Research Institute for Chromtaography, Kortrijk, Belgium, This email address is being protected from spambots. You need JavaScript enabled to view it.

Optimization of the separation performance of fluid-based separation techniques, and especially of liquid chromatography (LC), is essential for analysis of the complex samples encountered in today’s research (omics, food, environmental, natural product, etc. samples).

Around a decade ago, tremendous improvements were made in LC column technology (sub-2 µm porous particles, core-shell particles) and in instrumentation (pressures up to 1200 bar) opening new possibilities in terms of speed and resolution to LC practitioners for tackling complex samples. A now well-accepted metric of separation power in gradient elution, mandatory for samples of high complexity, is the peak capacity (nc). nc is the maximum number of peaks that can fit side-by-side between the first and last peak of interest with resolution 1. With state-of-the-art LC, peak capacities of ca. 600 (in ca. 1 h) in conventional one-dimensional LC (1D-LC) can routinely be obtained.

It is, however, wishful thinking that such peak capacities are sufficient to separate very complex mixtures. Peak capacity should significantly outstrip the number of components in a sample; the statistical theory of peak overlap (developed by Giddings) indicates to resolve 98% of randomly distributed sample components, nc should exceed the number of components by a factor 100 ! This means that a nc value of 10.000 corresponding to ca. 1x108 theoretical is needed to “chromatographically” resolve a sample containing 100 components.

One straightforward approach to increasing nc is multidimensional LC, especially two-dimensional LC or 2D-LC. In 2D-LC the sample is subjected to two different separation mechanisms and this in an off-line or on-line way. On-line 2D-LC is divided in heart-cutting LC (LC-LC), multiple heart-cutting or parking LC (mLC-LC) and comprehensive LC (LC×LC). LC-LC and mLC-LC are applied to better resolve components in a selected retention time window or windows, while in LC×LC, the entire sample is subjected to two separation mechanisms.

The recent developments in 2D-LC hardware design, software approaches and column configurations will be presented. The figures of merit of LCxLC will be discussed and illustrated with analysis of several complex matrices including some of QA/QC.

 The Molecular Foundry, E.O. Lawrence Berkeley National Laboratory, This email address is being protected from spambots. You need JavaScript enabled to view it.

Typically, we controlled chemistry and through it selectivity of our monolithic columns by using: (i) direct copolymerization of functional monomers, (ii) preparation of parent monolith with reactive functionalities and its post-polymerization functionalization, (iii) thermally or photoinitiated grafting of functional monomers on the pore surface, and (iv) attachment of gold and silver nanoparticles to the pore surface followed by reaction with functional thiols. Recently, a new approach was introduced that enables functionalization of pore surface of monoliths and involves application of metal-organic frameworks (MOFs). These frameworks are compounds consisting of metal ions or clusters coordinated to rigid organic molecules to form one-, two-, or three-dimensional structures that can be porous. We used two implementations: (i) admixing preformed MOF to the polymerization mixture followed by the thermally initiated free radical polymerization and (ii) forming the MOF within the pores applying layer-by-layer approach. The former technique will be demonstrated with the preparation of monolithic column designed for enantioseparation while the latter led to column for selective preconcentration of phosphopetides and to column for the separation of native fatty acids according to their length and degree of unsaturation.

Polymer Institute, Slovak Academy of Sciences, Dúbravská cesta 9, 84541 Bratislava, Slovakia, This email address is being protected from spambots. You need JavaScript enabled to view it.

Advanced synthetic polymers represent important group of modern, high-performance materials. Here belong complex polymers (COP) and complex polymer systems (CPS). COP exhibit more than one dispersity (distribution) in their molecular characteristics - molar mass, chemical structure and physical architecture. Their typical representatives are various kinds of copolymers and functional macromolecules. CPS comprise the two and multicomponent blends of macromolecules and block copolymers containing their parent homopolymers. Molecular characterization of both COP and CPS is an exacting analytical challenge because the analytes must be separated. At present, size exclusion chromatography/gel permeation chromatography (SEC/GPC) dominates the area. Though SEC/GPC is a marvelous method, it cannot give quantitative data on molecular characteristics of COP and of most CPS because the size of macromolecules in solution depends on all molecular characteristics of macromolecules to be separated. Moreover, the separation selectivity of SEC/GPC is fairly limited and both low detector sensitivity and sample capacity of SEC/GPC does not enable to characterize minor ( solve the problem, coupled methods of polymer HPLC were introduced, which combine exclusion – entropic, and interaction – enthalpic retention separation mechanisms. Two of the coupled methods of polymer HPLC, namely liquid chromatography under critical conditions (LC CC) and liquid chromatography under limiting conditions (LC LC) of enthalpic interactions will be presented, compared and critically evaluated in the contribution with the conclusion that LC CC is preferable for oligomers and for binary blends of macromolecules with molar mass below about 10 kg/mol whereas LC LC has no upper molar mass limit but its performance may be not sufficient for low oligomers. The sample recovery represents an important limitation of LC CC of macromolecules with high molar mass. In contrast to LC CC, LC LC is robust and user-friendly. LC LC enables to separate even four-component CPS in one single step and can be easily engaged in the two-dimensional polymer LC.

BIA Separations d.o.o., Mirce 21, Ajdovscina, SI-5270, Slovenia, This email address is being protected from spambots. You need JavaScript enabled to view it.

While traditional drugs are usualy small molecules, biopharmaceuticals, like proteins, plasmid DNA, viruses or virus-like particles, are large complex bioparticles, with heterogeneus physicochemical properties, sensitive to the environmental conditions (pH, I) and shear forces. Therefore, during manufacturing process of biological nanoparticles there are several issues which have to be considered. To satisfy the severe requirements placed on the purity of biopharmaceutical products demanding several-step purification processes have to be developed. An integral part of downstream processing is the chromatographic unit operation.

To gain as high productivity as possible chromatographic resin should exhibit high dynamic binding capacity and high purification yield meaning high selectivity and low degradation of target macromolecule should be achieved preferably at high linear velocity and low pressure drop. Convection-based transport is an extremely important feature that accelerates the separation and purification process, especially of large biomolecules with low mobility. In recent years chromatographic monoliths, also called Convective Interaction Media (CIM), appeared as a resin of choice for biopharmaceutics’ purification. Their specific porous structure enables the convective mass transport and high surface accessibility to large molecules. Therefore they allow very fast separations and exhibit very high binding capacities for extremely large bioparticles.

In this work key features of large biological nanoparticles that influence the downstream process of target compound will be discussed. The differences between classical matrixes for liquid chromatography and for biochromatography will be exposed and case studies of several biopharmaceuticals of different classes using special monolithic chromatography columns will be presented.

1 Bioprocess Engineering Laboratory, School of Engineering and Graduate School of Medicine, Yamaguchi University, Ube 755-8611, Japan

2 Faculty of Chemistry and Chemical Technology, University of Ljubljana,1000 Ljubljana, Slovenia, This email address is being protected from spambots. You need JavaScript enabled to view it.

Numerous techniques have been developed over the past decades to enhance the in-vivo stability of therapeutic proteins. In particular, PEGylation, known as the covalent binding of PEG chains to proteins, has become a technique of choice to extend their in-vivo circulation half-life. However, the side directed PEGylation reaction needed for human treatment still represents a challenge, due to consecutive competitive reactions with activated PEG, native and PEGylated protein. As a consequence, PEGylation produces positional isoforms and isomers which have different PEG chain number and further purifications steps are needed to isolate target positional isoform. For efficient development and production of PEGylated protein drugs, it is therefore essential to have fast analytical method for monitoring of PEGylation reaction either during development phase (QbD) or as a part of production process (PAT) where high frequency data are required for process control. As PEGylation alters surface protein charge distribution, ion-exchange chromatography seems to be a method of choice. Among different types of stationary phases monoliths were selected due to predominant convective transport providing efficient separation in short time at low pressure drop for such large protein molecules as PEGylated proteins.

In this work we developed different chromatographic methods for separation of PEGylated protein positional isoforms and isomers using a strong cation exchange SO3 CIM disk monolithic column (diameter =12 mm, thickness = 3 mm, volume 0.34 mL) and implementing either salt or pH gradient. While optimal separation using salt gradient was achieved with citric buffer pH 4.5, for pH gradient carbonate buffer from pH 10 to 12 provided optimal results. Despite different elution mechanism both methods provided separation of mono, di and tri PEGylated proteins as well as positional isomers but differing in resolution and elution order. Under optimal conditions, the analysis was completed within 3 min at flow rate of 10 mL/min and pressure drop below 1 MPa (10 bar).

University of Maribor, Faculty of Chemistry and Chemical Engineering, PolyOrgLab, This email address is being protected from spambots. You need JavaScript enabled to view it.

Emulsion templating method is frequently used for the preparation of macroporous polymeric monoliths (1). While the morphology of so prepared polymers (termed polyHIPEs) is generally composed of spherically shaped primary pores several micrometer in diameter and a number of secondary pores connecting primary pores, there are some methods enabling the manipulation of mesoporous and microporous structure of the polymer film in the monoliths. The lecture will focus on presentation of a combination of emulsion templating and post polymerisation hypercrosslinking in order to produce polyHIPE monoliths with a distinct bimodal porous structure. Furthermore, examples of chemical functionalization and solid phase extraction from aqueous sources will be given.

 1 I. Pulko, P. Krajnc, Macromol. Rapid Commun. 2012, 33, 1731

 University of Pécs, This email address is being protected from spambots. You need JavaScript enabled to view it.

The study of the details of mass transfer in liquid chromatography is of central interest. We apply the microscopic (or molecular dynamic) model of chromatography to study the reversed phase separation of small and large molecules. The microscopic theory of chromatography describes the evolution of a chromatographic peak as the random migration of the molecules along the column combined with adsorption–desorption processes that occur at random, too.

The molecular dynamic model is rather straightforward to comprehend and it can furnish direct answers when one tries to understand the development of chromatographic peaks. We show that the microscopic model can be rather simply used to estimate the fundamental characteristics of the separation process. We can estimate the rate a molecule is adsorbed on the surface of the stationary phase while it migrates along the column.

When combining the general rate model with the molecular dynamic model, one can consider and compare the kinetics of the transfer of solute molecules between the flowing and stagnant zones of mobile phase, the pore diffusion, etc. We analyze the peak shapes recorded under linear conditions, and can characterize the heterogeneity of the surface of the stationary phase. With a peak shape analysis that is based on the molecular dynamic model of chromatography, we can identify the presence of heterogeneous mass transfer or adsorption kinetics. We can, furthermore calculate the amount of retention due to the individual adsorption sites. The general rate model of chromatography, which is a macroscopic model, offers the most detailed description of the separation process. We compare the results provided by both microscopic and macroscopic analysis of the peak shapes and statistical moments. In this paper we discuss the influence of wide pore-size distributions. We present results obtained on nonporous, fully porous, and fused-core particles. Furthermore, mass transfer in supercritical fluid chromatography and reversed-phase liquid chromatography are compared.

 Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Studentská 573, CZ-53210 Pardubice, Czech Republic, This email address is being protected from spambots. You need JavaScript enabled to view it.

In the past ten years, LC on polar stationary phases with mobile phases containing organic solvent and less than 5–40% water (HILIC) has become preferred method for separation of polar compounds, namely pharmaceuticals or biopolymers. On polar columns, sample interactions, retention and separation selectivity may strongly change across the full composition range of aqueous-organic mobile phases, HILIC retention mechanism predominating in the organic-rich, and reversed-phase (RP) mechanism in more aqueous media. We determined the accumulation of water from acetonitrile-rich aqueous-organic mobile phases in the water-rich diffuse layer filling the pores of eighteen polar columns, using frontal chromatography combined with Karl Fischer titration. The sorption can be described by Langmuir isotherm and it strongly depends on the type of stationary phase, ranging from less than one monomolecular water layer up to 6 - 10 water layers equivalents. Some water present in polar solvents is accumulated even on non-polar columns. Considering the actual concentration of water in calculations largely improves the accuracy of prediction of gradient elution data in HILIC systems. The dual retention mechanism enables using a single polar column alternatively in the HILIC and RP retention modes of various polar sample compounds such as phenolic acids and flavones. This approach enhances separation selectivity and peak capacity in single- and two-dimensional HPLC.

 Thermo Fisher Scientific, This email address is being protected from spambots. You need JavaScript enabled to view it.

One of the most successful approaches to the retention and separation of polar compounds is hydrophilic interaction liquid chromatography (HILIC). HILIC is not a straightforward technique and one of its most common drawbacks during method development is the difficulty in identifying the most suitable column to use. The stationary phases used in HILIC are quite diverse, and systematic studies of their chemistries are relatively limited. Kumar et al. [1] re-developed early characterisation studies, incorporating the effects of changing a range of experimental parameters as well as a characterisation study on the stationary phases. This approach has been previously applied in RPLC [2, 3], but novel in HILIC and represents a good practical guide to adjustments of conditions that effect the selectivity of a separation.

The aim of this work was to develop these areas of critical understanding by testing a wider range of experimental parameters to determine the interactions between them and how these parameters affect the chromatography of a series of test compunds. Five main classes of HILIC columns were investigated in this study:

Bare silica, neutral bonded ligands, charged ligands, zwitterionic phases and mixed mode phases. A preliminary column characterisation investigation was performed. This highlighted fundamental differences in the column chemistries. In addition the following experimental factors were studied: effect of solvent content, effect of buffer concentration and effect of buffer pH. The work was finally summarised in a convenient work flow which describes in detail a generic approach to method development within the HILIC mode of chromatography.

[1] Kumar A., Heaton J.C. and McCalley D.V., 2013, J. Chromatogr. A, 1276, 33.

[2] Neue U. D., O’Gara J.E. and Mendez A., 2006, J. Chromatogr. A, 1127, 161.

[3] Neue U. D. and Mendez A., 2007, J. Sep. Sci., 30, 949.

 1 Thermo Fisher Scientific, Tudor Road, WA7 1TA, Runcorn, UK

2 University of Liverpool, Oxford Street, L69 7ZD, UK, This email address is being protected from spambots. You need JavaScript enabled to view it.

In recent years there has been a renewed interest in particle technology for both HPLC and UHPLC, due to the introduction of solid core technology. Prior to this there was very little novel development in HPLC particle morphology The patents associated with solid core technology have resulted in manufacturers and researchers having a much greater level of interest in the basic manufacture of silica particles, particularly since the current manufacturing approach is known to be a fairly involved process.

This has led to the development of a completely new type of particle which is manufactured using a one-step synthesis delivering near monodispersed particles and core shell morphology. The morphology of the particle has been designed to deliver the real advantages of the core-shell particles, with a range of parameters being varied to ensure the robustness of the manufacturing.

Careful investigation of some of the morphologies produced revealed that the structure of the particle produced resembled very closely a true fractal structure. Fractal structures have been associated with chromatographic media for some time, however they have been more associated with the concept of being a fractional dimension rather than having a degree of self similarity. These new particles demonstrate, not only a fractional dimension, but also a degree of self similarity. This opens up a possibility of having a surface topography which is essentially homogeneous to solute molecules, thus eliminating one of the issues associated with traditional porous structures in which molecules effectively do not experience the same surface topography resulting in different retention times, the so called ‘c’ term in the van Deemter equation. This is particularly pertinent for large molecules where diffusional processes related to the different pore depths can result in poor peak shape, either through equilibration effects or because molecules effectively get stuck between the walls of the pores.

Examples will be given of the different morphologies that can be manufactured and also how these can be applied to the analysis of large protein molecules.

Boreskov Institute of Catalysis SB RAS, Novosibirsk, Russian Federation, Pr. Lavrentyeva

Novosibirsk state University, 630090, Pyrogova st., 2, Novosibirsk, Russia, This email address is being protected from spambots. You need JavaScript enabled to view it.

Comprehensive two dimensional gas chromatography (GCxGC) is a high-effective technique for analysis of complex multicomponent mixtures.

An important advantage of GCxGC is that it allows analyzing of complex samples by separating the components in groups according to chemical classes. Frequently those samples contain high-boiling fractions of compounds. The examples of those samples are crude oil, products of petrochemistry, coal chemistry and etc. It is known that in order to obtain the best separation power in GCxGC one should use as polar phase as possible for one of the dimension. However, the thermal stability of common polar and high polar stationary phases is not sufficient to analyze high boiling complex samples.

Recent advances in the development of new liquid phases made it possible to use some classes of ionic liquids (ILs) as polar stationary phases [1]. There are works devoted to research of columns with imidazolium and phosphonium ILs for GCxGC in the literature [2]. However, the possibility of using ILs for high temperature GCxGC separations (up to 300°C) is not closely investigated. Our work is devoted to the research of imidazolium and pyridinium ILs as stationary phases columns for GCxGC. In our earlier studies we have shown that these classes of ILs allow to work at the temperature up to 300°C [3] and possess different range of selectivity [4]. Therefore, we consider that their use in GCxGC will allow to obtain the high selective separations at high temperature. In our work the series of columns with pyridinium and imidazolium ILs were prepared and were investigated as columns for GCxGC. These columns were shown to work at the temperature 280°C-300°C with good separation characteristics. The number of separation of multicomponent high boiling samples was obtained. Among them: crude oil and oil products, natural bitumen, high boiling mixtures of biological origin.

[1] С.A. Shamsi, N.D. Danielson, J. Sep. Sci. 30 (2007) 1729.

[2] С. Ragonese, D. Sciarrone, J. Chromatogr. A 1255 (2012) 130-144.

[3] M.V. Shashkov, V.N. Sidelnikov, Anal. Bioanal. Chem. 403 (2012) 2673-2682.

[4] M.V. Shashkov, V.N. Sidelnikov, J. Chromatogr. A 1309 (2012) 56-63.

 University of Tasmania, Australian Centre for Research on Separation Science, Private Bag 75, Hobart, TAS 7001, Australia, This email address is being protected from spambots. You need JavaScript enabled to view it.

Our aspiration was to design portable LC as a widely available ‘open platform’, moderate-pressure (over 100 bar), and importantly highly flexible modular system, based almost entirely on commercial off-the-shelf low-cost components. In this work, a modular portable LC has been designed, characterised and used, weighing under 2 kg (plus data acquisition). The backbone of the system was a breadboard assembled modular flexible microfluidic system (LabSmith), complemented with other off-the-shelf components, including an injection valve and on-capillary detectors, all operated through a PC. The system can accommodate six syringe pumps with 80, 20 and 5 µL syringe options, and maximum backpressures of up to ca. 150 bar, with two used for each A and B mobile phase in gradient operation and another pump for sampling and one in reserve for post-column derivatisation. The flow rate (typically 1 µL/min), which exhibited substantial pulsations due to the individual steps of the step motors driving the syringe pump, could be well dampened by using microbubbles of low-solubility inert gas (nitrogen, helium), automatically aspirated in each mobile phase draw cycle. Each of the two pairs of syringe pumps for mobile phases A and B were connected with microfluidic switching valves (two 4-port valves), and the A and B streams joined together with a Y-connector and a microfluidic pressure sensor, thus providing a system for low hold-up volume gradient formation. The mobile phase was led to a nano-LC sampling valve and to a capillary LC column. The detection was on-column (100 µm i.d. PTFE-coated fused silica) with an LED-based deep-UV-vis-NIR photometric detector, and end-column with an electrochemical detector (amperometric and/or potentiometric). The performance of the system was evaluated including the pumping accuracy, linearity of gradient formation, separation performance and sensitivity of detection. Typically analysis runs used 12-20 µL of solvent with overall reproducibility of 1 5 % and 3 6% RSD for retention times and peak area in gradient mode, respectively. Future avenues for miniaturisation of the data acquisition and system control are discussed.

 Department of Chemistry, Seoul National University, Seoul 151-747, Korea, This email address is being protected from spambots. You need JavaScript enabled to view it.

In molecule optics, a matter wave of molecules is manipulated by a molecule-optical component made of external, typically radiative, fields. The molecule-optical index of refraction, n, for a nonresonant IR laser pulse focused onto a molecular beam can be obtained from the energy conservation and wave properties of molecules. Experimentally measured values of n for benzene and nitric oxide agreed well with the calculated values. Since n depends on the properties of molecules as well as those of the laser field, a molecule prism composed of the focused nonresonant laser field can separate a multi-component molecular beam into several components according to their molecule-optical refractive indices n. We obtained a chromatographic resolution of 0.90 for the spatial separation of a mixture beam of benzene and nitric oxide using a focused Nd:YAG laser pulse as a molecule prism. In addition, ways of improving the efficiency of optical forces will be discussed.

 1 Babes-Bolyai University, Raluca Ripan Institute for Research in Chemistry, 30 Fantanele Street, 400294, Cluj-Napoca, Romania

2 Babes-Bolyai University, Faculty of Environmental Science and Engineering, 30 Fantanele Street, 400294, Cluj-Napoca, Romania, This email address is being protected from spambots. You need JavaScript enabled to view it.

