M. Zeisbergerova, Z. Hrotekova, V. Kahle, K. Slais
Dept. of Analytical Chemistry,Palacky University, Olomouc, Czech Republic Dept. of Biochemistry, Masaryk University, Brno, Czech Republic. Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, Brno, Czech Republic.

Amino acids (AA) play an important role in the metabolic processes of a living organism. The determination of physiological amino acid levels is of importance to the clinical diagnosis and treatment of hereditary diseases, kidney diseases or metabolic disorders. Capillary zone electrophoresis (CZE) is widely used to study amino acids. This technique has several advantages, such as quick analysis, high efficiency and low sample consumption. Since small sample amounts (often in trace concentrations) are injected into the capillary, a high-sensitivity detection system is required. Nowadays, the laser-induced fluorescence (LIF) detection is well-established detection technique with the lowest detection limits. LIF is suitable for detection in small volumes, so it is widely used for capillary separation methods and microchip-based separations. Here we present the miniaturized LIF detection cell for capillary separation methods based on utilization of liquid core waveguide capillary (LCW) [1,2]. LCW capillary is a fused silica capillary coated with special fluoropolymer (Teflon AF) with index of refraction lower than that of silica and even water. This fact gives the capillary ability to guide the light. Analytes from the CE capillary are transversely illuminated in the LCW cell by the optical fiber-coupled Ar+ laser. Emitted fluorescence is axially imaged at the LCW end via optical fiber joined to a compact CCD array spectrometer. Suggested LIF configuration has simple arrangement and easy optical alignment. The spectral resolution of CCD spectrometer allows omit filters and other optical elements. The whole device also offers a high degree of miniaturization. The detection system was used in capillary electrophoresis of derivatized amino acids. Amino acids in blood plasma were measured to compare the amino acid levels of healthy and pathological patients. The fluoresceinisothiocyanate (FITC) was used for derivatization of standard AA mixtures and plasma samples. For analysis of cysteine, homocysteine and gluthathione the 5-iodoacetamidofluorescein (5-IAF) was used. This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic, grant No. 3417/2005 and the Academy of Sciences of the Czech Republic, grant No. S4031201. [1] Wang S. H., Huang X. J., Fang Z. L., Dasgupta P. K., Anal. Chem 73 (2001), 4545-4549. [2] Kostal V., Zeisbergerova M., Slais K, Kahle V., J. Chromatogr. A 1081 (2005) 1081, 36-41.

Development of Affinity Analytical Magnetapheresis for Particle and Biochemical Analysis
C. Bor Fuh1, H.Y. Tsai2, F. H. Hsu1
1 Department of Applied Chemistry,National Chi Nan University 2 School of Applied Chemistry, Chung Shan Medical University

Affinity analytical magnetapheresis is based on the affinity interactions between particle surfaces in analytical magnetapheresis. In affinity analytical magnetapheresis, magnetic particles with antibody (Ab) [or antigen (Ag)] on the surface are flowed through a separation channel to form a zonal deposition matrix on the channel bottom plate under magnetic fields. Nonmagnetic particle samples with specific Ag (or Ab) on the surface are selectively captured to the predeposited zone and separated from those particles without specific Ag (or Ab) on the surface due to specific Ag-Ab affinity interactions as they flow through the channel. The deposited Ag-Ab complex can be used for examination and further analysis under the microscope. The predeposited matrix can be formed selectively and changed relatively easily as needed. Several examples of affinity analytical magnetapheresis were studied. The selectivities of affinity analytical magnetapheresis were 94 % higher than those of label-controlled samples. The running time was generally less than 10 min. The detection limits were around 10
-sup(-11) g. This method extends the applications of analytical magnetapheresis to nonmagnetic particles. It has a great potential to provide a simple, fast, and selective analysis for applications of particles, blood cells, and immunoassays.

