teaching and learning “analytical and · pdf file“atomic absorption spectometry...

MEDICAL UNIVERSITY, SOFIA CEEPUS PROJECT H – 76 UNIVERSITY OF SOFIA, FACULTY OF CHEMISTRY, SOFIA SOUTH WEST UNIVESITY, BLAGOEVGRAD INSTITUTE OF MOLECULAR BIOLOGY, BAS

5th INTERNATIONAL SYMPOSIUM AND COURSE

TEACHING AND LEARNING “ANALYTICAL AND BIOANALYTICAL MONITORING METHODS”.

Sofia, BULGARIA

31 May – 07 June 2004

THE INTERNATIONAL SYMPOSIUM AND COURSE IS ORGANIZED UNDER THE AUSPICES OF

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THE MINISTRY OF SCIENCE AND EDUCATION OF BULGARIA

LKB

ACM-2

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PROGRAM Sunday 30.05.2004 12.00 - 18.00 19:30 Arrival

Well come party (at the “House of Scientist)

SCIENTIFIC SESSIONS Monday 31.05.2004 9:00 – 9:30 Opening Symposium 9:30 – 10:15 Prof. Dr. Marek Trojanowicz (Warsaw University, Poland)

“BIOSENSING IN HIGH-PERFORMANCE CHEMICAL SEPARATIONS”

10:15 – 11:00 Prof. Dr. Dimiter Tsalev (University of Sofia, Bulgaria) “ATOMIC ABSORPTION SPECTOMETRY – BIOLOGICAL AND ENVIROMENTAL APPLICATIONS”

11:00 – 11:30 Coffee break 11:30 – 12:15 Prof. Dr. Ferenc Kilár (University of Pécs, Hungary)

„EVOLUTION AND DEVELOPMENT OF ISOELECTRIC FOCUSING“

12:15 – 13:15 Lunch break

13:15 – 14:00 Prof. Dr. Dusan Kaniansky, M. Masár, R. Bodor, E.Ölvecká, M.Danková (Comenius University, Bratislava, Slovakia) “CAPILLARY ELECTROPHORESIS AND MINIATURIZATION”

14:30 – 15:30 Visit at the Institute of Molecular Biology – BAS (National Archeological Museum)

Tuesday

01.06.2004

9:00 – 9:45 Prof. Dr. Vasil Simeonov (University of Sofia, Bulgaria) “CHEMOMETRICS IN ANALYTICAL CHEMISTRY”


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10:30 – 11:00 Coffee break


11:45 – 12:30 Prof. Dr. Reinhold Wintersteiger, K. Eggenreich (Karl Franzens University, Graz, Austria) “DEVELOPMENT OF SENSITIVE AND SELECTIVE DRUG DETERMINATION IN BIOLOGICAL MATERIALS”


13:30 – 14:15 Prof. Dr. Dimiter Tsalev (University of Sofia, Bulgaria) “VAPOUR GENERATION ATOMIC ABSORPTION SPECTOMETRY”

14:15 – 15:00 Prof. Ewa Poboży, Jacek Musiejowski (Warsaw University, Poland) “ON-LINE PRECONCENTRATION METHODS IN CAPILLARY ELECTROPHORESIS”

15:00 – 15:30 Coffee break 15:30 – 16:15

Poster session 19:30 Wednesday

Coordination Meeting of H-76 CEEPUS Network 02.06.2004

9:00 – 9:45 Prof. Dr. Martin Schmid, G. Gübitz (Karl Franzens University Graz, Austria) “ENANTIORESOLUTION BY CAPILLARY ELECTROCHROMA-TOGRAPHY. PACKED CAPILLARIES VERSUS PARTICLE LOADED MONOLITHS”

9:45 – 10:30 Prof. Dr. Gerald Gübitz, M. Schmid (Karl Franzens University Graz, Austria) “CHIRAL SEPARATION OF AMINO ACIDS AND DIPEPTIDES BY HPLC, CE AND CEC”


11:00 – 11:45 Prof. Dr. Dobrin Svinarov (Medical University, Sofia, Bulgaria) "PHARMACOKINETIC VERSUS PHARMACODYNAMIC DRUG MONITORING: THIOPURINE METHYLTRANSFERASE MEASUREMENT BY HPLC”

11:45 – 12:30 Prof. Dr. Ernst Lankmayr, M. Gfrerer (Technikal University, Graz, Austria)

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“CONSIDERATIONS TO ORGANIC TRACE ANALYSIS”


13:30 – 14:15 Prof. Dr. Florin Irimie, Csaba Paizs, Monica Tosa, Cornelia Majdik, Paula Moldovan (Babes-Bolyai University, Cluj, Romania) “TRICKS FOR IMPROVING THE SELECTIVITY OF ENZYMATIC REACTIONS”

14:15 – 15:00 Prof. Dr. Béla Tökés (University of Medicine and Pharmacy, Targu Mures, Romania) “PERSPECTIVES IN COMBINATORIAL ELECTROCHEMISTRY”


15:30 – 16:15 16:15– 17:00

Dr. Augostin Curticapean (University of Medicine and Pharmacy, Targu Mures, Romania) “INORGANIC COORDINATION COMPOUNDS AND CAPILLARY ELECTROPHORESIS”

Dr. M. Masár, R. Bodor, E. Ölvecká, M. Danková, D. Kaniansky (Comenius University, Bratislava, Slovakia) “ON-LINE COMBINATIONS OF ELECTROPHORESIS METHODS ON A COLUMN-COUPLING CHIP”

Thursday

03.06.2004

Excursion 8:30 – Departure 10:30 –12:30

Visit at University of Blagoevgrad (Visit at the Rila Monastery)

13:00

Lunch in pub 17:00 –

Departure

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Friday

04.06.2004

9:00 – 9:45 Dr. M. Marusteri (University of Medicine and Pharmacy, Targu Mures, Romania) “APPROACHES ON SIMULATION AND MODELING IN BIOANALYTICAL FIELD”

9:45 – 10:30 Prof. Dr. Gabor Dibó (Eötvös University, Budapest, Hungary) “APPLICATION OF FLUORESCENT TAGGING IN COMBINATORIAL CHEMISTRY ”


11:00 – 11:45 Dr. Gabriella Donáth-Nagy (University of Medicine and Pharmacy at Targu Mures, Romania) “PH-OSCILLATORS AND THEIR BIOLOGICAL IMPORTANCE”

11:45 – 12:30 Dr. J. Marák, V. Vaváková, D. Kaniansky (Comenius University, Bratislava, Slovakia) “SOFTWARE VALIDATION IN CAPILLARY ELECTROPHORESIS”

12:30 – 13:30 Closing of the meeting

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LIST OF POSTER PRESENTATION 1. Annamária Bui, Zoltán Péterfi, Ildikó Kustos, Béla Kocsis, Ferenc Kilár: ANALYZATION OF 8-AMINOPYRENE-1,3,6-TRISULFONIC ACID-LABELLED HYDROLIZED ENDOTOXINS BY CAPILLARY ELECTROPHORESIS 2. I. Bacskay, A. Takátsy, A. Elfwing, A. Ballagi, F. Kilár, S. Hjertén:

POLYACRYLAMIDE GELS AS “ARTIFICIAL ANTIBODIES” AGAINST PROTEINS AND BACTERIA 3. Marek Bujdoš, Ján Medveď, Jana Kubová: SOME PROBLEMS OF THE DETERMINATION OF CADMIUM BY HYDRIDE GENERATION AAS

4. C. Vari, Silvia Imre, Daniela Muntean, Maria Dogaru: PHARMACOKINETICS OF FLAVONOIDS 5. Anna Jankowska, Magdalena Biesaga, Krystyna Pyrzyńska, Przemysław Drzewicz, Marek Trojanowicz: DETERMINATION OF PHENOXY ACID HERBICIDES USING HPLC WITH PRECONCENTRATION BY SOLID-PHASE EXTRACTION

6. Tünde Konecsni, Leopold Kremser, Ferenc Kilár, Ernst Kenndler, Dieter Blaas: SEPARATION OF VIRUS-RECEPTOR COMPLEXES WITH DIFFERENT RECEPTOR OCCUPANCY: TWELVE INDIVIDUAL RECEPTOR MOLECULES ATTACH PER VIRAL PARTICLE OF HUMAN RHINOVIRUS SEROTYPE 2 7. E. Korom, Ibolya. Kiss, M. Biesaga, M. Trojanowicz, L. Gy. Szabó, Á. Farkas, F. Kilár: DETERMINATION OF FLAVONOIDS, CHLOROGENIC ACID AND CAFFEIC ACID IN APPLE AND PEAR SAMPLES USING HPLC 8. Jana Kubová, Marek Bujdoš, Peter Matúš, Ján Medveď: CHEMICAL PARTITIONING OF COPPER, IRON, MANGANESE, LEAD AND ZINC IN SOIL AND SEDIMENT REFRENCE MATERIALS AND ACID ATTACKED SAMPLES 9. D. TSEKOVA, G. GENCHEVA, G. GOCHEV, D. MECHANDJIEV*, G. TYULIEV*, P. R. BONTCHEV*: Synthesis and structure of two stable platinum(III) complexes with hematoporphyrin IX