Pharmaceuticals are micropollutants belonging to the main sources of water pollution due to their relative persistence and toxicity in the water body. These compounds are used to treat human and animal diseases and come in the surface waters from the industrial production, hospitals and incompletely metabolized drugs. During the last time an increased resistance of human and animal bodies to drugs was registered that leads to some unexpected health effects. This fact is caused by the uncontrolled input of pharmaceuticals in surface waters that determined the European Union to pay a special attention to this topic. Thus the European Parliament amending the Water Framework Directive (2000/60/EC) is preoccupied to introduce some anti-inflammatory drugs, namely diclofenac and 17beta-estradiol, in the list of emerging pollutants and to establish their maximum allowable concentration in surface waters. In this work, the authors present the results of one year monitoring (July 2014-June 2015) of some classes of pharmaceuticals (nonsteroidal anti-inflammatories, hormones, antibiotics, anticancers) widely used for the human and veterinary treatments, in Tisza River and some of its tributaries on Romanian territory. The isolation of studied pharmaceuticals from water matrices was achieved by solid-phase extraction (SPE) on Strata C18-U and Strata X cartridges. The pharmaceutical analysis was performed by high performance liquid chromatography coupled with diode array detector and/or mass spectrometer. The SPE-HPLC-DAD/MS procedures were used for the determination of the target compounds in river water samples collected from the Romanian Tisza River Watershed during four seasons. The developed procedures were validated and studies of statistical significance of the obtained results were done. Among the target pharmaceuticals found in the investigated river water samples were diclofenac and ibuprofen as anti-inflammatories; estrone and 17beta-estradiol as hormones and tetracycline, doxycycline, ceftazidime and ceftriaxone as antibiotics. The monitoring results show the importance of the study of pharmaceuticals in surface waters for a better risk assessment.

Acknowledgements: This work has been performed in the frame of NATO Science for Peace 984440/2014-2017 Project.

 Institute of Environmental Geochemistry of the National Academy of Sciences of Ukraine, Kyiv, Ukraine, This email address is being protected from spambots. You need JavaScript enabled to view it.

State pollution of rivers and other surface water reservoirs in Ukraine is under the regular control of the State. This control is carried out by the Ministry of Environment, Ministry of Health, other governmental departments and agencies. In Ukraine developed an extensive regulatory documentation, which determine the procedures waters monitoring and methods of measurement. Monitoring of water quality include the following mandatory chemical parameters as pH, the presence of ammonia nitrogen, heavy metals, content of acid residues as nitrites, nitrates, chlorides, sulfates, phosphates, oxygen content, chemical, and biological oxygen demand, phenols, surfactants. The control of organic components, such as pesticides, pharmaceuticals and others, conducted in areas where there is a threat of such pollution. Chromatographic methods primarily used in this case.

Assessment of surface water pollution is carried by a combined index - an index of pollution IWP, which is calculated by the formula :

IWP = ∑(C / MAC)/n

C – actual concentration

MAC - Maximum allowable concentration

n – number of indications

By this indicator are set 5 levels of contamination: allowable, reasonable; increased; high; very high. Most of the surface waters in Ukraine have a reasonable level of pollution. A high and very high levels of surface waters pollution is determined in the south and east of Ukraine. This is due to the presence in these areas, the mining industry and intensive agriculture. In the western regions of Ukraine, in particular in the Transcarpathian region the level of surface waters contamination is allowable. These areas belong to the recreational areas and the number of enterprises which pollute the environment on this territory is insignificant.

Acknowledgements: This work has been performed in the frame of NATO Science for Peace 984440 Project.

1 Vienna University of Technology, Institute of Chemical Technologies and Analytics, Getreidemarkt 9/164 AC, A-1060 Vienna, Austria

2 Austrian Institute of Technology, Vienna, Austria, This email address is being protected from spambots. You need JavaScript enabled to view it.

Rechargable Li-ion batteries have become an indispensable element of our daily life, as they power up laptops and mobile phones, consumer electronics, medical devices, and also electric and hybrid vehicles [1]. For electrical transportation they are typically used in much larger aggregates. The larger scale of these batteries increases the hazard arising from malfunctioning or misuse significantly, and has thus prompted detailed studies regarding the safety of Li-ion batteries and particularly their electrolytes under extreme conditions. This electrolyte, consisting of a mixture of organic carbonates with the addition of a suitable lithium salt, degrades already under conditions of normal use, but even more under extreme conditions of temperature or overcharging/overdischarging, as well as under mechanical stress, thereby forming degradation products that represent both a fire or explosion hazard, and also may be toxicologically relevant.

We report here on the chromatographic characterization of the organic electrolyte and its volatile degradation products under conditions of normal use as well as after exposure to thermal and electrochemical stress. The great variety of reaction products formed under these conditions requires the application of different chromatographic techniques for their characterization [2]. In particular, GC/MS and HPLC/MS are advantageously used for the characterization of the volatile degradation products and the electrolyte, respectively. Both an off-line and an on-line approach for the sampling of the volatile emissions from the headspace have been investigated, the latter also allowing the dynamic behavior of the emissions to be studied. Various experimental set-ups have been developed and applied to this end, and their performance and analytical potential will be presented and critically discussed [3,4].

Financial support of this work by the Austrian Research Promotion Agency (FFG) under Grant No. 835790 (SiLithium) is gratefully acknowledged.

REFERENCES:

1. J.B. Goodenough, K.-S. Park, J. Am. Chem. Soc.2013, 135, 1167-1176.
2. G. Gachot, P. Ribière, D. Mathiron, et al., Anal. Chem. 2011, 83, 478-485.
3. A. Amon, Master Thesis, Univ. of Vienna (2014).
4. C. Kanakaki, PhD Thesis, Vienna Univ. of Technology (in preparation, 2015)

 1 National University of Science and Technology "MISIS", Leninskiy prospekt 4, 119049, Moscow, Russia

2 State Scientific-Research and Design Institute of Rare-Metal Industry "Giredmet", B. Tolmachevsky lane, Building 5-1, 119017, Moscow, Russia, This email address is being protected from spambots. You need JavaScript enabled to view it.

New family of heterochain N,S-containing sorbents has been synthesized by thiomethylation of polyamins. Applicability of the sorbents for extraction and concentration of noble metals (NM) and platinum group elements (PGE) was tested in the presence of macroscopic amounts of mineral and secondary raw materials for their further study by atomic-spectral methods. Quantitative information about PGE and NM was obtained by high resolution continuum source atomic absorption spectrometry (HRCS-AAS).The properties of the sorbents were studied depending on the composition and structure, the number of complex-forming groups, solvent composition, and concentration and nature of the secondary components. In order to achieve better understanding of the sorption mechanism EXAFS investigation at Pt LIII edge was performed using ANKA synchrotron source, Karlsruhe. Preliminary data suggest that Pt atoms are sulphur bonded. Kinetics of PGE, Au, Ag sorption was studied at static conditions at room temperature and on heating. Capacity of the sorbent was established. For example, for the sorbent (S - 29.40%, N - 11.89%, C - 44.28%, H - 7.36 %) the capacity is 3 g Ag per gram of the sorbent. The sorbents were developed and tested in the analytical laboratory practice “Separation and concentration in the chemical diagnosis of functional materials and objects of the environment” of NITU MISIS and the test analytical and certification center of Giredmet Institute.

The authors gratefully acknowledge the financial support of the Ministry of Education and SRF in the framework of Increase Competitiveness Program of NUST «MISiS»(№ К1-2014-026), financial support of RFBR (13-03-00440).

 1 Babes-Bolyai University, Faculty of Environmental Science and Engineering, 30 Fantanele Street, 400294 Cluj-Napoca, Romania

2 Babes-Bolyai University, Faculty of Geography, 5-7 Clinicilor Street, 400006 Cluj-Napoca, Romania

3 Babes-Bolyai University, Raluca Ripan Institute for Research in Chemistry, 30 Fantanele Street, 400294, Cluj-Napoca, Romania, This email address is being protected from spambots. You need JavaScript enabled to view it.

Priority substances are defined according to the Water Framework Directive (WFD) as the substances identified to present a significant risk to or via the aquatic environment. These substances include 45 organic compounds from different classes and heavy metals, their determination representing the base of the characterization of the chemical status of water body.

In the frame of NATO SfP 984440 Project the authors wish to develop a methodology able to identify the River Basin Specific Pollutants from Upper Tisza. For this purpose, the analysis of the priority substances in the water body is required as a starting point.

This paper contains the results of eight months monitoring of different classes of priority substances such as pesticides, halogenated compounds, polyaromatic hydrocarbons, phthalates and heavy metals performed on river water samples collected from the Upper Tisza and its Romanian Tributaries.

For the quantification of these compounds different analytical protocols based on solid phase extraction subsequent by gas and liquid chromatography were used.

The obtained results show that the priority substances found in the studied area are p,p’-DDT, chloroform, naphthalene, anthracene, fluoranthene and benzo(b)fluoranthene as organic compounds, and lead, cadmium and mercury as heavy metals. Except of p,p’-DDT and cadmium, the concentration of the other found priority substances do not exceed the Environmental Quality Standard (EQS) requirements. Other compounds not being included on this list such as acenaphthene, fluorene, phenanthrene and pyrene have also been found.

For a better estimation of the aquatic medium contamination with priority substances, analyses of sediment and biota were carried out. Specific methods for the extraction based on Soxhlet or ultrasound assisted methods followed by gas and liquid chromatography were developed. These analyses were focused on organochlorine pesticides, polyaromatic hydrocarbons and phthalates. The obtained results confirm the presence of these compounds in the analyzed samples.

Acknowledgements: This work is performed in the frame of NATO SfP 984440 Project, Science for Peace and Security Programme.

 School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, 30, quai Ernest-Ansermet, CH-1211 Geneva, Switzerland, This email address is being protected from spambots. You need JavaScript enabled to view it.

Analysing metabolomes from various natural sources is a task that requires methods providing high chromatographic resolution for detailed metabolite profiling or high throughput for rapid fingerprinting for metabolomics [1,2]. Furthermore these methods should give on-line spectroscopic information for the identification of each individual metabolite for dereplication purposes. In this respect the introduction of UHPLC has allowed a remarkable decrease in analysis time and increase in peak capacity, sensitivity and reproducibility compared to conventional HPLC. In complement to this powerful chromatographic method, the introduction of benchtop high resolution MS instruments that provide sensitive detection and accurate MS and MS/MS information for dereplication has been key for metabolomics. Recently the development of neural network mining methods for metabolite identification have revolutionized conventional MS/MS database search for establishing link between metabolites having similar fragmentation pattern that greatly help metabolite annotation. For unambiguous de-novo identification, LC-MS targeted isolation together with at-line microNMR approaches provide low microgram level of metabolites sufficient for acquiring a full set of 1D and 2D NMR spectra. Such information complements well the search in MS and chemotaxonomic data bases for dereplication. In addition to these methods, HPLC activity-based profiling provides information on the bioactivity of metabolites of interest, directly at the analytical scale, speeding up the drug discovery process in natural product research. The impact of these technologies in NP research studies and future trends will be discussed. This will be illustrated by examples or our latest metabolomics and bioactivity-guided isolation studies on plants and microorganisms.