Laser Microdissection and Non-Contact Extraction of Cellular Material for Microscale Alzheimer Research
Bernd Sägmüller, Christian Sauber, Manuela Neumann, Hans A. Kretzschmar
Carl Zeiss / PALM Microlaser GmbH, Am Neuland 9+12, 82347 Bernried, Germany (Saegmueller) Agilent Technologies AG, Waldbronn (Sauber) Institut of Neuropathology, Ludwig Maximilians University, Munich, Germany (Neumann, Kretzschmar)

The molecular mechanisms involved in most neurodegenerative disorders, such as Parkinson’s and Alzheimer’s disease, are still unclear. So far, protein expression analysis is often performed on homogenized preparations of whole tissues which do not provide any information about relevant changes in specific cell types. A beneficial method to microscale bio-separation in extraction of material down to the volume of a single cell is non-contact laser microdissection and transport of the selected cell(s) into processing means. This allows delivering the biomaterial directly into the processes of CE, ICP/MS and HPLC/MS identification means or intermediate preparation steps. The aim of the following study was to examine whether laser-microdissected samples of single cell types from post-mortem brain tissue can be used for protein expression analysis. We illustrate this method by investigation of HE stained frozen sections (15 µm) from human post-mortem brain tissue and collected cell material selectively by laser-microdissection and pressure catapulting (LMPC) as combined in the AutoLPC-function of a MicroBeam instrument (Carl Zeiss / PALM Microlaser GmbH). Different amounts of material have been collected such, that the cells are readily fragmented and homogenized while being catapulted out of the brain section into the urea denaturation buffer (6M urea, 100mM Tris/HCl), which was placed in the center of a cap for sealed reaction tubes exactly on top of the desired areas for LMPC-cell-harvest. The tryptic digested protein mixture was subsequently analyzed with a nano-LC/MSMS ion trap system (Agilent Technologies). Database searching was done with SpectrumMill MS Proteomics Software. Applying this non-contact investigation method we were able to identify the glial fibrillary acidic protein by 25 distinct peptides, the myelin basic protein by 6 distinct peptides and a mutant beta-actin by 7 distinct peptides.

HPLC-CHIP/MS as an Analytical Tool for Injecting Sample Amounts in the Low Nanoliter Range
Martin Vollmer, Anabel Fandino, Jan Eickhoff, Bernd Glatz
Agilent Technologies, R & D and Marketing GmbH u. CoKG

Nano HPLC/MS and HPLC-Chip/MS have been proven to be a valuable tool for the analysis of highly complex proteome samples since separations performed on low volume columns with flow rates in the nanoliter per minute range result in excellent sensitivity in combination with nano electrospray. HPLC-Chip/MS adds a high degree of flexibility, ease of use and robustness to the technology. Since column volumes in Nano and HPLC-Chip technology are very small, usual injection volumes between 100 nl and a few microliters are far too large to obtain acceptable peak shapes or resolution. Therefore, an enrichment column is used to concentrate the analyte prior to transferal and separation on the analytical column. This process, however is only effective for relatively hydrophobic compounds. Analytes with low k’ with aqueous solvents such as small pharmaceutical molecules or hydrophilic peptides are frequently lost during the enrichment process. In contrast, direct injection without enrichment results in severe peak broading and tailing due to the unfavourable ratio of injection and column volume. In order to address this issue we developed an HPLC- Chip with an integrated sample loop allowing few nanoliters sample injections. The Chip demonstrates efficient separations of compounds with low and high k’. In addition we present an alternative method on an already commercially available HPLC-Chip using the groove of a “nano” valve that is part of the HPLC-Chip/MS interface as injection device which yielded similar results. Different analyses for samples from the pharmaceutical and proteomic area are presented in order to show proof of concept for a wide field of applications.

New Device For Protein Characterization.
G. Zilberstein, E.M. Baskin
Technion , Haifa, Israel

We present a novel device and method for protein characterization based on a specific dependence of standard chemical potential of any protein molecule and protein complexes on the pH value and ionic strength of the surrounding solution. This dependence is demonstrated theoretically in the framework of a simple model. It is shown that the dependence is defined by the pH-depended internal conformations of proteins. This approach presents encouraging prospects for new protein characterization and fingerprinting. In protein chemistry and biology pH dependence of standard protein chemical potential plays increasingly important role. We have developed simple theoretical model of method and device operation for measurement this parameter.