10. Peter Matúš, Jana Kubová: FRACTIONATION OF ALUMINIUM IN ENVIRONMENTAL SAMPLES ACIDIFIED BY MINING ACTIVITY 11. Peter Matúš, Jana Kubová, Marek Bujdoš, Ján Medveď: FRACTIONATION OF ALUMINIUM IN SOIL AND SEDIMENT REFERENCE MATERIALS AND IN ENVIRONMENTAL SAMPLES ACIDIFIED BY MINING ACTIVITY 12. Ján Medveď, Marek Bujdoš, Peter Matúš, Jana Kubová: DETERMINATION OF TRACE AMOUNTS OF GOLD BY ET AAS IN ACIDIFIED ENVIRONMENTAL SAMPLES

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13. V. Poór, S. Juricskay, A. Bufa, I. Bíró, E. Telegdy, T. Tényi, Á.Gáti, P.Osváth, F. Wilhelm: URINARY STEROID MEASUREMENTS IN SOME ENDOCRINE AND PSYCHIATRIC DISEASES 14. Dorota Szydłowska, Monica Campàs, Jean-Louis Marty, Marek Trojanowicz: PROTEIN PHOSPHATASE-BASED BIOSENSOR FOR DETERMINATION OF MICROCYSTINS

15. T. Konecsni, F. Kilár: STUDY OF THE LABELING OF HUMAN SERUM TRANSFERRIN USING FITC

16. Krystyna Pyrzyńska, Tomasz Wierzbicki: APPLICATION OF PORPHYRINS FOR PRECONCENTRATION OF VANADIUM USING SOLID-PHASE EXTRACTION 17. B. Zlateva, I. Kuleff, R. Djingova: CHEMICAL COMPOSITION OF ANCIENT RESIN OF ALEPPO PINE (PINUS HALEPENSIS): A PRELIMINARY REPORT 18. Csaba Paizs, Cornelia Majdik, Monica Toşa, Paula Moldovan, Liisa T. Kanerva, Florin Dan Irimie: CANDIDA ANTARCTICA LIPASE A IN THE DYNAMIC RESOLUTION OF NOVEL FURYLBENZOTIAZOL –BASED CYANOHYDRIN ACETATES 19. M. Masár, M. Danková, E. Ölvecká, A. Stachurová, D. Kaniansky, B. Stanislawski: DETERMINATION OF SULFITE IN WINE BY ZONE ELECTROPHORESIS WITH ON-LINE ISOTACHOPHORESIS SAMPLE PRETREATMENT ON A CHIP

20. M.Danková, S. Strašík, J. Jánošková, J. Pivovarská, D. Kaniansky: PEPTIDE MAPPING BY CAPILLARY ZONE ELECTROPHORESIS ON-LINE COUPLED WITH CAPILLARY ISOTACHOPHORESIS 21. S. Strašík, M. Danková, M. Molnárová, M. Lučaníková, D. Kaniansky: CAPILLARY ISOTACHOPHORESIS WITH DIODE-ARRAY DETECTION AND CHEMOMETRY DATA PROCESSING 22. E. Ölvecká, B. Pollák, D. Kaniansky, B. Stanislawski: SEPARATION OF PROTEINS BY ZONE ELECTROPHORESIS ON-LINE COUPLED WITH ISOTACHOPHORESIS ON A COLUMN-COUPLING CHIP WITH CONDUCTIVITY DETECTION 23. V. Vaváková, J. Marák, D. Kaniansky: COMPUTER ASSISTED CHOICE OF DISCRETE SPACERS FOR ISOTACHOPHORESIS SEPARATIONS 24. M. Spasova, G. Ivanova, T. Pajpanova, T. Milkova: SYNTHESIS OF NEW FERULOYLAMINO ACID DERIVATIVES

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L –1 BIOSENSING IN HIGH-PERFORMANCE CHEMICAL SEPARATIONS

Marek Trojanowicz

Department of Chemistry, Warsaw University, Pasteura 1, 02-093 Warsaw, Poland

[email protected]

The appropriate detector and effective procedure of sample pretreatment enabling the

elimination of matrix effects in analysis of complex samples are significant elements of each high-

performance separation methods of analysis. The application in high-performance liquid

chromatography and capillary electrophoresis of biosensors provides a possibility of improvement

of detection and in many cases allows to detected solutes non-detectable by common methods of

detection. For this purpose a homogeneous derivatization can be employed, flow-through reactors

with immobilized biomolecules or integrated enzymatic sensors or immunosensors. In gas

chromatography two area, where biosensing is employed are olfactometry and antennography. In

capillary electrophoresis numerous applications have been developed using on-line immunoassay

and also detection with a single cells.

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L - 2

ATOMIC ABSORPTION SPECTROMETRY - BIOLOGICAL AND ENVIRONMENTAL APPLICATIONS

Dimiter L. Tsalev

Faculty of Chemistry, University of Sofia, 1 James Bourchier Blvd., Sofia 1164, Bulgaria, Phone (00359-2)

8161 318, Fax (00359-2) 96 25 438, E-mail <[email protected]>

Atomic absorption spectrometry (AAS) [1] is one of the most valuable techniques in the vast

application area of biological and environmental analysis [1–6]. It is now a very well established,

reliable and cost-effective analytical tool in thousands of laboratories throughout the world for

quantitative determinations of chemical elements (up to 60–70 analytes) at trace levels - down to

nanograms (ng, 10-9 g) and picograms (pg, 10-12 g). Whereas flame AAS (FAAS) lacks sensitivity

at analyte concentrations below 0.1 and 10 µg/g in analyses of liquid and solid samples,

respectively, two modern AAS techniques with better detection power (10–1000 fold) are applied

in the lower concentration range: electrothermal atomic absorption spectrometry (ETAAS) [6] and

vapour generation AAS (VGAAS), viz. hydride generation AAS (HGAAS) and cold vapour

technique (CVAAS).

The scope, analytical methodology and applications of AAS in biological and environmental field,

its critical comparison with other methods for elemental analysis, and rational combination with

techniques for sample pretreatment, preconcentration and speciation are critically discussed. An

emphasis is placed in this overview on ETAAS, which offers potentialities for performing in situ

chemistry in the graphite atomizer by means of chemical modification [7]; its applicability to solid

or slurried microsamples, direct analysis of intact or simply diluted microsamples, high degree of

automation and other positive assets. This lecture is supplemented by numerous examples from the

author’s own research in this field, with applications to biological and environmental materials [2–

4].

More info at: <http://www.chem.uni-sofia.bg/depart/achem/staff/tsalev.htm>

References [1] Welz, B., Sperling, M. Atomic Absorption Spectrometry, 3rd edn., Wiley–VCH, Weinheim, 1998, ISBN 3-527-28571-7 [2] Tsalev, D.L., Zaprianov, Z.K. Atomic Absorption Spectrometry in Occupational and Environmental Health Practice”, Vol. I: Analytical Aspects and Health Significance, CRC Press, Boca Raton, Florida, 1983, pp. 1–252. ISBN 0-8493-5603-2 [3] Tsalev, D.L. Atomic Absorption Spectrometry in Occupational and Environmental Health Practice, Vol. II: Determination of Individual Elements, CRC Press, Boca Raton, Florida, 1984, pp. 1–295. ISBN 0-8493-5606-0 [4] Tsalev, D.L. Atomic Absorption Spectrometry in Occupational and Environmental Health Practice, Vol. III: Progress in Analytical Methodology, CRC Press, Boca Raton, Florida, 1995, pp. 1–349. ISBN 0-8493-4999-0 [5] Tsalev, D.L. Electrothermal AAS in occupational and environmental health practice - a decade of progress and establishment. Invited lecture. J. Anal. At. Spectrom., 1994, 9, 405–414 [6] Tsalev, D.L. Electrothermal AAS, in Encyclopedia of Environmental Analysis and Remediation, vol. III, Meyers, R.A. (Ed.), Wiley, New York, 1998, pp. 1583–1605 [7] Tsalev, D.L., Slaveykova, V.I. Chemical modification in ETAAS, in Advances in Atomic Spectroscopy, Vol. IV, Sneddon, J. (Ed.), JAI Press Inc., Greenwich, Connecticut, 1998, Ch. 2, pp. 27–150

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L - 3 EVOLUTION AND DEVELOPMENT OF ISOELECTRIC FOCUSING

Ferenc Kilár

Department of Analytical Chemistry, Faculty of Science and Institute of Bioanalysis, Faculty of

Medicine, University of Pécs, Pécs, Hungary

Isoelectric focusing has had a long evolutionary process. The development of this technique

was based on several original ideas and needed tremendous work. This lecture summarizes the

progression of events that led to successful preparative and analytical applications of isoelectric

focusing from the earliest “electrophoretic knowledge” until the modern theoretical advances.