1. Wolfender JL, Rudaz S, Choi YH, Kim HK. Plant metabolomics: from holistic data to relevant biomarkers. Curr. Med. Chem. 2013 20: 1056-90.
2. Wolfender J-L, Marti G, Thomas A, Bertrand S. Current approaches and challenges for the metabolite profiling of complex natural extracts. J Chromatogr A 2015 1382: 136-164.

 1 National Research Council, Institute of Chemical Methodologies, Area della Ricerca di Roma 1, 00015 Monterotondo, Rome, Italy

2 Molnár-Institute Schneegloeckchenstrasse 47, 10407 Berlin, Germany, This email address is being protected from spambots. You need JavaScript enabled to view it.

Plant secondary metabolites are organic compounds produced by plants, which are not involved in the growth, photosynthesis, reproduction and other primary functions of the plants but are believed to have a role in defence mechanisms, such as protection against herbivores, pests and pathogens. Phenolic compounds are one of the main classes of secondary metabolites and because are widely distributed in the plant kingdom form an integral part of human diet. In addition to confer specific sensorial characteristics to plant-derived foods and/or beneficial effects on human health, phenolic compounds are also biomolecules with pharmacological activity employed in phytotherapeutic medicine. The identification and quantification of these target compounds in plants tissues and agro-food matrices is a challenging task, continuously requesting the development of more robust, efficient and sensitive instrumental analytical techniques. This communication discusses fundamental and practical aspects of both reversed phase high performance liquid chromatography (RP-HPLC) and capillary zone electrophoresis (CZE) tailored for the analysis of plant secondary metabolites. The communication reports the results of our recent studies carried out to evaluate the influence of various operational parameters and experimental conditions employed in CZE and in RP-HPLC on the separation performance of phenolic compounds and other secondary metabolites in plant tissues and agro-food matrices. Because both RP-HPLC and CZE require the use of a liquid phase, whose composition plays a fundamental role on the separation mechanisms and performance, most of our investigations has been focused on the selection of the proper composition of the liquid phase, which, in the case of RP-HPLC, has been optimized by a quality by design approach.

 The Weizmann Institute of Science. PO.BOX 76100, Rehovot, Israel, This email address is being protected from spambots. You need JavaScript enabled to view it.

Metabolomics and Lipidomics, involve targeted and untargeted analysis of various endogenous biomolecules, in complex samples estimated 5000-20,000 metabolites in a single plant. Thus multiple modes of analysis are needed to enable identification as well as quantitation to get a better understanding of these thousands Metabolites.

NMR, GC/MS, UHPLC/MS, USFC/MS are currently used for metabolome analysis, lately advancements in Ion Mobility separation, (a gas-phase separation tool) namely Traveling-Wave Ion Mobility (TWIM) combined with high-sensitivity mass spectrometry QTOF-MS, have been used to set the stage for a myriad of metabolomics studies, in plants and other organisms.

Here we describe the use of UHPLC - TWIM- QTOF-MS, to generated, Retention Times, Drift times, and accurate Mass. For the polar and semi polar metabolites. In addition, we used Super Critical Fluid Chromatography USFC-TWIM-QTOF-MS for the non-polar and semi polar metabolites analysis.

Plant and Algae samples data, were analyzed by a dedicated software tool, using multiple parameters such as, Retention Time, exact mass, MS/MS fragments, isotope distribution, and drift times, in order to achieve a three dimension resolution and more reliable data. That enables searching external databases, such as: LipidBlast, ChemSpider, METLIN, Elemental Composition, and our own database.

With the added parameters and the software tool, we found many more lipids in our samples, than with our previous method. We hope to verify some of these in the near future.

 1 University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Department of Chemistry and Biochemistry, Mănăstur 3-5 Cluj-Napoca, 400372, Romania

2 Università degli Studi di Messina Dipartimento di Scienze dell'Ambiente, della Sicurezza, del Territorio, degli Alimenti e della Salute, Viale F. Stagno d'Alcontres, 3198166, Messina, Italy, This email address is being protected from spambots. You need JavaScript enabled to view it.

Fruits and berries contains large amount of xanthophylls which are often esterified with various fatty acids [1]. The saponification procedure, usually performed prior chromatographic separation, also removes the acyl moieties of xanthophyll esters and unable the proper characterization of native carotenoid extracts from plant materials.

The fruits of Hippophae rhamnoides (sea buckthorn), Physalis alkekengi (Chinese lanterns) and Prunus armeniaca (apricot) fruits were used for the analysis of esterified xanthophylls. Characterization of unsaponified extracts was performed by HPLC-DAD-APCI-MS on C18 and C30 RP columns. Pure esters of zeaxanthin (Z) and β-cryptoxanthin (BCR) with myristic (M), palmitic (P), oleic (O) and linoleic (L) acids were obtained by semi-synthesis and used for identification and stability studies. The major xanthophylls in all three fruits are Z and BCR, small amounts of lutein being identified only in sea buckthorn berries. In the sepals of Physalis alkekengi Z and β-cryptoxanthin was almost completely esterified with M and P acids. Sea buckthorn berries of various cultivars were analyzed and found to contain a significantly higher number (14) of both mono- and diesters. Z was mainly esterified with P acid but esters with O and palmitoleic acids were also found. Interestingly, lutein was the only xanthophyll esterified with polyunsaturated fatty acids (L, hexadecadienoic). Apricot fruits are rich sources of β-carotene and the only important xanthophyll is BCR. Investigations on several cultivars showed a high variability in carotenoid and esters content and profile. The major fatty acids esterifying BCR are P, stearic and lauric acids but small amounts of esters with O, L and linolenic acids were also detected. Stability tests performed on pure esters and on sea buckthorn fruits showed a better stability of esterified xanthophylls compared to the free form during thermal treatment and storage.

1. This work was supported by a grant of the Romanian National Authority for Scientific Research CNCS-UEFISCDI, project PN-II-ID-PCE-2011-3-0721

References

1. Weller, P., Breithaupt, D.E., J. Agric. Food Chem., 2003, 51, 7044-7049

 1 Irish Separation Science Cluster (ISSC), Department of Chemistry and the Analytical &Biological Chemistry Research Facility (ABCRF), University College Cork, Western Road, Cork, Ireland.

2 Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec, Canada H4P2R2, This email address is being protected from spambots. You need JavaScript enabled to view it.

Increasingly nanoscale particles, pores and surfaces, are being tailor-made to provide high performance separation and detection. New superficially porous (core shell) silica particles for liquid chromatography are a good example in separation science, where nanoscale control over shell thickness enhances column efficiency. Nanoparticle incorporation in the separation and/or sensing roles, can also be an attractive means of enhancing selectivity and detectability.

In this work, sensitive and selective detection of disease indicating biomarkers is demonstrated using capillary electrophoresis with embedded gold and silica nanoparticles and amperometric detection at a boron doped diamond (BDD) electrode. The BDD electrode has emerged to play an important role in improving sensitivity in electrochemical detection. Luong et al.1 reviewed the application of the BDD electrode for sensitive detection in bioanalysis. Chen et al.2 reviewed the application of the BDD electrode for electrochemical detection in capillary electrophoresis.

By coating the fused silica capillary with gold nanoparticles (AuNPs) embedded in a cationic polymer poly (diallyldimethylammonium) chloride (PDDA), efficient separation and sensitive detection was achieved for analysis of 3-indoxyl sulphate (IXS), vanillylmandelic acid (VMA), homovanillic acid (HVA) and tryptophan (TRP) in urine3. The application of this separation and sensing approach is further applied in the field of bacterial cell-to-cell communication in the analysis of cell signalling molecules in the Pseudomonas Quinolone Signal (PQS) quorum sensing system4. P. aeruginosa is best known as an antibiotic resistant human pathogen associated with hospital-acquired infections and is the primary cause of morbidity and mortality in cystic fibrosis sufferers.

References:

1. J.H.T. Luong, K.B. Male, J.D. Glennon, Analyst, 2009, 134, 1965.
2. G. Chen, Talanta, 2007, 74, 326.
3. L. Zhou, J.D. Glennon, J.H.T. Luong, Anal Chem, 2010, 82, 6895.
4. L. Zhou, J.D. Glennon, F.J. Reen, F. O’Gara, C.M. McSweeney, G.P. McGlacken, Chem Comm, 2011, 47, 10347.

 1 Department of Chemistry, Laboratory of Analytical Chemistry, P.O. Box 55, 00014 University of Helsinki, Finland

2 Department of Engineering and Chemical Sciences, Karlstad University, 651 88 Karlstad, Sweden

3 Wihuri Research Institute, Haartmaninkatu 8, FIN-00290 Helsinki, Finland

4 National Institute for Health and Welfare, Public Health Genomics Unit, Biomedicum, Haartmaninkatu 8, FI-00251, Helsinki, Finland, This email address is being protected from spambots. You need JavaScript enabled to view it.

Although microsystems and miniaturized instrumental techniques, which can be considered key technologies for future progress in biochemistry, biotechnology, and medicine, have inspired many scientists toward further methodological developments, there continues to be a great need for analytical systems that enable dynamic and real-time monitoring of biological processes. Among many instrumental techniques and tools employed in exploring the role of biomolecular interactions in physiological and pathological phenomena, biosensors, capillary electromigration techniques, nano liquid chromatography and microscale thermophoresis enable small sample size, low reagent consumption, and label-free analysis, all of great advantage relative to more traditional approaches such as ELISA and affinity chromatography. The strength of the interactions is determined as affinity constants, partition coefficients, retention factors, and reduced mobilities.

In this talk versatility of capillary electromigration techniques, including adsorption energy calculations, that allow the differentiation of even small differences in the binding processes, will be introduced. Furthermore the exploitation of molecularly imprinted polymers in the isolation of human biomolecules will be demonstrated. Other techniques and approaches, such as microscale thermophoresis, quartz crystalline microbalance and molecular dynamics simulation calculations provide supportive and complementary insight into the interaction mechanisms.

[1] K. Lipponen, P. W. Stege, G. Cilpa, J. Samuelsson, T. Fornstedt, M.-L. Riekkola, Anal. Chem. 83 (2011) 6040.

[2] K. Lipponen, Y. Liu, P. Wanda Stege, K. Öörni, P. T. Kovanen, M.-L. Riekkola, Anal. Biochem.424 (2012) 71.

[3] K. Lipponen, S. Tähkä, M. Kostiainen, M.-L. Riekkola, Electrophoresis 35 (2013) 1106.

[4] K. Lipponen, S. Tähkä, J. Samuelsson, M. Jauhiainen, J. Metso, G. Cilpa-Karhu, T. Fornstedt, M. Kostiainen, M.-L. Riekkola, Anal. Bioanal. Chem. 406 (2014) 4137.