The Molecular Pinball Machine: A novel nanofluidic device for single-molecule analysis
G.O.F. Parikesit, A.P. Markesteijn, O. Piciu, V.G. Kutchoukov, J. Westerweel, A. Bossche, Y. Garini, I.T. Young
TU Delft, Delft, The Netherlands

We develop a new electrophoresis technology for detecting and manipulating single biological molecules in solution inside nanofluidic devices. The goal is to unravel parameters of individual molecules, which are usually hidden in conventional measurements on molecule-ensembles. In particular, we develop a nanofluidic device with a branched U-turn geometry [1], loosely dubbed as 'The Molecular Pinball Machine'. Using the device, we aim to characterize and sort single biological molecules. The channel depth is only 150 nm, while the channel length and width are in the micrometer scale. Hence it is a 2D-like nanofluidic device, where the probability of multiple molecules located on top of each other is reduced, and every single molecule is always located in optical focus. Electrokinetics is used to manipulate the fluid solution and to sort the molecules, while quantitative microscopy is employed to characterize the molecules and to detect their passage. A possible application of the device is the analysis of individual DNA molecules, a key issue in genomic studies. LPCVD a:Si is deposited on top of a glass wafer. Etching 150 nm into this layer creates the patterned fluidic channels. Some part of the a:Si layer is doped with As+, and activated using excimer laser annealing, forming built-in electrodes. They are partly coated with Al to further reduce the resistance. Anodic bonding is performed between the remaining a:Si layer and a capping Borofloatglass wafer, forming the closed channels [2]. We use 110-nm fluorescent beads to characterize the electroosmotic flow in the device, and to study the pathlines and the velocity distribution prior to molecule sorting [3]. Numerical simulation is done using FLUENT. Deviation between measurement and simulation is explained by Brownian motion (not included in simulation). Using MATLAB, we perform numerical simulation on electrophoretic (EP) and dielectrophoretic (DEP) forces [4,5]. The result shows that manipulation and sorting of single-molecule is feasible, using the built-in and external electrodes. With DEP, polarizable molecules are attracted to (or repulsed from) the highest electric field density. With EP, various force fields can be induced, and (positively) charged molecules can be pushed up, down, right, or left in the device. In conclusion, a 2D-like nanofluidic device is developed for single-molecule analysis. The setup allows quantitative microscopy for single-molecule detection. The electroosmotic flow (pathlines and velocity distribution) in the device has been characterized. Simulation shows the feasibility of single-molecule manipulation using electrokinetical (electrophoretic and dielectrophoretic) forces. References: [1] G.O.F. Parikesit, Proc. of SPIE Vol. 5515 (2004). [2] V.G. Kutchoukov, Sensors and Actuators A, 114:521-527 (2004). [3] G.O.F. Parikesit, Lab on a Chip, DOI:10.1039/B505493A (2005). [4] G.O.F. Parikesit, Proc. of SPIE Vol. 5718 (2005). [5] G.O.F. Parikesit, Proc. of MicroTAS (2005).

On-column Labeling for Capillary Electrophoretic Analysis of Individual Mitochondria Directly Sampled from Tissue Cross Sections
Hossein Ahmadzadeh, LaDora Thompson, Edgar A. Arriaga
Department of Chemistry, California State polytechnic University, Pomona, CA, 91768 USA

This work reports on a new individual organelle analysis procedure to (i) sample mitochondria from a tissue cross section into a fused silica capillary, (ii) on-column label these organelles with a mitochondrion-selective probe, and (iii) analyze the labeled organelles by capillary electrophoresis with post-column laser-Induced fluorescence detection (CE-LIF). In this procedure the bright field visualization and positioning of the sampling end of the capillary is not compromised by the fluorescence of a labeling reagent in the tissue cross section. In addition, on-column labeling allows for better control of the reaction time and probe concentrations. As a proof-of-principle, we show results of mitochondria from rat gastrocnemius muscle cross sections that were on-column labeled with 10-N-nonyl acridine orange, a mitochondrion-specific probe that stoichiometrically binds to cardiolipin. The CE-LIF results (i.e., organelle number, electrophoretic mobility of individual organellar events, and relative cardiolipin content of individual organellar events) for the gastrocnemius cross sections are similar when labeling is performed either on-column or in-tissue, further confirming the utility of the procedure. On-column labeling of organelles not only makes more practical the CE-LIF analysis of muscle mitochondria in aging and exercise physiology studies, but may easily be extended to the analysis of individual organelles in other biological samples.