A worthwhile prelude to the evolution of successful isoelectric focusing is a tracing of the

history or electrophoresis in general. “Electrophoresis” has a long history, which probably starts as

early as 600 B.C. in ancient Greece. Although there is no written record from that time, we know,

as A. Kolin – a pioneer of the technique – pointed out in his lecture about “Evolution of Ideas in

Electrophoretic Developments”1, that Thales of Miletus – generally considered to be the father of

Greek science – had knew of the special properties of amber (“electron” in ancient Greek).

Since then a lot of steps were necessary to develop the electrophoretic techniques and

especially isoelectric focusing that is a one of methods with the highest resolution.

The names that will be mentioned include Reuss, Helmholtz, Gouy, Chapman, Kohlrausch,

Tiselius, Raymond, Weintraub, Davis, Orstein, Hjertén, Ikeda, Suzuki, Kolin, Svensson (later

called Rilbe), Vesterberg, Bjellqvist, Righetti and many others who contributed to the innovative

development of this method.

Isoelectric focusing has run through a long and intensive developmental process. As it

always is with any instrumental techniques, the progress of IEF possesses maxima and minima at

times when either the technique was in favor above others or it suffered from difficulties in

effective applications. The convenience of searching electronic databases for following the number

of scientific papers published on isoelectric focusing as a “concept” from the beginning to date

allows us to view these “ups and downs”. Certainly, the citation index does not provide the entire

number of publications or even the number of cases when the technique was employed, however, it

can be useful as a measure of interest in and importance of the technique. It is apparent that after a

very short, very intensive increase in the late sixties (when the technique itself was born) and after

the renewed interest (the introduction of two-dimensional gel electrophoresis) towards the mid-

eighties, a slow decrease can be seen from the nineties. However, the overall annual production of

publications is still high.

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L - 4

CAPILLARY ELECTROPHORESIS AND MINIATURIZATION

D. Kaniansky*, M. Masár, R. Bodor, E.Ölvecká, M.Danková

Department of Analytical Chemistry, Faculty of Natural Sciences, Comenius University, Mlynská Dolina CH-2, SK-84215 Bratislava, Slovakia

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L – 5

CHEMOMETRICS IN ANALYTICAL CHEMISTRY V. Simeonov

Chair of Analytical Chemistry, Faculty of Chemistry, University of Sofia “St. Kl. Okhridski”, 1164

Sofia, J. Bourchier Blvd. 1, Bulgaria

The aim of the present lecture is to present the main multivariate statistical methods used in

various fields of Analytical and Environmental Chemistry in order to estimate, design, and

optimize analytical methods and experiments, to classify, model, and interpret analytical and

ambient datasets.

Following topics will be discussed and illustrated with case studies:

Classical statistics to evaluate errors in analytical practice with respect to accuracy,

precision (uncertainty), detection limit, sensitivity, selectivity, signal drift etc.;

Factorial designs to range and optimize analytical input factors and output signals;

Multivariate statistics in Analytical Chemistry: cluster analysis, principal components

analysis, time-series analysis, partial least squares regression, Tucker 3 modelling,

chemical mass balance modelling, QSAR and QSRR modelling etc.;

Application of chemometric and environmetric methods in real analytical studies, mostly in

the field of environmental analytical chemistry (rainwater, surface water, sea and lake

water, airborne particulate, marine sediments, soils etc.).

The duration of the course of lectures will be 3 academic hours.

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L - 6

DEVELOPMENT OF SENSITIVE AND SELECTIVE DRUG DETERMINATION

IN BIOLOGICAL MATERIALS

R. Wintersteiger, K. Eggenreich

Institute of Pharmaceutical Sciences, Karl-Franzens-University of Graz, Austria

Determination of drugs and metabolites in biological materials like plasma, serum, urine, cervical

mucus or tissue may sometimes be rather complicated due to the numerous by-products being

present in such matrix. which often may be responsible for interferences. Since furthermore the

compounds under investigation have to be determined in the nano-, pico- and even femtomol

range, highly selective and sensitive methods including efficient sample preparation techniques are

needed.

Therefore, successful strategies are discussed by means of different substances with different

functinal groups in order to analyze them in different materials by GC/MS or mainly HPLC in

combination with UV, electrochemical, fluorescence or MS detection.

Teicoplanin, a glycopeptide antibioticum, which is added prophylactically to bone cement before

application in order to minimize infections can be quantified for pharmakokinetic studies in tissue

after derivatization with fluorogenic agents. Similar techniques have been developed for the

determination of aminocresol hair dyes and its metabolites in human keratinocytes and a specific

rat liver fraction or for prostaglandins and isoprostans in human plasma which there indicate cell

demage or inflammatory processes.

As further examples the selective and sensitive quantification of clonidine, recently discovered as

an effective analgesic, in serum by GC/MS and of chlorophenoxy carboxylic acids in drinking

water by electrochemical detection following to photolysis are reported. Also an example for the

enzyme activity determination of indolamine 2,3-dioxygenase via kynurenine which is a metabolic

product of tryptophan metabolism using HPLC and UV detection is given. As pre-cleaning and

pre-concentration techniques solid-phase extraction and column switching are shown.

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L - 7 VAPOUR GENERATION ATOMIC ABSORPTION SPECTROMETRY

Dimiter L. Tsalev

Faculty of Chemistry, University of Sofia, 1 James Bourchier Blvd., Sofia 1164, Bulgaria, Phone (00359-2)

8161 318, Fax (00359-2) 96 25 438, E-mail <[email protected]>

The current state of vapour generation atomic absorption spectrometric techniques (VGAAS), in

particular hydride generation AAS (HGAAS) [1] and cold vapour technique (CVAAS) is presented

and critically discussed: scope, modern methodology, merits and limitations.

VGAAS techniques are occupying an important niche in modern analytical determinations of ppb

and sub-ppb levels of several trace elements and some of their molecular/redox species. They are

readily available, more robust and less expensive as compared with some modern, more sensitive

approaches such as atomic fluorescence spectrometry (AFS) and inductively coupled plasma mass

spectrometry (ICPMS).

An emphasis is given on some current trends in VGAAS [2,3]: (i) sample pre-treatment in

continuous flow (CF) and flow injection (FI) systems by means of automation, scaling-down and

speeding-up; (ii) intensification of sample attack (pressurised decomposition, microwave heating

(MW), on-line UV photooxidation); the automated on-line UV photolytic decomposition of some

environmentally relevant organoaresenic and organotin compounds is presented with a view to both

coupling HPLC with HGAAS detection (HPLC–UV–HGAAS) and species-independent

quantitation of the total arsenic or tin in samples containing various organic species (arsenite,

arsenate, monomethylarsonate, dimethylarsinate, arsenobetaine, arsenocholine, Sn(IV), Me2Sn,

Me3Sn, Et3Sn, Pr3Sn, Ph3Sn, BuSn, Bu2Sn, Bu3Sn, Bu4Sn); in some respect this approach is more

robust than the on-line microwave decomposition (FI-MWD–CVAAS and FI-MWD–HGAAS);

(iii) on-line enrichment by VG with in-atomizer trapping of hydrides in a heated graphite atomizer

in the presence of permanent chemical modifiers (Zr–Ir; W–Ir), the integrated platform of the

Transversely Heated Graphite Atomizer (THGA) being pre-treated with Zr (0.1–0.25 mg) or W

(0.2–0.5 mg) and then with Ir (2–20 µg) for permanent modification; (iv) speciation analysis in

flow-injection and hyphenated systems (e.g. HPLC–UV–HGAAS), etc. [2,3]. Examples from the

authors’ own research on trace element determinations (As, Bi, Cd, Hg, Sb, Se, Sn) in

environmental and biological materials (water, soils, sediments, plant, urine, etc.) [1,4] by direct

(CF-HGAAS, FI-HGAAS and CF-HGAFS) and hyphenated VG techniques (HG–ETAAS, FI-

MW–HGAAS, FI-MW–CVAAS, HPLC–UV–HGAAS) are given.

More info at: <http://www.chem.uni-sofia.bg/depart/achem/staff/tsalev.htm>

References [1] Dedina, J., Tsalev, D.L. Hydride Generation Atomic Absorption Spectrometry, Wiley, Chichester, 1995, 526 pp., ISBN 0 471 95364 4

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[2] Tsalev, D.L. Vapour generation or electrothermal atomic absorption spectrometry?—Both! Spectrochim. Acta Part B, 2000, 55, 917–933 [3] Tsalev, D.L. Hyphenated vapour generation atomic absorption spectrometric techniques. J. Anal. At. Spectrom., 1999, 14, 147–162 [4] Tsalev, D.L. Atomic Absorption Spectrometry in Occupational and Environmental Health Practice, Vol. III: Progress in Analytical Methodology, CRC Press, Boca Raton, Florida, 1995, 349 pp., ISBN 0-8493-4999-0

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L - 8

ON-LINE PRECONCENTRATION METHODS IN CAPILLARY

ELECTROPHORESIS

Ewa Poboży and Jacek Musiejowski

Department of Chemistry, Warsaw University, Pasteura 1, 02-093 Warsaw, Poland [email protected]

Enhancement of detectability of analytical response represents a major challenge in the

field of capillary electrophoresis (CE). In most common measuring systems a poor detection limits

are caused mainly by small sample injection volume and short optical path length for on-capillary

detection. Several on-line strategies have been proposed to improve detection limits such as the use

of capillaries equipped with extended detection path length (Z-shape, multi-reflection and bubble

cell), the use of more sensitive detection methods (laser-induced fluorescence and electrochemical

detection), as well as on-line preconcentration methods using solid sorbents, membranes and

microreactors.