[5] J. Witos, J. Samuelsson, G. Cilpa-Karhu, J. Metso, M. Jauhiainen, M.-L. Riekkola,” Analyst, 2015, DOI: 10.1039/C5AN00210A.

 M. Curie-Skłodowska University, M. Curie-Skłodowska Sq.3, 20-031 Lublin, Poland, This email address is being protected from spambots. You need JavaScript enabled to view it.

Plants are unlimited source of biologically active substances used in the treatment of many human and animal diseases. They are also area of interest in searching new drugs that can be used to treat cardiovascular problems, diabetes, Alzheimer, cancer and other civilization illnesses. Because of increasing bacterial resistance against commonly used antibiotics there is also the urgent need for new antibacterials.

Chromatography is the most popular tool in the analysis of plant constituents. While high-performance liquid chromatography (HPLC) gives the biggest separation and detection possibilities, it does not provide information on biological properties of analytes. This problem can be solved by thin-layer chromatography –direct bioautography (TLC-DB) which can be used for searching biologically active substances in very complex matrices, such plants [1]. The principle of TLC-DB is that separation, biological detection (e.g. antimicrobial) and visualization are performed directly on a TLC layer [2]. In more detail, a developed TLC plate is dipped in a suspension of microorganisms growing in a nutrient broth and incubated in a humid atmosphere. The microorganisms grow directly on the surface of a TLC plate excluding spots of antimicrobials. Visualization is usually carried out by spraying a plate with tetrazolium salt, such as MTT, which is converted by living bacteria into the purple formazan. Creamy spots appearing against a colored background, so-called inhibition zones, point to the presence of antimicrobial agents. TLC-DB was used as a bio-guiding method to point substances with antibacterial activity in Matricaria recutita L., Achillea millefolium L., Salvia officinalis L., Thymus vulgaris L., Chelidonium majus L. and Hypericum perforatum L. extracts. The targeted substances, found by TLC-DB in analytical scale, were isolated in bigger amounts using TLC semi-preparative fractionation and then subjected to LC-MS analysis for structural evaluation.

[1] Marston, A. J.Chromatogr. A 2011, 1218, 2676–2683

[2] Choma, I. M.; Jesionek, W. Effects-Directed Biological Detection: Bioautography. In Instrumental Thin-Layer Chromatography; Elsevier: Amsterdam, 2014

 JLU Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany, This email address is being protected from spambots. You need JavaScript enabled to view it.

On the one hand, questions such as “Which compounds of the thousands of compounds in a complex sample are effective?” can hardly be solved with a rational effort. On the other hand, direct bioautography, which could answer such a question, led to diffuse zones and was not convincing analysts. Recently, it was shown that sharply-bounded bioactive zones were available after several hours of incubation, and in combination with targeted mass spectrometry, led to a streamlined methodology that is able to answer such effect-directed questions within 15 min per sample [1-3].

The first part of the streamlined hyphenated method is an effect-directed screening of up to 22 raw extracts in parallel. Thus, this part (HPTLC-UV/Vis/FLD-(bio)assay) can be described as a non-targeted, effect-directed detection of single or also coeluting effective compounds of the complex sample. The second part is a highly targeted characterization of the discovered effective compounds via the direct link to structure elucidating techniques (HPTLC-HRMS or NMR or ATR FTIR). The zone of interest is selectively eluted via a short orthogonal column into the HRMS or into a microvial for NMR or ATR FTIR recordings. Thus, information on effective compounds in a complex sample and their sum formulae can be obtained from a single chromatographic run.

Depending on the bioassay or enzymatic assay selected, for example, œstrogens, antibiotics, xanthine oxidase inhibitors, acetylcholinesterase inhibitors or tyrosinase inhibitors are discovered in complex samples. However, every technique has its limitation, and for oxidation-prone or volatile compounds, this streamlined methodology is limited in the information content. Nevertheless, it may serve as a survey on effect-directed components in complex samples. Benefits may result from the side-by-side sample comparison, the matrix-tolerance, the avoidance of carry over and of discrimination, the always fresh adsorbent, the comparatively low-tech workflow as well as the multifold evaluation of the separated sample saved on the plate.

Literature:

[1] G.E. Morlock, ACS Syposium Series 1185 (2013) 101-121.

[2] G.E. Morlock, I. Klingelhöfer, Anal. Chem. 86 (2014) 8289–8295.

[3] I. Klingelhöfer, G.E. Morlock, J. Chromatogr. A 1360 (2014) 288-295.

 ¹ School of Pharmacy and Applied Science, La Trobe Institute of Molecular Sciences, La Trobe University, Edwards Rd, Bendigo 3550, Australia

² Physiology Department, Medical School, Universiti Teknologi Mara, Selangor, Malaysia, This email address is being protected from spambots. You need JavaScript enabled to view it.

This study describes a simple high performance thin layer chromatographic (HPTLC) method for the quantification of apigenin, chamazulene, bisabolol and parthenolide in leaf and flower head extracts from feverfew, German chamomile and marigold and comparison of their free radical scavenging activities. Feverfew (Tanacetum parthenium) leaves have been traditionally used in the treatment of migraine with parthenolide being the main bioactive compound. However, due to similar flowers, feverfew is sometimes mistaken for the German chamomile (Matricaria recutita). Bisabolol and chamazulene are the main components in chamomile essential oil. Marigold (Calendula officinalis) was included in the study for comparison, as it belongs to the same Asteraceae or Compositae (commonly known as daisy) family.

Parthenolide was found to be present in all leaf extracts but was not detected in calendula flower extract. Chamazulene and bisabolol were found to be present in higher concentrations in chamomile and Calendula flowers. Apigenin was detected and quantified only in chamomile extracts. Antioxidant activity in sample extracts was compared by superimposing the chromatograms obtained after post chromatographic derivatization with DPPH free radical and post chromatographic derivatization with anisaldehyde. It was found that extracts from chamomile flower heads and leaves have the most prominent antioxidant activity, with bisabolol and chamazulene being the most effective antioxidants.

Molnár-Institute for Applied Chromatography, Schneeglöckchenstr. 47, 10407 Berlin, Germany, This email address is being protected from spambots. You need JavaScript enabled to view it.

Last year the Nobel Prize for Chemistry was granted to scientists, who were modeling physico-chemical interactions in biochemistry. Although we are modeling since 1985, the recognition of the importance of modeling for the whole Life Science is a great motivation for the Molnar-Institute to continue in finding the best column and the best separation in HPLC, by modeling a large number of possible chromatograms.

It is 40 years ago, that Csaba Horváth discovered in 1975 the reason for the success of Reversed Phase Chromatography at Yale – changes in the structure of water as part of the eluent. Complex calculations were required, but computers were just starting to emerge. He used a PDP11 “cabin” to describe, how acids dissociation might be modeled by equations.

Today we are modeling separation choices in a multi-variable Design Space to the most robust method for industrial drug production. With a computer generated separation model at hand, one can calculate the influence of various experimental parameters on the separation and, therefore, also model the robustness of a separation. This is extremely useful if a method should be used for a long time where one has to take into account day-to-day variations in method conditions.

The talk will demonstrate on case studies, how modeling software can work together with modern instruments to produce the best separation instead of what used to take months, in as fast as a few hours.

1 Restek Corporation, weerhaan 9, 4336KT, Middelburg, The Netherlands

2 Restek Corporation, PA, USA, This email address is being protected from spambots. You need JavaScript enabled to view it.

For several reasons, there is interest to replace helium for a different carrier gas in gas chromatography. Hydrogen is the obvious choice, but there is a concern on safety and reactivity. It is also possible to use nitrogen, but this is often not considered because it has a low optimal flow and velocity.

By using method translation and chromatogram modeling it became clear that the loss of efficiency using nitrogen could be perfectly compensated by using a smaller ID capillary of a shorter length. By replacing a 30m x 0.25 for a 20m x 0.15mm, it is demonstrated that separations under nitrogen are exactly the same as obtained with Helium, in the same analysis time, while using exact the same conditions for oven programming. Even the inlet pressures are very close.

The only price that has to be paid, is a loss in loadability, which means that this concept will not work for every application, but for many, it will. Besides the guaranteed availability, using nitrogen offers a big advantage in the cost and consumption volume of carrier gas, meaning cost per analysis will also benefit significantly.

Department of Chemistry, University of Parma, Parco Area delle Scienze 17/A, 43124 Parma, Italy, This email address is being protected from spambots. You need JavaScript enabled to view it.

Food products may come in contact with polymeric materials during processing by kitchen equipment and tableware, and storage and/or heating in plastic containers. This contact can be responsible for the migration of several compounds depending on food and plastic chemical properties, temperature and time of contact.

Polycarbonate is one of the most studied food contact material; migration of its monomer bisphenol A, and additives such as UV-absorbers or antioxidants, employed to prevent polymer degradation, has been reported to occur in food simulants (1). Ageing and light exposure are responsible for polymer degradation; besides, mechanical or chemical abrasion occurring during use and/or washing can damage the surface leading to release of oligomers and genesis of degradation products belonging to the so-called non-intentionally added substances (NIAS).

In this context, the development of suitable analytical methods to identify and to determine even low concentrations of possible contaminants is essential for safety assessment. High resolution and accurate mass spectrometry has been demonstrated to be a very promising technique in this field (2). The present study was aimed at developing and validating a method based on UHPLC coupled to Orbitrap technology to be employed for the identification and the monitoring of several compounds through a both targeted and untargeted approach. The investigation was focused on tableware of different age. Qualitative and quantitative analysis of bisphenol A and common additives employed in plastic production was carried out. Besides, the identification of colouring agents and polymer products of degradation was performed by means of the high mass accuracy. Statistical analysis permitted to correlate age, degree of surface damage, and bisphenol A migration, and allowed to discriminate new and old samples by evaluating the occurrence of a different pattern of degradation products.

References

1. Gao Y, Gu Y, Wei Y (2011) J Agric Food Chem 59, 12982.
2. Bignardi C, Cavazza A, Corradini C, Salvadeo P (2014) J Chromatogr A 1372, 133.

Institute of Chemistry, University of Silesia, 9 Szkolna Street, 40-006 Katowice, Poland, This email address is being protected from spambots. You need JavaScript enabled to view it.

Nowadays, protein and peptide analysis is on top of the research agenda of molecular biochemists, neurologists, geneticists, etc. At the same time, the enantioseparation of D/L amino acid pairs is on the one hand still a challenge and on the other, it is widely neglected. Consequently, a considerable confusion exists in various different areas of life sciences related to such issues, as homochirality of mammalian and human amino acids, geochronology making use of the amino acid clock, etc. Amino acid analysis with use of GC needs pre-column derivatization of amino acids to enhance the volatility thereof. Depending on pre-column derivatization type performed, the GC enantioseparation of amino acid pairs can be done with use of commercially available non-chiral or chiral capillary columns (i.e., in the so-called indirect or direct approach). The HPLC enantioseparation of amino acids is done mostly with use of regular non-chiral chromatographic columns (e.g., packed with C18, C8, etc.), yet upon pre-column amino acid derivatization with the Marfey’s reagent (or its analogues), to obtain respective diastereomers. For direct enantioseparation of underivatized amino acids, an absolutely limited spectrum of commercially available chiral HPLC columns is available from the market.