Capillary batch injection: A novel injection concept for microfluidic systems
A. Stevens, F.M. Matysik
Institute of Analytical Chemistry, University of Leipzig, Leipzig, Germany

A new concept termed capillary batch injection (CBI) is applied as a sample introduction technique for capillary electrophoresis (CE) in very short capillaries (capillary length ranging between 5 and 10 cm). This approach can be considered as an alternative to chip electrophoresis with a comparable speed of separation. The CBI-CE system is coupled to electrochemical detection (ED) with offers selective and sensitive determinations. A mixture of ascorbic acid, noradrenaline and L-dopa in a 10 mM borate buffer (pH = 9.5) served as model system. The injection was controlled by a syringe motor with a 10 µL syringe coupled to a 75 µm capillary. The analytical performance was optimized regarding several parameters such as capillary dimension, distance between injection and separation capillary and convection within the injection cell. Separations of cationic, neutral and anionic species could be performed in less than 100 s applying a separation voltage of 750 V. The complete system has the potential to be used for point-of-care analysis owing to its small dimensions and low energy consumption.

Mutation detection by Amplification Refractory Mutation System (ARMS) and microchip electrophoresis
Marina Cretich1, Marcella Chiari1, Stefania Stenirri2, Barbara Foglieni2, Laura Cremonesi2, Maurizio Ferrari234, Ivan Rech5, Alessandro Restelli5, Massimo Ghioni5, Sergio Cova5
1: Istituto di Chimica del Riconoscimento Molecolare (ICRM) – C.N.R.- Milano, Italy 2: Unit of Genomics for Diagnosis of Human Pathologies, IRCCS H San Raffaele, Milano, Italy. 3: Diagnostica e Ricerca San Raffaele S.p.A, IRCCS H. San Raffaele, Milano, Italy. 4: Universitŕ Vita-Salute, H. San Raffaele, Milano, Italy. 5: Politecnico di Milano, DEI, Dipartimento di Elettronica e Informazione, Milano, Italy.

Genetic tests, with their enormous scope of applications in biotechnology and medicine provide a very large demand for simple and economical analytical systems. The use of micro-machined devices for capillary electrophoresis (CE microchip) is rapidly increasing due to the low reagent consumption and rapid analysis. We describe here a new fully automated microchip electrophoresis instrument based on single photon avalanche detectors (SPAD) with dual-wavelength LIF detection, able to detect CY 5 and CY 5.5. The apparatus is equipped with a software interface, thermal control and automatic optical alignment. The electrophoresis instrument allows the simultaneous detection of the two fluorescence colours with good reproducibility and reliability assigning the genotype in less than 1 minute. This system was used for the identification of the main mutation causing cystic fibrosis, named deltaF508, by the Amplification Refractory Mutation System (ARMS). In this protocol each sample is amplified in two separate PCR reactions, one containing a primer specifically designed to amplify only the wild-type allele, the other a primer to amplify the mutated one. Since the two primers are differently labelled, the sample genotype is deduced based on the fluorescence of the amplicons run in the CE microchip. Electrophoretic separations were carried out in simple cross geometry microchips (channels have a width of 50um and length of 35 mm and 10 mm respectively) coated by dynamic adsorption of dimethylacrylamide and allyl glycidil ether (EPDMA) copolymer for an efficient control of the electroosmotic flow. The PCR fragments were separated by using a high molecular mass polyacrylamide produced by inverse emulsion polymerization as the sieving matrix.

Coupling High Speed Liquid Chromatography and Laser-induced Fluorescence: Opening the Way to Superb Resolution and Sensitivity via High Speed LC. Application to the Separation, in less than 5 minutes, of 18 Amino Acids at the nM level.
F. Couderc, N. Siri
ERT1046, Laboratoire IMRCP, Université Paul Sabatier, 31062 Toulouse Cedex France. Picometrics, 10 avenue de l’Europe, 31520 Toulouse, France.