Several techniques have been described to enhance sensitivity by on-capillary sample

concentration during or just after injection. These methods are based on the field strength

differences between the sample zone and the running electrolyte. In general these techniques are

designed to compress analyte bands within the capillary thereby increasing the volume of sample

that can be injected without loss of CE efficiency.

In this lecture two on-line sample concentration techniques, sample stacking and sweeping

in micellar electrokinetic chromatography (MEKC) will be presented. Sample stacking occurs as

ions cross the boundary that separates regions of the high electric field sample zone and low

electric field background solution zone. Sweeping is defined as the picking and accumulating of

analytes by the pseudo-stationary phase that penetrates the sample zone devoid of micelles ground

conductivity.

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L - 9

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L – 10 CHIRAL SEPARATION OF AMINO ACIDS AND DIPEPTIDES BY HPLC,

CE AND CEC

G. Gübitz, M.Schmid,

Institute of Pharmaceutical Chemistry, Karl Franzens University Graz, Universitätsplatz 1, A-8010 Graz, Austria

The enantiomers of amino acids can exhibit different biological activities. Although L-amino acids

are predominant, also D-amino acids have been found in nature and have been recently shown to

play a role in human physiology. Many amino acids have pharmacological relevance and there are

several examples where one enantiomer is pharmacologically active while the other form shows

MARUSTER side effects or even toxic effects. The detection of traces of the unwanted enantiomer

in the presence of a high excess of the other enantiomer is therefore of great importance. Amino

acids and dipeptides are building blocks of proteins. Chiral separation methods are needed for

control in peptide synthesis for racemization processes an for the investigation of protein

hydrolysates. Furthermore, the determination of amino acid enantiomer ratios is of interest with

archeologic cavations and with investigation of fossils and meteorites. The development of

methods for chiral separation has been subject of intensive studies all over the world during the

past two decades.

The aim of this lecture is to give an overview of the state of art in chiral separation of amino

acids and peptides and to present some examples of our own activities in this field. The first

approach to be discussed is the principle of ligand exchange which was applied in HPLC, CE and

CEC. We developed chiral HPLC phases on the basis of L-amino acid Cu(II) complexes

chemically bonded to silica gel. These phases showed excellent enantioselectivity for amino acids

and dipeptides. For CE, L-hydroxyproline and N-alkyl-L-hydroxyproline derivatives were used as

chiral selectors in form of their Cu(II) complexes. For CEC, new polymeric monolithic LE phases

were developed prepared by in-situ polymerization directly in the capillary. Another chiral

separation principle practised in HPLC and CE is the stereoselective inclusion of primary amines

into the cavities of chiral crown ethers. The third separation principle to be discussed is the use of

macrocyclic glycopeptide antibiotics as chiral selectors in CEC.

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L - 11 PHARMACOKINETIC VERSUS PHARMACODYNAMIC DRUG

MONITORING: THIOPURINE METHYLTRNSFERASE MEASUREMENT BY HPLC

Dobrin A. Svianrov

Laboratory of TDMCT, Alexander Hospital, Medical University, Sofia, Bulgaria Therapeutic Drug monitoring (TDM) is a multidisciplinary endeavour that links laboratory

medicine and clinical pharmacology to provide permanent improvement of patient care. The

development of TDM brings the era of its renaissance featuring the following charactreristics: 1)

The classic concept of therapeutic concentration range is supplemented by setting of new

pharmacokinetic and or pharmacodynamic targets, utilizing goal-oriented, model-based

individualization for each patient; 2) Direct links between TDM and pharmacoeconomics are

demonstrated by specific prospective cost-benefit and cost-effectiveness studies; 3) New strategies

for pharmacokinetic monitoring significantly improve patient outcome in problem fields, such as

immunosuppressive TDM; 4) Pharmacodynamic monitoring is introduced into clinical practice

with critical impact on antineoplastics, immunosuppressants, corticosteroids, and anti-HIV agents.

This new approach includes determination of enzyme activities and/or their products,

immunophenotyping, and molecular biology techniques. It will be illustrated by our recently

developed method for the determination of thiopurine methyltransferase (TPMT) activity in

isolated human erythtocites (Ery).

Genetic polymorphism of the S-methylation pathway catalysed by TPMT is known to be

responsible for variation in the metabolism, toxicity, and therapeutic efficacy of thiopurine drugs.

The method is based on a new, simple, nonradioactive HPLC procedure, based on the conversion

of 6-mercaptopurine (pH 7.5, 37°C) to 6-methylmercaptopurine (6-MMP) using S-adenosyl-L-

methionine as methyl donor. Ery lysates were prepared as previously described and incubated in

appropiate mixture for TPMT raction. The incubation step was stopped by a mixture of

trichloracetic acid/ acetonitrile containing the internal standard 4-aminoacetophenone. 6-MMP was

quantified by absorbance at 290 nm after chromatographic separation on a Zorbax SB-Phenyl

column (5 µm, 4.6x250mm) using mobile phases (flow rate 1.1 mL/min) consisting of acetonitrile,

phosphate buffer pH 3.0, triethylamine and dithiothreitol. The assay was linear up to 50 nmol/(mL

Ery*h) and the detection limit was 0.3 nmol/(mL Ery*h). The extraction efficiency of 6-MMP was

95-103% (n=3) and its analytical recovery ranged between 98.4% and 101.1% (n=12). The within-

day imprecision using pooled human erythrocytes (n=12) was 4.4% at a TPMT activity of 14.3

nmol/(mL Ery*h) and 4.9% at a TPMT activity of 6.5 nmol/(mL Ery*h). The between-day

imprecisions (n=12) were 6.8% and 7.5% nmol/(mL Ery*h), respectively. A very good agreement

was found between TPMT activity determined with this method (y) and a widely used

radiochemical procedure (x), (r=0.94; n=130; y=0.502+0.946x; p<0.05). Genotype analysis of all

21

individuals with TPMT activity under 12.5 nmol/(mL Ery*h) revealed a genotype/phenotype

correlation of 85%. The new HPLC method for determination of TPMT activity in Ery is a simple,

rapid and reliable non-radioactive procedure, which can successfully be used for both research and

routine clinical analysis. It is suitable for pharmacodynamic therapeutic drug management of

thiopurine medications.

The future of TDM is challenged by the exponential growth of pharmacogenomics and

proteomics, both of which open new horizons for full understanding of the mechanisms of

individual variability in drug therapy and drug safety.

22

L – 12 CONSIDERATIONS TO ORGANIC TRACE ANALYSIS

E. Lankmayr, M. Gfrerer

Institute for Analytical Chemistry and Radiochemistry, Technical University Graz, Austria

Due to usually complex matrix composition of bioenvironmental and bioanalytical samples, a

dedicated sample preparation is a mandatory step for organic trace analysis. In general, samples

need to be extracted from the sample matrix to obtain the analytes of interest in solution for further

analysis. A number of criteria needs to be considered for a selection of an appropriate methodology

to obtain accurate and precise results with reasonable effort. The application of high performance

extraction techniques such as fluidized bed-, accelerated solvent- or microwave assisted extraction

helps to reduce both, analysis time and solvent consumption. The advantages and disadvantages of

these advanced techniques are discussed together with some representative examples.

In order to enhance selectivity and frequently also detectability, many analytes can be transferred

into stable chemical derivatives. Also this step can be successfully enhanced and speeded up by the

dedicated use of microwave energy. The proper selection of suitable solvents with high loss

tangents and polar reagents permits for very rapid and efficient chemical reactions.

23

L – 13

TRICKS FOR IMPROVING THE SELECTIVITY OF ENZYMATIC REACTIONS

Florin Dan IRIMIE*, Csaba PAIZS, Monica TOSA, Cornelia MAJDIK and Paula

MOLDOVAN

Department of Biochemistry and Biochemical Engineering, Babeş-Bolyai University, Arany János 11, RO-3400 Cluj-Napoca, Romania; [email protected]

Driven by the increased demand for chiral drugs in enantiomerically pure form, the search for

novel methods for enantiopure compounds prepartions is a major topic in contemporary organic

synthesis. In this context, the use of biocatalysts has found widespread application in preparative

organic chemistry over the last decade.

From the three principles of biocatalytic reactions where chiral molecules are involved, i.e. (i)

transformation of (natural) chiral building blocks (ii) desymmetrization of meso- and prochiral

compounds and (iii) kinetic resolution of racemates, 5 the latter is astonishingly dominant in the

majority of applications which is probably due to the fact that meso- and prochiral substrates are

less easily synthesized than racemates. Despite its widespread application, kinetic resolution is

impeded by several inherent disadvantages for practical applications, in particular on an industrial

scale.