TLC is a flexible analytical tool which permits direct enantioseparation of amino acids, without a need to derivatize them prior to the analysis proper. Apart from commercial stationary phases used for direct enantioseparation of amino acids, a possibility exists of an in-home preparation of different chiral stationary phases and of specifically modified mobile phases which facilitate such enantioseparation. In most cases, molecular mechanism of direct amino acid enantioseparations by means of TLC is based on complexation of transition metals. Special attention will be given in this talk to selected examples of successful amino acid enantioseparations with use of TLC. In conclusion, on certain occasions direct enantioseparation of amino acids performed with aid of TLC can provide unequivocal results in shorter time and at a lower cost than the fully instrumental GC and HPLC technique.

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11000, Belgrade, Serbia, This email address is being protected from spambots. You need JavaScript enabled to view it.

Blood-Brain Barrier (BBB) plays an essential role in protecting Central Nervous System (CNS) from harmful agents present in the bloodstream. In order to estimate central nervous delivery of compounds a lot of useful in vitro models have been developed. The most used are Parallel Artificial Membrane Permeability Assay (PAMPA)[1], Biopartitioning Micellar Chromatography (BMC) [2] and cell based assays. BMC is a mode of micellar liquid chromatography that uses micellar mobile phases of Brij35 in adequate experimental conditions. It can be useful in mimicking the drug partitioning process into biological systems, because the characteristics of BMC system are similar to biological barriers and extracellular fluids. In this work, the BBB penetration of α-adrenergic/imidazoline receptor ligands and related CNS active compounds were examined using BMC system. Obtained retention factors were correlated with permeability coefficients (logPe) obtained using PAMPA [3], as well as retention factors obtained using Reversed-Phase High-Performance Liquid Chromatography (RP HPLC). Further, Partial Least Square (PLS), Multiple Linear Regression (MLR) and Artificial Neural Networks (ANN) models were developed using retention data from BMC system and molecular parameters calculated for the optimized compounds using Chem3D Ultra 9.0.1, Dragon and ADMET predictor software. Formed models could be used as time and cost efficient screening tool for evaluation of blood-brain barrier permeation of novel α-adrenergic/imidazoline receptor ligands, as promising drug candidates for treatment of hypertension or neurological diseases.

Acknowledgment

This work was supported by the Ministry for Science of the Republic of Serbia (Contract No. 172033). The COST Actions CM1103 and CM1207 are also kindly acknowledged.

References

[1] Avdeef A, 2005. The rise of PAMPA. Expert Opin. Drug Metab. Toxicol. 1, 325-342

[2] Molero-Monfort M, et al., 2000. Micellar liquid chromatography for prediction of drug transport. J. Chromatogr. A 870, 1-11

[3] Vucicevic J, et al., 2015. Prediction of BBB permeation of α-adrenergic and imidazoline receptor ligands using PAMPA technique and QSPR analysis. Eur. J. Pharm. Sci. 68, 94-105

1 Laboratory of Pharmaceutical Quality Control-UFRGS, Av. Ipiranga 2752, 90610000, Porto Alegre, Brazil

2 Faculty of Pharmacy-UFRJ, Av. do Aloízio, 27930560, Macaé, Brazil, This email address is being protected from spambots. You need JavaScript enabled to view it.

Identification of pharmaceutical degradation products (DP) used to be mostly performed by tiring and time-consuming separation/isolation techniques (e.g. preparative column and thin layer chromatography), followed by purification processes, and finally analysed by spectroscopic methods. Development of more sensitive and precise mass analysers and improvement of liquid chromatography coupled to mass spectrometry (LC-MS), outcome on elimination of isolation and fraction collection steps from DPs characterization process. Atmospheric pressure ionization, especially electrospray ionization (ESI), is the leading nebulizing method in analysis of pharmaceutical drugs. Several methods have been developed with ion trap as mass analyser but most recently, quadrupole time-of-flight has been widely employed to analyse DP mass fragments due its high resolution and measurements accuracy. This work aims to review later trends regarding characterization and identification of drug impurities upcoming from degradation processes using LC-MS, practical approach will be set throughout the presentation of studies performed by our research group. Delapril (DEL) and manidipine (MAN) hold synergic antihypertensive effects. DEL (alkaline medium) and MAN (UVA radiation) exposed to stress conditions presented three and two DPs, respectively. Identification analyses were conducted on a LC-ESI triple quadrupole MS. Another combined drug formulation studied was vildagliptine-metformine, type 2 Diabetes manager. Due to divergent physicochemical properties, two separation techniques had to be performed (reverse phase and HILIC columns) in order to identify the main DP of each compound. Anticoagulant rivaroxaban (RIV) under stress state yield emerge of three DP, although they had different structures, m/z values, and retention times, it was possible to find similarity between fragmentation patterns. LC-MS/MS technique allows reliable drug DP analysis in short time and with less chemicals, therefore reducing costs and environmental exposition. DP elucidation enables major access and knowledge as to drug stability, increasing quality control tools and safer treatments.

Acknowledgment: PPGCF-UFRGS.

Technische Univesrität München, Chair of Urban Water Systems Engineering, Am Coulombwall 8, 85748, Garching Munich, Germany, This email address is being protected from spambots. You need JavaScript enabled to view it.

Most environmental and food samples have complex matrices and contain compounds with a wide range of polarity.

Reversed phase liquid chromatography (RPLC) has been established and frequently used for the separation of various apolar (logD>1) and mildly polar (logD>0) compounds.

On the other hand, very polar compounds (logD<0) can effectively be separated by Hydrophilic interaction liquid chromatography (HILIC). This technique became more convenient since the mechanisms have been understood better. The use of either RPLC or HILIC separation technique in compound screening, generally results in a loss of a large fraction of the analytes present to a sample. The serial on-line combination of HILIC and RPLC lead to an easy, robust and relatively fast screening technique, which combined with atmospheric pressure ionization (API) and mass spectrometry (MS) constitute a unique tool in the monitoring of real samples [1-3].

The successful analysis of phenols in wine will be presented as an example for the wide separation ability of the HILIC-RP coupling [1], followed by some screening results of environmental interest. We present for the first time new impressive projects with RPLC-HILIC-API-MS resulting in highly informative screening details over a broad range of polarity.

[1] G. Greco, S. Grosse, and T. Letzel: HILIC-RP HPLC-API-ToF MS for the determination of polar and apolar organic molecules in wines. Journal of Separation Science 2013, 36 (8), 1279-1388.

[2] G. Greco and T. Letzel: The hyphenation of HILIC, RP- HPLC and API – MS. LCGC N. America and Spectroscopy supplement: Current Trends in Mass Spectrometry 2012, May, 40-44.

[3] G. Greco, S. Grosse, T. Letzel: Robustness of a method based on the serial coupling of reversed-phase and zwitterionic hydrophilic interaction LC/MS for the analysis of phenols. Journal of Separation Sciences 2014, 37 (6), 630-634.

1 “Scienze del Farmaco e Prodotti per la Salute” Department, University of Messina, viale Annunziata, 98168 – Messina, Italy

2 “Scienze dell’Ambiente, della Sicurezza, del Territorio, degli Alimenti e della Salute” Department, University of Messina, Viale F. Stagno d’Alcontres 31, 98166 Messina, Italy

3 Chromaleont s.r.l., c/o “Scienze del Farmaco e Prodotti per la Salute” Department, University of Messina, viale Annunziata, 98168 – Messina, Italy

4 University Campus Bio-Medico of Rome, Via Álvaro del Portillo, 21 - 00128 Rome, Italy", This email address is being protected from spambots. You need JavaScript enabled to view it.

Lipidomics is a branch of metabolomics and aims to study all the lipids within a living system or in complex biological samples. The abundance and differences of individual lipid molecular species in biological samples may be indicative as a clinical tool for risk assessment and disease monitoring. In this study, we developed a practical workflow for lipid profiling of biological samples by using several analytical techniques such as one- and multidimensional chromatography coupled to mass spectrometry (MS). Specifically, liquid chromatography (LC) separation is necessary for preliminary identification of lipid molecular species, because it can increase sensitivity for low abundant analytes by separating them from matrix components which might produce ion suppression; moreover, it can provide more detailed information on lipid isomers compared to direct infusion mass spectrometry. Unlike gas chromatography (GC), LC techniques offer a wide variety of separation mechanisms, thanks to a higher number of stationary phases. Different approaches in multidimensional liquid chromatography were developed for an increased resolving power compared to one-dimensional methods. The analysis of intact lipid constituents was carried out with both hydrophilic interaction liquid chromatography (HILIC) mode (for a class-type separation) and reversed-phase (RP) mode (for lipid species separation). GC analyses provided information on fatty acid profile of biological samples investigated (whole blood, plasma, erythrocyte membrane, muscle).

The main focus of this work is a lipidomic study of different biological samples, in order to perform a full characterization of lipid molecular species.

1 University of Florence, Via della Lastruccia 3 - 13, 50019 Sesto Fiorentino (Florence), Italy

2 ABSciex, Viale Lombardia, 218, 20047 Brugherio, Monza Brianza, Italy

3 Department of Plant, Soil and Environmental Science, Viale delle Idee, 30-50019 Sesto Fiorentino, Florence, Italy

4 Department of Chemistry, Via Pietro Giuria 5, 10125 Turin, Italy, This email address is being protected from spambots. You need JavaScript enabled to view it.

Vaccinium myrtillus berries (wild bilberry) are an important economic resource in various European regions, including Italian mountain areas, owing to their use as supplement of anthocyanins in the human diet. Berry anthocyanins are in fact recognized as anti-allergic, anti-inflammatory, antihypertensive, antimicrobial and anticancer molecules [1]. Recently, the presence of a different Vaccinium species, namely V. gaultherioides, has been increasingly observed in the zones traditionally populated by V. myrtillus, such as Tuscan Apennines. The phenotype of V. gaultherioides berry is very similar to the one of V. myrtillus and the two berries can be confused by the harvesters involved in the production chain of transformed bilberry. This aspect is potentially awkward since no data regarding the primary and secondary metabolic profiles of V. gaultherioides berries, including anthocyanins, are reported in literature.