The recent development of new column phases and innovative HPLC instrumentation leads to the development of extremely rapid separations. As the speed of the separation is increased, the peak becomes sharper (narrower) and the use of a conventional fluorescence detector becomes significantly more difficult. This difficulty arises because the size of the cell becomes significant with respect to the peak volume and the resolution is severely reduced. An additional concern with the use of ultra high pressure chromatography is that the pressure at the outlet of the column is relatively high, and may damage the flow cell in the detector. LIF detection is commonly used to provide highly sensitive “in capillary” detection, in Capillary Electrophoresis as well as with in capillary and nano LC systems. In the latter approach, a piece of silica capillary is simply plugged at the outlet of the column. This technique is also used with the new, very rapid LC techniques, as the capillary itself is the cell. The laser excitation is achieved through a “window” (removed coating of the capillary) while the rest of the length of the capillary provides a counter pressure. In this paper we present the separation of 18 amino acids (all the common amino acids, except Pro and Lys) labelled with naphthalene-2,3-dicarboxyaldehyde (NDA) using an Acquity UPLC™ system (Waters, Inc., Milford MA) with a 1 mm micro-column and detection with a ZetaLIF Laser Induced fluorescence detector. The linear velocity of the solvent in the capillary is optimized and subnanomolar LODs are reached. We believe that these are the fastest LC separations amino acids using fluorescence detection at such low concentrations, opening the way to high throughput amino acids analyses in areas ranging from the foodstuff industry to neuropharmacology… The results described in this paper, albeit preliminary, clearly indicate that the coupling of High Performance LC to LIF detection is very promising: From the LIF detection of a broad range of compounds using various available laser sources, to the coupling of LIF and mass spectrometry detections, for instance in proteomic assays after nano High Speed LC separations, the fields to explore remain numerous and highly exciting.

Quantitative Physiology of Microbes
L. M. Blank, G. Ionidis, B. Bühler, A. Schmid
University of Dortmund, Dortmund, Germany

White Biotechnology serves an ever growing industry that delivers chemical products for such diverse applications as agricultural chemicals, organics, plastic material and drugs, to name just a few. Advantages for the production of pharma intermediates are the high specificity (chiral, chemical, regional) that minimize or excludes unwanted byproducts. Companies involved often exploit processes based on whole cells that have the advantage of self replicating catalysts and, importantly, regeneration capacity for cofactors that are necessary for a wide variety of enzyme based reactions e.g. redox reactions. The limitations of whole-cell biocatalysis are however numerous, include substrate/product toxicity, low biocatalytic activity, low biocatalyst concentration, and technical challenges such as oxygen transfer, and are traditionally addressed by optimizing process conditions. With the advent of “omes”, the focus shifts slowly to the smallest catalytic- and production unit, the cell itself. Important limitations are enzyme availability, cell metabolism and heterogeneity of cell populations. Here, we describe the application of whole-cell biocatalysis for the production of pharmaceutical synthons. We will show results indicating that biocatalyst efficiency is coupled to the energy metabolism of the host strain. Studying the metabolic network by mass spectroscopy-based Fluxome and Metabolome analyses holds high potential for future strain and process optimization. This will be exemplified by solvent induced intracellular carbon flux changes in Pseudomonas strains. Finally, we give an outlook on chip-based dielectrophoresis single-cell experiments for whole cell biocatalyst design.

Building Blocks for Sample-limited Lipidomics
M.E. Johnson, T. Fahrenholz, R. Johnson, K. Adams
Duquesne University, Pittsburgh, Pennsylvania, USA