The most obvious drawbacks are as follows: (i) The theoretical yield of each enantiomer can never

exceed a limit of 50%. (ii) Separation of the product from the remaining substrate may be

laborious, in particular for those cases where simple extraction or distillation fails and

chromatographic methods are required. (iii) In the majority of processes, only one stereoisomer is

desired and there is little or no use for the other. In some rare cases, the unwanted isomer may be

used through a separate pathway in an enantioconvergent fashion, but this requires additional labor

and a highly flexible synthetic strategy. (iv) For kinetic reasons, the enantiomeric purity of

substrate and/or product is depleted at the point where separation of product and substrate is most

desirable from a preparative standpoint, i.e. 50% conversion. As a consequence, alternatives to

resolution techniques that can deliver a single isomer from a racemate are highly advantageous.

24

L – 14 PERSPECTIVES IN COMBINATORIAL ELECTROCHEMISTRY

Béla Tőkés

Dep. of Physical Chemistry, University of Medicine and Pharmacy, Tg-Mureş, ROMANIA

In design and synthesis of pharmaceutical compounds, beside de chemical synthesis

methods, are succesfully used numerous electrochemical ones, frequenthy more efficient compared

with the traditional procedures. This statement is the most convincing at the qualitative and

quantitative analysis of all compounds participating in the technological processes from the raw

materials to the final products. The information obtained by means of these methods contributes at

the same time towards the elucidation of the reaction mechanisms what can be utilized finally in

their optimization.

Since in the complex mechanism of electrode processes may be included one or more

chemical steps, the great majority of these transformations are chemical/electrochemical reactions.

On the other hand, in the last decades a new an very efficient branch of organic synthesis was

developped - that is the combinatorial chemistry, realized both in solution and on solid support.

The rapid development of the combinatorial chemistry is due to the high demand for new drug-like

substances triggered by emergence of high-throughput screening techniques. The authors

demonstrate [1] that the role of solide support may particularly carre out the electrode of the

electrolyser cell, too. The electrode as a reaction partner has a very complex role. Namely, besides

de electron-transfer, the orientation effect in the electrode field (about 107 V/m), the adsorbtion and

the Wien-effects are important. It is a particularity of electrochemical syntheses that on their place

of development – electrode surface – the double electric layer has a strong influence on

participating molecule state. Considerable potential gradient existing in this layer perturbes

molecules, modifying the valence angles and the atomic distances, moves the electron densities.

From standpoint of combinatorial electrochemistry especially the role of the added substances must

emphasize. The products of the electrode process can transform in the desired final product

depending on this factor, as well. Because bothe depolarizers and parteners added may be used

simultaneously in a large variety to realize combielchemically a product, the number of

compounds synthesized may be as great as it was foreseen [2]. All electrochemically active

compounds may be qualitatively and quantitatively characterized by the current-potential curves

and the optimal synthesis conditions established. The chosen compound will be synthesized on

preparative scale under these conditions. Other information, properties, and parameters may be

obtained from Hammett, Taft, Zuman, Hansch etc. type correlations. In polarography research one

of the variables is the half-wave potential. The authors have synthesized and studied in this way,

directly in polarographic cell, over 300 nitrones.

25

Nowadays, combinatorial electrochemistry is at the beginning. In the last years have been

developed some modern high-performance synthesis methods and high –throughput screening

techniques (ex the spatially addressable electrolysis platform and others). The experimental results

and their theoretical consequences are extremely promising, and open new possibilities for a

rational optimization of the studied reactions, i. e. increasing the efficiency of new drug and

chemical syntheses [3,4]. References:

1. Tőkés, B., Suciu, G., Nagy, G. Pharmazie 2002, 57, 122 ; 2. Baizer, M. M. (ed) Organic electrochemistry. New York, Marell Dekker Inc., 1973; 3. Sin, T., Li, W., Yudin, A. K. J. Comb. Chem. 2000, 2, 545 ; 4. Yudin, A. K., Sin, T. Curr. Opin. Chem. Biol. 2001, 5, 260

26

L - 15 INORGANIC COORDINATION COMPOUNDS AND CAPILLARY

ELECTROPHORESIS

Augustin CURTICAPEAN

U.M.F. Tg-Mures, Gh. Marinescu str., No. 38, Tg-Mures, ROMANIA [email protected]

Metal-Oxygen Clusters as Inorganic Coordination Complexes have specific structures but

also specific and very interesting properties. The structures building by polycondensation of

different oxoions covers a large variety of shapes with ratios characteristic for several types of

clusters: Anderson, Keggin, Dawson-Wells etc. Many properties depend not only on the structures

but also on the nature of the heteroatoms and addeada metal cations. A small “slice” from the entire

number of properties, is aimed on their electrophoretic characteristics. Using various chelating

agents there were issued interesting results and observations were published lately concerning the

out comes.

The Molybdenum blue Mo(VI)-Mo(V) method has been used for determination of inorganic

oxoanions by colorymetry [1]. Also the Keggin and Dawson-Wells structures were mainly studied

[2, 3].

Others have studied the system V(V), V(IV) by complex formation with phosphomolybdic

reagent [4]. Using CH3CN as auxiliary solvent there has been developed a sensitive capillary

electrophoresis determination for P(V) [5].

Finally, the studies were re-oriented on metal-oxygen clusters formation, especialy on

complexation of some metal cations with lacunary polyoxometalates [6].

Keywords: Metal-oxygen clusters; Capillary electrophoresis; Polyoxometalates

References 1. F., Lucena-Conde; L, Prat; Anal. Chim. Acta, 1957, 16, 473. 2. T., Hori; M., Sugiyama; S., Himeno; Analyst, 1988, 113, 1639. 3. S., Himeno; T., Ueda; H., Niiya; I., Iwai; T., Hori; Anal. Sci., 1997, 13, 369. 4. I., Kitazumi; Y., Nakashima; S., Himeno; J. Chromatogr. A, 2001, 939, 123-129. 5. Y., Nakashima; T., Goto; I., Kitazumi; S., Himeno; Electrophoresis, 2001, 22, 3377. 6. S., Himeno; N., Ishio; J. Electroanal. Chem., 1998, 451, 203.

27

L - 16

ON-LINE COMBINATIONS OF ELECTROPHORESIS METHODS ON A COLUMN-COUPLING CHIP

M. Masár*, R. Bodor, E.Ölvecká, M.Danková, D. Kaniansky


28

L – 17 APPROACHES ON COMPUTER ASSISTED SIMULATION AND

MODELING IN THE BIOMEDICAL FIELD

Marius Stefan MARUSTERI

Medical Informatics Dept.

University of Medicine and Pharmacy, Tg-Mureş, ROMANIA

This lecture will present the main concepts regarding computer assisted simulation and

modelling in the biomedical field, starting from the basic tehniques (iconic models, symbolic

models) to advanced tehniques which are able produce „real-life ” models.

An important part of the lecture will be focused on simulation and modelling examples

produced on our Medical Informatics Dept.. We developed simulations and models of W/O

interface, iconic models of evolution in a vegetal tissue on a one year period using morphing

techniques, QSAR/QSPR analisys of new compounds generated using „games-like” simulation etc.

We use those approaches to develop also new bioanalytical methods such as a new way to

control the stability of a pharmaceutical emulsion using low-cost digital image processing hardware

and software, a new method to estimate the gravity of experimental ulcer on rats using computer

assisted morphometry etc.

Because for a long time we were focused to find low-cost solutions for a computer assisted

biomedical practice and research, the lecture will present a new way to use free but powerful and

easy to configure Linux distributions, ready to run a lot of Open Source Software for biomedical

purposes (IBM Data Explorer, NAMD, VMD and a lot of bioinformatics software). We use a

special kind of bootable Linux CD which has preinstalled a lot of above mentioned software

together with a version of OpenMosix cluster solution, which allow scientists to build Linux

clusters (“cheap” supercomputers) in minutes.

References:

1. Marusteri M., Csedő C., Szocs D., Haas A. Botanical Notes 1999, XXV, 94-99. 2. Marusteri M., PhD dissertation, 2001, University of Medicine and Pharmacy Tg.

Mures 3. The Bio-Linux Project - http://envgen.nox.ac.uk/biolinux.html

29

L - 18 APPLICATION OF FLUORESCENT TAGGING IN COMBINATORIAL

CHEMISTRY

Gabor Dibó

Department of Organic Chemistry, Eötvös University, Budapest, Hungary

30

L - 19 pH-OSCILLATORS AND THEIR BIOLOGICAL IMPORTANCE

Gabriella Donáth-Nagy

Department of Physical Chemistry, Faculty of Pharmacy

University of Medicine and Pharmacy at Targu Mures, ROMANIA Gh. Marinescu Street No. 38, RO-4300 Tg-Mures

One of the most important and interesting oscillators are those in which the pH-value

changes periodically between two levels (pH-oscillators). They show oscillations in batch,

semibatch and flow conditions as well, depending on their initial composition [1,2]. A newly

discovered pH oscillator is the bromate-sulfite system, and its mechanism was thoroughly studied

[3]. In these systems the concentration of dissociated form of some ionizable drugs increase and

decrease periodically, so it may be possible to realize a periodic drug-delivery through a membrane

[4]. However, acidic drugs can attenuate the pH oscillations by a simple buffering mechanism, so

the rhythmic drug delivery has been demonstrated only under constrained circumstances.