For this reason, the polyphenolic composition of V. myrtillus and V. gaultherioides berries has been comparatively investigated using liquid chromatography coupled with mass spectrometry. Different stationary phases (e.g. C18 and pentafluorophenyl) and particle sizes (2.6 and 1.7 µm) have been tested in order to enhance the chromatographic selectivity. Target tandem and non-target high-resolution mass spectrometric approaches have been investigated.

Furthermore, antiradical (DPPH method) and antioxidant (FRAP method) activities have been determined for the two berry species. The results showed that the two species exhibit very different abundances of phenolic substances and antiradical/antioxidant activities. More in detail, V. myrtillus fruits had much higher contents of phenolic compounds and antiradical/antioxidant activities than those of V. gaultherioides. The anthocyanin profiles of the two species was different as well, being V. myrtillus and V. gaultherioides berries dominated by delphinidin/cyanidin glycosides, and by malvidin derivatives, respectively.

Based on these results, it is of paramount importance to carefully distinguish between the two species during the harvesting activities.

This research was funded by "PRAF 2012-2015, Misura 1.2. e)" of Regione Toscana.

Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Kralovopolska 135, 61265, Brno, Czech Republic, This email address is being protected from spambots. You need JavaScript enabled to view it.

DNA structure, its structural changes and interactions can be studied using DNA probes and markers, e. g. osmate adducts. Short synthetic oligodeoxyribonucleotides (ODNs) serve as model molecules for studies of properties of such adducts and of reactions leading to their formation [1].

For the research of ODNs derivatives it is important to study the mother ODNs first. From the existing literature (e.g. Refs. 2, 3) it is not possible to create any consistent notion of the electrophoretic behaviour of ODNs. At pH values 8 – 11, which dominate in CE separations of nucleotides and their oligomers, ODNs migrate anionically because of their surplus negative charge. This fact led to expectation that electrophoretic behaviour and properties of ODNs are determined exclusively by their negative electrophoretic charge.

The presented study was motivated by the expectation stated above. We investigated the influence of background electrolyte (BGE) pH, composition and concentration on the electrophoretic transport of short ODNs (mostly 5 – 10 nucleotide units long) in fused silica capillary. ODNs mobilities, which changed with changes in all three BGE characteristics, cannot be systematically explained even if the BGE characteristics in addition to acid-base properties of ODNs are considered. Even more complex are separation efficiency variations. Our experiments indicate that the electrophoretic properties and behaviour of short ODNs in aqueous solutions are more complex than it has been presented until now. Probably there are other processes relevant for CE separations of short ODNs. We show that separation efficiencies higher than 400 000 theoretical plates can be reached.

This work was supported by the Czech Science Foundation (grant P206/12/G151).

References:

1. Fojta M., Kostečka P., Pivoňková H., Horáková P., Havran L.: Curr. Anal. Chem. 7, 35 (2011).

2. Dolník V., Liu J., Banks F. B., Jr., Novotný M.V., Boček P.: J. Chromatogr. 480, 321 (1989).

3. McKeown A. P., Shaw N., Barrett D. A.:Electrophoresis 22, 1119 (2001).

Jagiellonian University, Department of Analytical Chemistry, Ingardena 3, 30-060 Krakow, Poland, This email address is being protected from spambots. You need JavaScript enabled to view it.

Ergot alkaloids are a group of ergoline derivatives, such as ergine (LSA), ergometrine, lysergic acid α-hydroxyethylamide (LSH), lysergol and others. Many of those substances exhibit biological activity in human, including psychedelic effects [1].

Ergot alkaloids are produced by Clavicipitaceae fungi and can be found in their plants hosts, including Convolvulaceae family, known under common name Morning Glory. Due to their hallucinogenic properties and wide, legal availability, Morning Glory cultivars are used recreationally, but with a risk of poisonings [2]. Therefore, the assessment of ergot alkaloids concentration in plants from the Convolvulaceae family is highly important for toxicological and forensic purposes.

A simple and rapid method has been developed for determination of ergine and ergometrine, and identification of other ergot alkaloids in Morning Glory seeds from as little as 10 mg of sample. For the purpose of the analysis, three extraction techniques: microwave-assisted extraction (MAE), ultrasound-assisted extraction in ultrasound bath (UAE-B) and sonotrode extraction (UAE-S) have been optimized in terms of time, temperature and solvent, using experimental design. Optimal conditions were established and it was assessed that UAE-B method was the most suitable, enabling complete extraction of ergine and ergometrine without degradation of labile analytes. The optimized extraction parameters were: 60°C, 30 min, in 10 mL of MeOH:water 7:3 (v:v). The extracts were filtrated and analysed directly by HPLC-Q-TOF-MS in scan mode for quantitative and MS/MS mode for qualitative analysis. The optimized UAE-B/LC-MS method was validated and applied for Morning Glory samples. Ergine and ergometrine were determined in samples of Ipomoea purpurea and “Morning Glory” cultivars; other alkaloids (such as LSH, chanoclavine, penniclavine, lysergol) have been identified.

[1] A. Paulke et al., “Identification of legal highs - Ergot alkaloid patterns in two Argyreia nervosa products”, Forensic Sci. Int., 242, 62–71, 2014.

[2] H. B. Klinke et al., “Two cases of lysergamide intoxication by ingestion of seeds from Hawaiian Baby Woodrose”, Forensic Sci. Int., 197, 1–5, 2010.

Department of Chemistry, Aristotle University of Thessaloniki, 54124, Thessaloniki, Greece, This email address is being protected from spambots. You need JavaScript enabled to view it.

In the present study we explore the capabilities of a universally available software, Microsoft Excel. We demonstrate that Microsoft Excel 2010 or 2013 can be used in the whole procedure of an optimization scheme in liquid chromatography. With our package we take the advantage of its use in the whole procedure of separation optimizations in HPLC for modelling multilinear gradient retention data obtained in single and double gradient elution mode by changing organic modifier(s) content and/or eluent pH. For this purpose, ten chromatographic models were used and four methods were adopted for their application. The methods were based on a) the analytical expression of the retention time, provided that this expression is available, b) the retention time estimated using the Nikitas-Pappa approach, c) the stepwise approximation, and d) a simple numerical approximation involving the trapezoid rule for integration of the fundamental equation for gradient elution. For all these methods, Excel VBA macros have been written and implemented using two different platforms; the fitting and the optimization platform. The fitting platform calculates not only the adjustable parameters of the chromatographic models, but also the accuracy of these parameters and furthermore predicts the analyte elution times. The optimization platform determines the gradient conditions that lead to the optimum separation of a mixture of analytes by using the Solver evolutionary mode, provided that proper constraints are set in order to obtain the optimum gradient profile in the minimum gradient time. Undoubtedly, this environment will be a powerful tool to the application of systematic optimization strategies by the plethora of chromatographers.

Acknowledgment

The project is realized in the framework of the Operational Program “Education and Lifelong learning” and is co-funded by the European Union (European Social Fund) and National Resources as part of the Grant Standardizing Metabolomics (Excellence II: Metabostandards 5204).

Department of Pharmaceutics, Semmelweis University, Hőgyes Endre St. 7., H-1092, Budapest, Hungary, This email address is being protected from spambots. You need JavaScript enabled to view it.

The lecture sums up the modern tools of the radiochromatography in preclinical and clinical pharmacokinetic and drug metabolism research. The essential pharmacokinetic and drug metabolism information of different species (mouse, rat, dog, rabbit and human) contribute to the final drug registration process.

The high sensitivity (pg/ml, fg/ml, at/ml) and highly selective hyphenated techniques (LC/Triple Quad-Jet Stream-ESI-MS and GC/MS-MS, etc.) required for the pharmacokinetic studies had replaced the conventional methods of detections such as GC and HPLC.

Nowadays in the course of drug development the radioactive isotopes (beta and gamma single and/or double source) labeled (³H, ¹⁴C, ⁹⁹Tc, ¹³¹I) pharmacokinetic studies combined with the new generation of triple-quad MS techniques (LC, CE, OPLC) are essential. A number of related case studies will be presented.

The former Imaging Techniques (DAR, PIT) and the new generation of in vitro – in vivo Imaging Techniques (MALDI Imaging, nanoScan, PET/MRI in animal and human studies) will also be presented.

A complex multi-step process will be illustrated from separation, purification, isolation to structure elucidation (GC-MS, LC-MS/MS, LC-NMR) of minor and major metabolites derived from animal and human biological matrices. The addition of the above systems to the off-line and on-line separation and radioactivity detection possibilities of OPLC-DAR/PIT, OPLC-RD, HPTLC-DAR-MS and GC-RD, HPLC-RD and the combined multi hyphenated techniques, OPLC-DAD-RD-MS/MS, OPLC-DAD-RD-NMR as well as LC-DAD-RD-MS/MS and LC-DAD-RD-NMR resulted in a new, flexible and rapid high-performance complex solution in the metabolism research.

1 University of Castilla-La Mancha, Campus Universitario s/n, 02071, Albacete, Spain

2 Spanish Agency for Health Protection in Sport. Calle Greco s/n. 28040 Madrid. Spain, This email address is being protected from spambots. You need JavaScript enabled to view it.

Boldenone (androsta–1,4–dien–17β–ol–3–one, Bo) is an anabolic steroid known to have been used in cattle breeding or equine sport as a doping agent for many years. Although not clinically approved for human application, Bo or its main metabolite 5β-androst-1-en-17β-ol-3-one (BoM) were detected in several doping control samples (1). The possibility of endogenous Bo production in human beings has been discussed. This is a challenging issue for doping control laboratories as Bo belongs to the list of prohibited substances of the World Anti-Doping Agency (WADA) and is usually found at low concentrations. Anti-doping laboratories must have available methods capable of detecting synthetic steroids at concentrations below 10 ng mL-1 and, at the same time, be able to quantify endogenous steroids with accuracy. When atypical concentrations of endogenous steroids are measured, a complementary analysis using gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) must be carried out and besides the unequivocal identification of the steroids must be carried out by GC-MS (2). In this communication we describe a new method base on a first separation of unacetyl-steroids by RPLC followed by on-line coupled liquid chromatography-gas chromatography (LC-GC) using the Through Oven Transfer Adsorption Desorption (TOTAD) interface and mass spectrometer (MS) detector to analyze Bo and BoM. By this means, all the analyte eluted in the LC stage is automatically transferred to the GC, avoiding the contamination associated with manipulation of the sample. A fraction collector permits the two fractions eluted in LC to be stored and sent individually to the GC by means of the TOTAD interface, once the sample is derivatized, is completely automatic. The detection limits attained, measured in GC-MS in SCAN mode, is below 0.5 ng/mL for Bo and below 0.05 ng/mL for BoM. The presented LC-GC-MS method could be used to confirm the identity of the compounds analyzed by LC-GC-C-IRMS (3).