Situations that limit the amount of sample that is available for profiling a lipidome put stringent requirements on how the lipid extract is obtained and how the subsequent sample is prepared. Lipids can be particularly problematic because there is a high probability of sample loss due to surface adsorption, and the use of microscale architectures, with their inherently high surface area to volume ratio, exacerbates the problem. However, microfluidic platforms provide a promising venue for efficient sample preparation prior to a final capillary separation step and mass spectrometry because they can, in principle, offer low dead volume and versatile configurations. In this poster, we will describe a number of building blocks leading towards "sample to MS" lipidomics. Namely, we will discuss progress in neutral lipid fractionation on microchips using silica stationary phases, strategies for on-chip lipid extraction, and capillary electrophoretic separation of several lipid classes. Neutral lipids can be fractionated relatively efficiently on hand-packed glass microchips for subsequent analysis, but close attention must be paid to surface treatment and chip design for low dead volume. Lipids can be extracted using standard modified Folch-Pi methodology, though the use of on-chip cell lysis and lipid capture is more efficient and chemically more benign, and can work with much smaller samples. Finally, we will discuss separation strategies for neutral lipid separation: capillary electrochromatography for fatty amines, nonaqueous capillary zone electrohphoresis for fatty acids, and LC/MS of phospholipids and primary fatty acid amides. These techniques, together, provide several of the building blocks necessary for constructing a means of automating lipidomics on small sample.

Metabolomic Analysis of Cerebrospinal Fluid by Capillary Electrophoresis
R. Ramautar, G.W. Somsen, G.J. de Jong
University Utrecht, Utrecht, The Netherlands

Metabolomics is the comprehensive analysis of low-molecular weight metabolites in a biological system, such as cells and urine. Capillary zone electrophoresis combined with mass spectrometry (CZE-MS) has demonstrated to be a valuable tool for large scale metabolite analysis. The aim of the present study was to apply and optimize a CZE method for metabolomic analysis of cerebrospinal fluid (CSF). Metabolomic analysis of CSF is important for the diagnosis of diseases related to the central nervous system. The capillary has been coated using the procedure of Katayama et al. resulting in a stable cation-modified capillary with a pH independent electro-osmotic flow [1]. Due to the presence of proteins and high salt concentrations in CSF, optimization studies were focused on the influence of these matrix effects on the performance of the CZE method using some model compounds. Also the influence of the separation voltage and electrophoresis buffer on the performance of the system have been studied. Metabolomic analysis of CSF samples was primarily focused on organic acids because these compounds function as biomarkers for various diseases. The results of the optimization studies and electropherograms of CSF samples will be presented. References [1] H. katayama, Y. Ishihama, N. Asakawa, Anal. Chem., 70 (1998) 5272.

Novel derivatization reagents of amino acids for quantitative determination and mass distribution analysis in LC-MS/MS
K. Shimbo, A. Yahashi, M. Nakazawa, D. Iwahata, N. Kageyama, K. Hirayama, S. Iwatani, Y. Usuda, K. Matsui, H. Miyano
Ajinomoto Co., Inc., Kawasaki, Japan

High sensitive pre-column derivatization reagents for qualitative and quantitative analysis of intracellular amino acids using high performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) have been developed. These reagents, activated carbamates react briefly with amino group to form urea compounds (R-NH-(C=O)-NH-R’) under the mild condition. 4-(Trimetyammonium)anilyl-N-hydroxysuccinimidyl carbamate iodide (TAHS), which are designed to enhance ionization efficiency and to give sensitive product ions, achieved atto-mole level detection of amino acids. These adducts also provide characteristic cleavage at urea bond that is the binding position between reagent and amino group in the collision cell of triple quadrupole mass spectrometer and produce characteristic product ions derived from the reagent skeletons. Though many derivatization reagents for GC-MS or LC-MS were used in the stable isotope labeled feeding experiments, such as metabolomics studies, flux analysis or biosynthetic analysis to obtain isotope ratio, it was difficult to presume the cleavage position and complicated calculation procedures were needed. The unique cleavage position of these adducts between R-NH- of amino compounds and -(C=O)-NH-R’ of reagent, provide isotope ratio of amino compounds without any correction by selected reaction monitoring mode analysis of tandem mass spectrometry.