A general scheme for achieving perodic pulsatile drug release through a membrane was

described, with application to the delivery of hormonal agents and drugs. The permeability of the

membrane to a substrate of an enzymatic reaction is assumed to be dependent on the concentration

of the product of that reaction in a manner that displays product inhibition. Under certain

conditions this negative feedback can lead to oscillations in membrane permeability to the

substrate. If the membrane permeability to a drug is simultaneously affected, then this will lead to

oscillatory drug release [5].

Rhythmic drug delivery was observed recently through a hydrogel membrane, which is

independent of external modulation or physiological stimulation [6].

Key-words: pH-oscillators, membrane oscillators, periodic drug delivery [1] Rábai, Gy., Hanazaki, I. J. Phys. Chem. 1996, 100, 10615 [2] Hanazaki, I., Rábai, Gy. J. Chem. Phys. 1996, 105, 9912 [3] Okazaki, N., Rábai, Gy., Hanazaki, I. J. Phys. Chem. 1999, 103, 10915 [4] Misra, G.P., Siegel R.A.: J. Pharm. Sci.., 2002, 91, 2003 [5] Siegel, R.A., Pitt, C.G. J. Controlled Release., 1995, 33, 173 [6] Misra, G.P., Siegel R.A.: J. Controlled Release., 2002, 81, 1

31

L - 20

SOFTWARE VALIDATION IN CAPILLARY ELECTROPHORESIS J. Marák*, V. Vaváková, D. Kaniansky

Department of Analytical Chemistry, Faculty of Natural Sciences, Comenius University, Mlynská Dolina CH-2, SK-842 15 Bratislava, Slovakia

32

P - 1

ANALYZATION OF 8-AMINOPYRENE-1,3,6-TRISULFONIC ACID-LABELLED HYDROLIZED ENDOTOXINS BY CAPILLARY

ELECTROPHORESIS

Annamária Buia, Zoltán Péterfib, Ildikó Kustosb, Béla Kocsisb, Ferenc Kilára

a Institute of Bioanalysis, Faculty of Medicine, University of Pécs, 12 Szigeti u., Pécs H-7624, Hungary

b Department of Medical Microbiology and Immunology, Faculty of Medicine, University of Pécs, Pécs, Hungary

In this work the monosaccharide-composition of three different bacterial endotoxins (obtained from

E.coli O111, Salmonella adelaide O35 and Proteus morganii bacterial strains) was examined by

capillary electrophoresis applying laser-induced fluorescence (LIF) detection. Endotoxins are

complex macromolecules present on the outer cell walls of Gram negative bacteria. They consist of

lipid and polysaccharide (that is why their alternative name is lipopolysaccharide). The

polysaccharide part gives an immundeterminant role to the molecule.

After extracting and hydrolizing the polysaccharide chains, the obtained monosaccharides were

labelled with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) fluorescent dye. The three different

samples showed individual electropherograms. The peaks were identified with spiking with known

standard monosaccharides. As several bacterial endotoxins show serological cross-linkage - which

means they have to have some of their components in common, probably some kind of sugar -, this

method can be a fast and sensitive process for examining and identifying them.

33

P - 2

POLYACRYLAMIDE GELS AS “ARTIFICIAL ANTIBODIES” AGAINST PROTEINS AND BACTERIA

I. Bacskay1,2, A. Takátsy2,3, A. Elfwing4, A. Ballagi4, F. Kilár1,3, S. Hjertén2

1Institute of Chemistry, Faculty of Science, University of Pécs, Ifjúság útja 6. H-7624 Pécs, Hungary

2Department of Biochemistry, Uppsala University, Biomedical Center P. O. Box 576, SE-751 23 Uppsala Sweden

3Bioanalytical Institute, Faculty of Medicine, University of Pécs, Szigeti út 12. H-7624 Pécs, Hungary 4Center for Surface Biotechnology, Uppsala University, Biomedical Center P. O. Box 576, SE-751 23

Uppsala Sweden Synthesis of “artificial antibodies”. Hemoglobin (Hb) is added to the monomer solution. Following

the polymerization the gel is granulated and the hemoglobin is removed. The cavity formed has

both a shape and binding sites complementary to those of the hemoglobin molecule and, therefore,

recognizes this protein, but not other proteins.

Detection of a selectively adsorbed protein by staining. Gel granules were synthesized in the

absense of Hb (blank), (2) in the presense of Hb, followed by removal of the protein by trypsin; (3)

as in (2) followed by application of Hb and washing with buffer; (4) as in (3), but albumin and

phycoerythrin were added instead of Hb. In these experiments only gel (3) could be stained with

Comassie Brillant Blue.

Conclusions: Gels can be synthesized to be selective for a given protein (in this case hemogobin);

staining is a simple detection technique.

Detection of a selectively adsorbed protein by emission spectra. One could expect the maxima of

these spectra to change upon an interaction of a protein with the polyacrylamide gel, provided that

the interaction is strong enough. In some experiments we have observed such a shift in maximum.

The intensity of the emission peaks give a good estimation of the amount of protein adsorbed.

Selectivity of artificial antibodies, as studied by capillary electrophoresis. Selectively adsorbed

bacteria make the neutral polyacrylamide gel granules negatively charged. The eletrophoretic

migration velocity is, therefore, a measure of the amount of bacteria adsorbed (Fig. 1). This

experiment indicates that polyacryamide gels can be made selective also for particles, such as E.

coli bacteria.

34

0

10

20

30

40

50

60

0 5 10 15Time (min)

Dis

tanc

e (m

m)

Fig. 1.

gel selective for E. coli

blank gel

35

P - 3 SOME PROBLEMS OF THE DETERMINATION OF CADMIUM BY

HYDRIDE GENERATION AAS

Marek Bujdoš, Ján Medveď and Jana Kubová Comenius University of Bratislava, Faculty of Natural Sciences, Geological Institute, Mlynská dolina G, 842

15 Bratislava, Slovakia, e-mail: [email protected]

Cadmium is one of the most hazardous of elements to human health. For this reason, the

determination of this element in very low concentration is very important. Direct determination of

Cd in soils and soil extracts by atomic absorption spectrometry (AAS) is difficult because the high

salt concentration, arising from the original extract and major elements extracted from soil, leads to

spectral and chemical interferences that are difficult to remove.

The generation of volatile analyte species from aqueous solutions is a specialized form of

sample introduction in atomic spectrometry. Chemical pre-separation of the analyte from matrix,

more efficient sample introduction than conventional pneumatic nebulization of solutions, good

detection power and high sensitivity are the main advantages of the technique. HG AAS with

sodium tetrahydroborate(III) reductant is the most widely used vapour generation technique.

However, the number of elements forming volatile hydrides is limited (e.g. Se, As, Sb, Bi, Ge, Te,

In, Sn). Fortunately, in recent years, the determination of cadmium by hydride generation has

become feasible (Matusziewicz et al., 1997, Zyrnicki, 2003).

The optimization of Cd determination by HG AAS was performed. Concentrations of acid

(HNO3) in sample solution, NaBH4 and NaOH in the reductant solution were carfully adjusted to

achieve best sensitivity and analytical performance. The effect of the molar ratios of [BH4⎯]/[H+]

and [BH4⎯]/[HNO3] on the Cd vapor generation efficiency was studied (Lampugnani et al, 2003).

An extensive interference study was done and the influence of Mg, Ca, Al, Mn, Fe, Zn, Cu, Ni, Pb,

Se, Sb, As, Hg, NO3⎯, SO4

2⎯ and PO43⎯ was checked. Zn2+, Cu2+, Ni2+ and Pb2+ ions were found to

interfere with CdH2 generation. The partial elimination of their negative effect was achieved by

using of KCN in the solution of reductant, but especially in case of Ni2+ and Cu2+ ions is the

improvement insufficient. The suppressive effect of EDTA was also documented. Co2+ ions ant

thiourea were checked as often recommended sensitivity improvers (Hwang and Shiuh-Jen, 1997,

Vargas-Razo and Tyson, 2000), but only thiourea had positive effect and it was used in

concentration of 0.5 % w/v in further experiments. The cobalt ions caused interference and

suppressed the cadmium absorption signal.

The suitability of HGAAS for the deremination of Cd in three commonly used soil extraction

agents (0.43 M acetic acid, 0.05 M EDTA and 0.01 M CaCl2) was successfully tested. The

procedure was then used for the determination of Cd in these extracts from different soils and soil

reference materials. The results of the procedure was compared with results of ETAAS. The

36

achieved detection limit (3σ) was 0.05 ng ml-1 and the precision (RSD of 10 replicate analyses) was

up to 15%.

Hwang, T.J., Shiuh-Jen, J. J. Anal. At. Spectrom., 1997, 12, 579. Lampugnani, L., Salvetti, C., Tsalev, D.L., Talanta, 2003, 61, 683. Matusziewicz, H., Kopras, M., Sturgeon, E. Analyst, 1997, 122, 331. Vargas-Razo, C., Tyson, JF. Fresenius J. Anal. Chem., 2000, 366, 182. Zyrnicki, W. ICP Inf. Newsl., 2003, 28, 1057. This work was supported by Science and Technology Assistance Agency under the contract No. APVT-20-042002.