References:

(1)          Piper, T et al. Drug. Test. Analysis, 2 (2010) 217

(2)          WADA Technical Document – TD2014IRMS

(3)          Toledano, R.M. et al. J. Chromatogr. A, 1370 (2014) 171

Toxicological Center, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium, This email address is being protected from spambots. You need JavaScript enabled to view it.

Ethylphenidate is a new psychoactive substance (NPS) that recently appeared on the drug market as an alternative for methylphenidate, and it was already found responsible of a human fatality. The identification of (human) metabolites is a necessary task, for example to confirm the consumption of a particular drug when the drug is metabolized and, sometimes, not observed in urine. The aim of this study was to investigate the in vitro Phase-I and Phase-II metabolites of ethylphenidate using human liver microsomes (HLMs) and human liver cytosol (HLCYT). The metabolites formed were identified by liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) using a quadrupole/time-of-flight (QTOF) as analyser. Six Phase-I, but no Phase-II metabolites were detected. Ethylphenidate underwent hydroxylation forming two primary mono-hydroxylated metabolites and, subsequently, dehydration and ring opening with an additional hydroxylation, forming secondary metabolites. In addition, ritalinic acid and methylphenidate were formed, but through non-enzymatic hydrolysis and subsequent methylation, and, therefore, they are not considered metabolites of ethylphenidate. The involvement of different human cytochrome P450 (CYP) enzymes in the formation of ethylphenidate metabolites showed that rCYP2C19 was the most active enzyme being involved in the formation of all the six ethylphenidate metabolites detected, although also other rCYPs (rCYP1A2, rCYP2B6, rCYPC9, rCYP2D6, and rCYP3A4, but not rCYP2E1) participated in the metabolism of ethylphenidate.

All metabolites identified in the present study are candidate specific biomarkers for monitoring studies, including toxicological screening. However, the presence of methylphenidate and ritalinic acid cannot be solely associated to ethylphenidate intake because methylphenidate is a drug itself and ritanilic acid can be formed also from methylphenidate by hydrolysis.

Acknowledgements

A-TEAM (#316665) and SEWPROF (#317205) Marie Curie Training Networks are acknowledged. N.N. and A.L.N.v.N. acknowledge the University of Antwerp and FWO Flanders, respectively, for their postdoctoral fellowships.

Faculty of Chemistry, Maria Curie-Skłodowska Univ, Pl. M. Curie Skłodowska 3, 20-031 Lublin, POLAND, This email address is being protected from spambots. You need JavaScript enabled to view it.

Determination of free drug fraction in human serum is a very important issue in pharmacokinetics research. Drugs can reversible binding with a plasma proteins (especially HAS and AGP) formation complexes. Only unbounded part of the drug can caused a therapeutic effect. There are a lot of factors influencing on the bound a given drugs with a serum proteins – so the researches are very needed. There are a lot of method for determination of free drug fraction. Some problem with a method is isolation of proteins from the analytical system. The operations can change a free drug fraction value. Liquid chromatography methods are vide used for determination of the free drug fraction in systems without proteins. We proposed use Micellar Liquid Chromatography (MLC) for determination of the free drug fraction in two cases:

Fist as a method for determination of the free form of the drug in deprived of proteins system, second as direct sample injection analysis (DSI). MLC is a chromatographic method friendly to environment due to small consuming organic solvents. Surfactants used in MLC are biodegradable. Replacement RP-LC chromatography MLC is advantageous for natural environment . In the second hand, due to ability to solubilisation of the proteins and theirs complexes in micelles MLC can be used as DSI of samples containing proteins. In presented investigations results of MLC-DSI analysis were compared with a result obtained by adsorption method. Two adsorption systems, with a two adsorbents can be tested, and free drug (ampicillin) fraction can be determined by spectrophometric, RP-HPLC and MLC method. MLC systems, with a different stationary phase (RP-8e and RP-CN) and surfactants (SDS and CTAB) were tested for DSI analysis. The system consists of CTAB and RP-8e column which proved to be the most useful in the evaluation of the concentration of the free ampicillin fraction in HSA solutions (the obtained values were the similar to the values obtained by the use of adsorption method: RP-HPLC and spectrophotometric analyses).

1 “Scienze del Farmaco e Prodotti per la Salute” Department, University of Messina, viale Annunziata, 98168 – Messina, Italy

2 Chromaleont s.r.l., c/o “Scienze del Farmaco e Prodotti per la Salute” Department, University of Messina, viale Annunziata, 98168 – Messina, Italy

3 University Campus Bio-Medico of Rome, Via Álvaro del Portillo, 21 - 00128 Rome, Italy, This email address is being protected from spambots. You need JavaScript enabled to view it.

The present research is focused on the development of a new versatile multidimensional MDGC preparative system coupled, whenever required by the complexity of the sample, to an LC pre-separation step operated in normal phase. The system demonstrated to be capable of collecting sample components in a wide range of concentrations, in a short time period, while maintaining a high degree purity for the collected components. When necessary, the LC dimension allows the injection of higher sample amounts and the transfer of simplified sub-samples to the MDGC system equipped with a programmed temperature vaporizer injector (PTV). Furthermore, thanks to the LC pre-separation step it was possible to reduce the matrix effect due to the transfer of selected LC chromatographic bands, diluted in a pure organic solvent (the LC mobile phase), characterized by low viscosity, simultaneously reducing the possibility to introduce in the GC injector and columns non-volatile components. The system versatility enhances the collection of pure components from complex samples in a green manner thanks to the reduced number of runs required. In fact, depending on the complexity, as well as to the relative concentration of the components to be collected in the whole sample,the system can be operated in LC-GC-GC or in GC-GC-GC mode. Finally, by using a commercially available collection station with a 10-position carousel it was possible to exploit a multi collection step per run.

University of Zagreb, Faculty of Chemical Engineering and Technology, Marulićev trg 19, 10000 Zagreb, Croatia, This email address is being protected from spambots. You need JavaScript enabled to view it.

A priori knowledge of the retention time of a given analyte simplifies the determination of separation conditions therefore quantitative structure retention relationship (QSRR) modelling might be considered a reasonable selection. The first problem in QSRR modelling is to select the most informative descriptors from among a large number of mutually correlated descriptors, while the second one is to build the core model of isocratic and/or gradient elution retention. A lot of conventional methods have been elaborated that are mainly based on different types of regression and simple variable selection methodology (i.e. principal component analysis), showing rather questionable prediction ability. This work reveals recent results on development of artificial intelligence (AI) hybrid methodology implementing all three AI paradigms: artificial neural networks, genetic algorithms and fuzzy logic. The developed models were fully optimized and validated with external set of compounds showing significant improvement of generalization ability. Furthermore, recent demands for increasing the productivity using the gradients, in combination with ever growing complexity of analyzed samples, are introducing an additional request on the analytical system – beside being fairly separated, the peaks are required be as “smoothly” shaped as possible to ensure their precise quantification. In other words, the analysts are becoming interested in peak shapes and peak shape modelling as well. This work also discusses recent developments is peak shape modelling based on QSRR modelling. The developed models are based on generalized logistic distribution and hybrid AI systems. The external validation results show promising predictive ability, but still indicate that there is much to be done before QSRR based optimization strategy could be efficiently built into a useful commercial software.

1 Thermo Fisher Scientific GmbH, Im Steingrund 4-6, D-63303 Dreieich

2 Leopold-Franzens University Innsbruck, Innrain 80-82, A-6020 Innsbruck, This email address is being protected from spambots. You need JavaScript enabled to view it.

HPLC applications based on capillary columns have seen an impressive progress in the last decade. As an analytical tool capillary HPLC offers the following advantages:

• Easier hyphenation with mass spectrometers
• Significant reduction of eluent consumption
• Higher mass sensitivity in comparison to conventional HPLC
• Ideal for applications with limited sample volume

Introduced in 2010, capillary ion chromatography with electrolytic eluent generation offers similar advantages, although the advantages listed above have a different significance for ion chromatography applications. The most significant advantage of ion chromatography in the capillary format is the marked reduction of eluent consumption, which allows continuous operation over a long period of time. In conventional water analysis, sample volume is usually not the issue, but users investigating corrosion phenomena or metabolic pathways are benefitting from much smaller sample volumes (<< 1 µL) used in capillary IC. The higher mass sensitivity in capillary IC in comparison to conventional IC has a huge impact on trace analysis, because the same concentration sensitivity can be achieved with much smaller sample volumes. Last but not least, capillary IC operating with very small flow rates improves the compatibility with modern mass spectrometers.

The expanded pressure tolerance of electrolytic eluent generation in capillary IC systems up to 34.5 MPa (5000 psi) allows the use of higher linear velocities of the mobile phase and thus much faster analysis times. On the other hand, it also facilitates high-resolution separations of complex samples through the use of packing materials with smaller particle sizes. In this presentation, examples of high-throughput and high-resolution separations of inorganic and organic ions from different application areas will be shown, utilizing resin-based as well as monolithic stationary phases.

Chair of Environmental Chemistry and Bioanalytics, Faculty of Chemistry & Centre for Modern Interdyscyplinary Technology Nicolaus Copernicus University, Gagarin 7, 87-100 Toruń, Poland, This email address is being protected from spambots. You need JavaScript enabled to view it.

Rapid detection and identification of microorganisms is a challenging and important aspect in many areas of our life, beginning with medicine, ending with industry. Unfortunately, classical methods of microorganisms’ identification are based on time-consuming and labor-intensive approaches. Screening techniques require rapid and cheap grouping of bacterial isolates, however modern bioanalytics demands comprehensive bacterial studies on molecular level. The new approach to the rapid identification of bacteria is to use the electromigration techniques, especially capillary zone electrophoresis (CZE). CZE is an important technique used in the analysis of microorganisms. However, the analysis of microbial complexes using this technology still encounters several problems – uncontrolled aggregation and/or adhesion to the capillary surface. One way to resolve this issue is the CZE analysis of microbial cell with surface charge modification by bivalent metal ions (e.g. Ca2+aq, Zn2+aq). Under the above conditions, bacterial cells create compact aggregates, and fewer high-intensity signals are observed in electropherograms. The capillary electrophoresis of microbial aggregates approach with UV and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) detection has been presented.

The aim of this study was to characterize the charge and surface of the bacteria in order to determine their role in adhesion and aggregation phenomena during the electrophoretic separation. The use of experimental techniques, including electrochemical and electrophoretic allowed the description of the relationship between the acid-base properties of pathogens and their behavior. In this studies was performed the identification of bacterial via spectrometric techniques.

Acknowledgments:

This work was supported by the National Science Centre (NCN, Warsaw, Poland) projects No. 2013/08/W/NZ8/701 – Symfonia-1, Maestro-6 (2015-2017) and by European Social Found, Polish National Budget, Kuyavian-Pomeranian Voivodeship Budget (within Sectoral Operational Programme Human Resources) “Krok w przyszłość 2014-2015”