Fast monitoring of metabolites showing the therapeutic effect of antibiotics in exhaled air using ion mobility spectrometry
J.I. Baumbach, V. Ruzsanyi
ISAS - Institute for Analytical Sciences, Department of Metabolomics, Bunsen-Kirchhoff-Straße 11, 44139 Dortmund, Germany

Volatile Metabolites occurring in human exhaled air are correlated directly to different kinds of diseases. An ion mobility spectrometer (IMS) coupled to a multi-capillary-column (MCC) was used to identify and quantify volatile metabolites occurring in human breath down to the ng/L- and pg/L-range of analytes within less than 500 s and without any pre-concentration. The IMS investigations are based on different drift times of swarms of ions of metabolites formed directly in air at ambient pressure. The effect of drug delivery on a patient showing Angina lateralis is presented to show the potential of the method developed in the field of detection of pathways, effective dosage and decision of effective time intervals to deliver pharmaceuticals. The IMS-chromatogram shows two different main bacteria involved in the present case. The identification was realised by measurements on pure bacteria cultures and signal processing of main peaks obtained. The effect of Amoxicillin application during the first 4 days was studied. IMS-chromatograms show the reduction of metabolite concentrations directly in the exhaled air. The vision of the project is to show, that metabolites in exhaled human breath are carrier of information of the health situation in addition to traditional methods using blood or urine.

Bacterial differentiation by ion mobility spectrometry. - First results of a pilot study
J.I. Baumbach1, P. Litterst2, M. Westhoff2, L. Freitag2, V. Ruzsanyi1
1 ISAS - Institute for Analytical Sciences, Department of Metabolomics, Bunsen-Kirchhoff-Straße 11, 44139 Dortmund, Germany 2 Lung Hospital Hemer, Theo-Funccius-Str. 1, 58675 Hemer, Germany

Purpose: Early diagnosis and specification of bacterial airway infection is of importance, especially in patients who are at high risk for respiratory failure, invasive or non-invasive ventilation and a prolonged hospital stay. Methods: Ion mobility spectrometer (IMS) coupled to a multi-capillary-column (MCC) identifies and quantifies volatile metabolites down to the ng/L- and pg/L-range of analytes within less than 500 s and without any pre-concentration. The IMS investigations are based on different drift times of swarms of ions of metabolites formed directly in air at ambient pressure. Head space over selected microbiological cultures was directly sampled for IMS-chromatograms. Results: In this in-vitro study IMS-chromatograms of different bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus agalactiae, Haemophilus influenzae, Klebsiella pneumoniae, Escherichia coli, Serratia marcescens, Pseudomonas aeruginosa, Enterobacter cloacae) and Candida albicans were obtained. The selected bacteria and Candida albicans could be defined and distincted by different metabolites. Conclusion: Ion mobility spectometry seems to provide a tool for precise bacterial analysis. The results of this pilot study have to be proved by an in-vivo study, especially in patients with airway infections as COPD-exacerbation and pneumonia. A future aspect might be the implementation of a beside test.

Quantitative Analysis of Metabolites from Penicillium Chrysogenum using LC-MS/MS
R. Maleki Seifar, U.D.M. Nasution, J.C. van Dam, J.J. Heijnen
Delft University of Technology, Delft, The Netherlands

Analysis of the intracellular metabolites because of the chemical complexity requires sophisticated analytical techniques such as LC-MS/MS. Using MS, it is possible to identify a single analyte out of a very complex mixture. Here, LC-MS/MS technique has been used for quantitative analysis of intracellular metabolites Pen G, PIO, 6-APA, OPC, HPA, PAA, O-OH-PAA during fermentation process of Penicillium Chrysogenum culture.

Metabolomic analysis of yeast and rice using capillary electrophoresis- mass spectrometry and their changes in response to chemical stress
Y. Tanaka, T. Higashi, R. Rakwal, S. Wakida, H. Iwahashi
Human Stress Signal Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Osaka, Japan