37

P – 4 PHARMACOKINETICS OF FLAVONOIDS

C. Vari, Silvia Imre, Daniela Muntean, Maria Dogaru

Faculty of Pharmacy, University of Medicine and Pharmacy Tg. Mures

Gh. Marinescu 38, RO-540139 Romania Email: [email protected]

Flavonoids absorption data after oral administration are questionable, some researchers denying the

efficiency of any oral treatment with natural flavonoids because of their insufficient

biodisponibility. The aim of this study was to evaluate the absorption of quercetol after intra-

peritoneal administration of unique dose of 100 mg/kg body in rats. The plasma levels of quercetol

were quantified by a validated HPLC-UV method. The plasmatic peak appeared after 20 minutes

with cmax = 3118,6±112.9 ng/ml. Other pharmacokinetic parameters were: t1/2 = 37.3 min,

AUC0→∞=44411.9±1960.5 ng/ml min, Kel=0.018±0.0005 min-1. Three metabolites were separated

from quercetol and their peaks persisted even after quercetol was not quantifiable. The remanent

mean time of quercetol about 32 minutes is characteristic to the substances with low absorption and

distribution but with a rapid elimination. The experimental results demonstrated the low absorption

of quercetol even after intra-peritoneal administration.

Bibliography

1. Cristea A.N. Farmacologie (note de curs), Editura Medicala, Bucuresti, 2001. 2. Dobrescu D. Farmacologie practica, vol. II, Editura Medicala, Bucuresti, 1989. 3. Stroescu V. Bazele farmacologice ale practicii medicale, Editura Medicala, Bucuresti,

2001. 4. Manach C., Morand C., Demigne C., Textier O., Regerat F., Remesy C. Bioavailability of

rutin and quercetin in rats, FEBS Letters, 1997, 409, 12-16. 5. Morrica P., Marra M., Seccia S., Ventriglia M., Moro O. HPLC determination of flavone in

human serum, International Laboratory, 1998, 28(1): 6L-6P.

38

P - 5 DETERMINATION OF PHENOXY ACID HERBICIDES USING HPLC

WITH PRECONCENTRATION BY SOLID-PHASE EXTRACTION

Anna Jankowska1, Magdalena Biesaga1 , Krystyna Pyrzyńska1, Przemysław Drzewicz2,

Marek Trojanowicz 1,2

1Warsaw University, Department of Chemistry, 02-093 Warsaw, Pasteura 1

[email protected] 2Institute of Nuclear Chemistry and Technology, 03-195 Warsaw, Dorodna 16

Phenoxy acidic herbicides are widely used for control of the growth of broad-leaves weeds

in crops because of their cheapness and effectiveness. Their increasing agricultural use leads to

greater contamination of environment with these species and their toxic degradation products like

chlorophenols and phenols. For this reason there is a constant need for improvement of analytical

methods of their determination in environmental samples.

The determination of herbicides and chlorophenols in one chromatographic run was the

goal of this work. The C18 analytical column was used and spectrophotometric detection at 280

nm. The citric and acetic acid with different acetonitrile concentration were examined as possible

eluents. The best results of chromatographic separation of five phenoxy acidic herbicides dicamba,

2,4-D, 2,4-DP, 2,4,5 –T, MCPP, five chlorophenols and three other phenols were obtained with

43,7 mM acetic acid and 40% of acetonitrile. The linear range for calibration curve was from 0.05

ppm to 15 ppm. Detection limits were 20 ppb for herbicides and 0.1 ppm for chlorophenols without

preconcentration step. The comparison of solid phase extraction (SPE) procedure with C18, phenyl,

anion-exchange and polymeric sorbents have been also carried out for preconcentration of phenoxy

herbicides. Before concentration step samples were acidified. The best results were obtained on

polymeric sorbent, over 85% for every phenoxy compound.

The developed method was applied in determination of herbicides in three commercial

preparations and in river water samples. This method was also applied in determination of the

degradation products of 2,4-D, MCPA and dicamba after radiolytic treatment with increased

radiation dose.

39

P - 6 SEPARATION OF VIRUS-RECEPTOR COMPLEXES WITH DIFFERENT

RECEPTOR OCCUPANCY: TWELVE INDIVIDUAL RECEPTOR MOLECULES ATTACH PER VIRAL PARTICLE OF HUMAN

RHINOVIRUS SEROTYPE 2

Tünde Konecsni 1,2, Leopold Kremser 1, Ferenc Kilár 2, Ernst Kenndler 1, Dieter Blaas 3

1) Institute for Analytical Chemistry, University of Vienna, Vienna, Austria 2) Institute for Bioanalysis, Faculty of Medicine, University of, Pecs, Pecs, Hungary 3) Max F. Perutz Laboratories, Department of Medical Biochemistry, University of Vienna,

Vienna, Austria The crystallographic icosahedral symmetry of the human rhinovirus particle dictates the presence

of 60 identical, symmetry related surface structures that are available for antibody and receptor

binding. X-ray crystallography has indeed shown that 60 individual very-low density lipoprotein

receptor modules can bind to HRV2. Their arrangement around the five-fold axes of the virion

strongly suggested the possibility that oligomers of such a module could attach simultaneously to

several symmetry-related sites. Using an artificial recombinant concatemer of five identical

receptor modules (V3 from the very-low density lipoprotein receptor) we demonstrate that virus-

receptor complexes with different occupancy can be separated by capillary electrophoresis. This is

probably due to extensive modification of charges at the virus surface becoming masked upon

receptor binding. Extrapolation of the measured mobilities to those observed at saturation of all

binding sites indicates that indeed 12 molecules of the concatemer can bind to one single virion.

40

P - 7 DETERMINATION OF FLAVONOIDS, CHLOROGENIC ACID AND

CAFFEIC ACID IN APPLE AND PEAR SAMPLES USING HPLC E. Korom1, Ibolya. Kiss1, M. Biesaga2, M. Trojanowicz2, L. Gy. Szabó1, Á. Farkas1, F. Kilár3

1University of Pécs, Institute of Biology, Department of Botany, 7624 Pécs, Ifjúság u. 6.

2University of Warsaw, Department of Analytical Chemistry, 02-093 Warsaw, ul. Pasteur 1. 3University of Pécs, Dept. of Analytical Chemistry, 7624 Pécs, Ifjúság u. 6.

Hungary

In this study the main phenolic compounds were determined by HPLC from the fruit skin

of three apple and two pear cultivars. All samples were collected during summer and autumn in

2002, every 3 weeks. The main objective was to find the best conditions for the separation of

quercetin and quercetin-3-O-glycosides, caffeic and chlorogenic acids, and determine how their

concentration changes in the apple and pear samples.

Chlorogenic acid, caffeic acid, quercetin and quercitrin were found in all three apple

cultivars. Rutin wasn’t found, and hyperoside was present only in Jonathan apple. The amount of

chlorogenic acid, caffeic acid and quercetin during the experiment was between 0-0,1 mg/g dw,

and the changing of the dry weight was not significant. In Golden Spur the presence of the above

phenolic compounds was higher in the first two samples than later, while in Jonathan apple there

was a sudden increase in dry weight in October. Quercetin showed lower amount than the

quercetin-glicosides (quercitrin and hyperoside). Quercitrin was the highest amount component.

After the higher level of this glycoside at the beginning, there was a decrease in Idared and Golden

Spur apples, whereas a continuous increase could be observed in Jonathan. Following an increase

during the summer period, the amount of quercitrin decreased again in September in each apple

cultivar.

Chlorogenic acid, caffeic acid quercetin and quercitrin were found in Williams and

Bosckobak pears, but no rutin and hyperoside. The amount of chlorogenic acid increased between

the first and the last sample collection. In August there was a sudden decrease in both pears. The

amount of caffeic acid and quercetin did not change significantly during the collection period, but

in Bosckobak the dry weight of quercetin was higher almost ten times (0,8-0,9 mg/g dw) than in

Williams (0,1-0,2 mg/g dw). The amount of quercetin was smaller than that of its glycoside,

quercitrin. Quercitrin was the greatest amount component, similarly to apple. The curve of

quercitrin is similar in the two pear cultivars, its amount increased until mid-summer, then

decreased in late summer, early September, and finally increased again in the second half of

September.