Metabolomics is an emerging field in analytical biochemistry and can be regarded as the end point of the "omics" cascade. The advantage of metabolomic analysis is that the biochemical consequences of mutations and stress-response mechanisms can be observed directly. Although several methods such as GC and HPLC are used to separate metabolites according to various chemical and physical properties, most of the metabolites are polar and ionic compounds. Capillary electrophoresis-mass spectrometry (CE-MS) is the most powerful tool for the analysis of charged species. The major advantages of CE-MS are that it exhibits extremely high resolution and can be sorted on the basis of mass-to-charge ratio. Soga et. al. have already proposed three CE-MS conditions for the comprehensive and adequate analysis of cationic, anionic and nucleotide metabolites [1,2]. The aim of this research was to investigate qualitative and quantitative difference of metabolites in response to chemical stress using model organisms such as yeast (Saccharomyces cerevisiae) cells and rice (Oryza sativa L.) leaves. For example, it is well known that the proteome analysis of the cadmium response in yeast showed a strong induction of enzymes of the sulfur metabolic pathway, consistent with a strong increase of glutathione (GSH) synthesis. In this study, therefore, a metabolomic analysis of the sulfur pathway based on the three CE-MS conditions was performed. We have also investigated stress-response metabolites from jasmonic acid-treated rice (cv. Nipponbare) leaves. Based on our experiments in rice, we propose a new CE-MS method for the analysis of flavonoids in plants. When the above-mentioned CE-MS mode for anionic metabolites was used, kaempferol was not detected probably due to the wall adsorption of SMIL-coated capillary. In addition, isomeric separation of apigenin and genistin could not be obtained under the separation conditions. The separation was successfully achieved with an untreated fused-silica capillary and an electrolyte solution composed of 50 mM ammonium acetate buffer (pH 8.5) and acetonitrile. As a preliminary result, remarkable changes in stress-induced metabolite concentrations will be introduced. [1] T. Soga, Y. Ohashi, Y. Ueno, H. Naraoka, M. Tomita, T. Nishioka, J. Proteome, Res., 2, 488-494 (2003). [2] S. Sato, T. Soga, T. Nishioka, M. Tomita, Plant J., 40, 151-163 (2004).

C. Draghici1, H. Billiet2, J. van Dam2, G. van Dedem2, Gh. Coman1, M. Badea1, S. Gocan3
1Transilvania University of Brasov, Romania, 2Delft University of Technology, The Netherlands, 3Babas-Bolyai University of Cluj-Napoca, Romania

Due to the fact that nucleotides are entities involved in the pathways of energy transfer in the living bodies, the separation studies of this bioactive compounds show more and more interest among the separation science researchers. In this work mixtures of three adenine nucleotides (AMP, ADP, and ATP) and four Nicotinamide nucleotides (NAD, NADH, NADP, and NADPH) were studied by use of different capillary zone electrophoresis techniques. CZE was performed with Tricine solution as background electrolyte and organic additives, while SDS was introduced as micellar agent for MEKC determinations. Taking into account the adenine nucleotides and nicotinamide coenzymes molecular structure and their possible linkage brakes, the stability was studied in different CE systems, so that both negatively charged and neutral degradation components should be identified. Different identification methods were applied to facilitate the observation in changes of the adenine and nicotinamide nucleotides stability: effective mobility, spiking by CZE, as well as capacity factors by MEKC. Starting with the observation that calculated effective mobility showed AMP and ADP as decomposition substances of NADH, together with not-identified compounds, the study focused on other decomposition compounds observed in different standard mixtures: the three adenine nucleotides as A1, the four nicotinamide nucleotides as A2 and all seven as A1+2. The study in CZE system, was performed with different Tricine concentrations (60, 80 and 90 mM), at pH 7.25 and 20 kV. Some observed degradation processes were: 1. release AMP, ADP and/or ATP from NADH, NADP and NADPH 2. equilibrium between NAD and NADH 3. equilibrium between NADP and NADPH Besides these possible processes involving the seven studied nucleotides, three other unknown substances were observed (by mean of the calculated mobility) and theoretically allocated as followes: • NI1 possible nicotinamide nucleoside monophosphate (NMP) • NI2 possible nicotinamide nucleoside diphosphate (NDP) • NI3 possible adenozine-2’,5’,5’-triphosphate (ATP’). Due to the fact that NMP was a possible degradation compound, this anionic standard was also investigated. The use of MEKC technique (90 mM Tricine, pH 7.25, 8.5 mM SDS, 20 kV) aimed to identify the neutral degradation compounds, so that new standards were investigated: nicotinamide (NA), adenine (ADE), adenosine (ADO), ribose (R) and nicotinamide dinucleoside (NAN). ADE and ADO had low capacity factors, and migration times very close to the EOF. The following degradative processes were observed: 1. NAD migration showed the presence of NAN, as degradation compound 2. NADH migration emphases the AMP, NMP and NAN formation.