41

P - 8 CHEMICAL PARTITIONING OF COPPER, IRON, MANGANESE, LEAD

AND ZINC IN SOIL AND SEDIMENT REFRENCE MATERIALS AND ACID ATTACKED SAMPLES

Jana Kubová, Marek Bujdoš, Peter Matúš, and Ján Medveď

Comenius University in Bratislava, Faculty of Natural Sciences, Geological Institute, Mlynská

dolina 1, 842 15 Bratislava, Slovakia

An optimised BCR three steps sequential extraction procedure (BCR SEP) and several single

extractions with KCl, NH4Cl, Na4P2O7 and 0.5 mol L–1 HCl were used for the fractionation of Al,

Cu, Fe, Mn, Pb and Zn in CRMs and in samples from a mining area with sulphidic deposits. A

good interlaboratory comparability was obtained for Cu, Pb and Zn in CRM 483, CRM 701,

SRM 2710 and SRM 2711 by BCR SEP. The reliability of the results obtained is also very

satisfactory. Some differences were found between our results and the indicative data for Al and Fe

fractionation. However, serious discrepancies were found for Mn, not only for individual steps of

the fractionation, but for the data obtained overall (sum of 1–3 steps), and for the total

concentration as well. Our results could be utilized as a contribution to the existing indicative

values for CRM 483, SRM 2710 and SRM 2711 for interlaboratory study. Moreover, data for the

fractionation of elements mentioned above for CRM 701 are first presented here.

A high correlation between 0.5 mol L–1 of HCl-extractable amounts of the elements studied, and

the sum of the three steps of BCR SEP in acid sulphatic weathering products and naturally acidified

soils was established, which allows us to suggest this rapid and cost-effective single extraction

procedure as a valuable tool in contamination assessment.

References:

Kubová, J., Streško, V., Bujdoš, M., Matúš, P., Medveď, J. Anal. Bioanal. Chem., in press.

THIS WORK WAS FINANCIALLY SUPPORTED BY THE VEGA SLOVAK GRANT AGENCY, GRANT

NO.1/0031/03.

42

P - 9 SYNTHESIS AND STRUCTURE OF TWO STABLE PLATINUM(III)

COMPLEXES WITH HEMATOPORPHYRIN IX

D. Tsekova, G. Gencheva, G. Gochev, D. Mechandjiev*, G. Tyuliev*, P. R. Bontchev*

Sofia University, Faculty of Chemistry, 1, J. Bourchier Avn., 1164 Sofia, Bulgaria *Bulgarian Academy of Sciences, G. Bontchev Avn., 1113 Sofia, Bulgaria

Rosenberg’s discovery of the cell-division inhibiting activity exerted by Cisplatin (cis-

diamminedichloroplatinum(II)) toward E. coli bacteria has initiated numerous fundamental

studies of the coordination chemistry of platinum compounds with different kind of ligands. The

major focus of this research is the synthesis of new Pt compounds with improved antitumor activity

and low toxicity. Nowadays the use of the two most widely explored chemoteraupeutic agents

Cisplatin and Oxaliplatin is confined by natural and acquired cell-resistance as well as by the side

effects, such as nephrotoxicity, gastrointestinal problems, ototoxicity etc. The strong side effects

could be limited by selective concentration of the platinum moiety in the tumor tissue. The

porphyrin ligands are promising agents for the transport of the cytostatic platinum drug into the

tumor cell. Two aspects make them suitable for cancer treatment: the marked enrichment of the

drug in the tumor cells and the phototoxic effect after irradiation with red light. Other factor of

great importance for the enhancement of antitumor activity and circumvention of the drug

resistance is the oxidation state of platinum in the antitumor drug.

In the present study we report the synthesis and structural characterization of two stable

Pt(III) complexes of hematoporphyrin IX (8,13-bis(1-hydroxyethyl)-3,7,12,17-tetramethyl-

21H,23H-porphyn-2,18-dipropanic acid). The complexes were obtained in the course of Pt(II)-

hematoporphyrin interaction in alkaline aqueous medium and aerobic conditions. The redoxy

interaction which complicates the complexation was investigated by the EPR method and

potentiometric measurements. An elongated octahedral structure with (dz2)2(dx2-dy2)1 ground state

was proposed for the first so called “sitting atop” complex cis-[Pt(III)(NH3)2(Hp-3H).2H2O].H2O.

Two of the N-atoms in the PtN4-coordination plane belong to hematoporphyrin pyrrolic cycles and

the other two come from the NH3-molecules coordinated in cis-position. Two water molecules are

located in the axial positions. The second complex [Pt(III)(Hp-3H).2H2O].H2O was isolated after

lowering of pH. In this complex Pt(III) is coordinated via four N-atoms of the porphyrin

framework. An elongated octahedral structure with two water molecules along the z-axis was

proposed for this complex. The structure of the complexes was studied by different instrumental

methods such as UV-VIS, IR, ESCA, EPR and magnetochemical measurements.

43

P – 10

FRACTIONATION OF ALUMINIUM IN ENVIRONMENTAL SAMPLES ACIDIFIED BY MINING ACTIVITY

Peter Matúš and Jana Kubová

Comenius University in Bratislava, Faculty of Natural Sciences, Geological Institute, Mlynská dolina 1, 842 15 Bratislava, Slovakia

The chelating ion-exchange was utilized for the fractionation of aluminium in acidified surface

water samples. Two chelating resins Iontosorb Oxin and Iontosorb Salicyl (cellulose resins

containing covalently bound 8-hydroxyquinoline and salicylic acid functional groups) were used

for the determination of reactive aluminium forms. These species were retained on the resins as Al

oxinate or Al salicylate complexes. The accurate time for sorption of reactive aluminium forms on

the resins were found after the study of aluminium distribution in synthetic solutions (pH range

3.0–7.0) which simulate the actual sample composition; they content some inorganic and organic

ligands. Especially the organic substances (humic and fulvic acids, low molecular weight organic

acids) have a negative influence on the reactivity of aluminium in sample. The water samples were

filtered before the sorption through 0.40 µm nitrate cellulose membrane in order to separate

insoluble aluminium species (high molecular weight organic or polymerized inorganic

compounds). The relatively high amounts of reactive Al (1–401 mg l-1; 64–89% of total soluble Al)

were found in four water samples. This fact was explained by extremely low pH (2.2–4.2) and high

EC (590–6100 µS cm-1) values and by the absence of organic matter in the samples.

The single extraction procedures with H2O and CaCl2 as extracting agent were used for

determination of active aluminium forms in acidified rock, soil, and sediment samples. The

amounts of active Al in samples ranged in the intervals 12–263 mg kg-1 (H2O extracts) and 3–346

mg kg-1 (CaCl2 extracts). The H2O extracts were filtered again through 0.40 µm nitrate cellulose

membrane. The values of active Al in CaCl2 extracts correlate better with active Al in filtered H2O

extracts.

The flame atomic absorption spectrometry FAAS and optical emission spectrometry with

inductively coupled plasma ICP OES were used for the aluminium quantification. The reliability of

the used methods was investigated. The precision of the all procedures was satisfactory (RSD 1–8

%). The accuracy of extraction procedures was checked by the use of the reference materials.

The work was financially supported by the VEGA Grant No 1/0031/03.

44

P - 11 FRACTIONATION OF ALUMINIUM IN SOIL AND SEDIMENT

REFERENCE MATERIALS AND IN ENVIRONMENTAL SAMPLES ACIDIFIED BY MINING ACTIVITY

Peter Matúš, Jana Kubová, Marek Bujdoš, Ján Medveď


The work presented describes the application of different analytical approaches for study of

aluminium mobility in rock, soil, and sediment samples affected by mining activity (secondary

quartzites with sulfidic deposits). For this purpose we used a combination of the single extractions,

the optimized BCR three-step sequential extraction procedure (SEP), and reactive aluminium

determination after chelating ion-exchange on Ostsorb (Iontosorb) Salicyl by a batch technique

with flame atomic absorption spectrometry quantification. The single extraction agents H2O, KCl,

NH4Cl, and BaCl2 were found to be the best for the quantitative estimation of the aluminium

mobility in rocks, soils, and sediments caused by acidification of the environment. This fact was

confirmed by reactive aluminium determination in the same samples. The vast majority of the

aluminium content of samples after application of the optimized BCR three-step SEP is in the

residues. The available fraction of aluminium extracted by dilute CH3COOH in the first step of this

procedure correlates with the reactive aluminium content. The amounts of aluminium released in

the second and the third steps and the sums from steps 1–3 of this procedure are closely associated

with the aluminium content values obtained by the single dilute HCl leach. The accuracy of results

obtained was verified with only informative values for individual fractions of the BCR three-step

SEP because of the absence of suitable certified or standard reference materials. The amounts of

the reactive aluminium determined in samples was in the range 12–82% of total soluble Al in the

filtered H2O extracts. It was confirmed that the acidified polluted samples contain the most of

reactive Al content, which is responsible for its toxicity.

References:

Matúš, P., Kubová, J., Bujdoš, M., Streško, V, Medveď, J. Anal. Bioanal. Chem., in press.

The work was financially supported by the VEGA Grant No 1/0031/03.

45

P - 12 DETERMINATION OF TRACE AMOUNTS OF GOLD BY ET AAS IN

ACIDIFIED ENVIRONMENTAL SAMPLES

Ján Medveď, Marek Bujdoš, Peter Matúš, and Jana Kubová


A method for determination of trace amounts of gold in environmental samples (rocks, soils,

sediments, and waters) by atomic absorption spectrometry with electrothermal atomization (ET

AAS) after preconcentration using a chelating sorbent Spheron Thiol 1000 is described. The

method accurately determines gold between 0.001 and several tens of grams per ton in samples

having complex variations in mineralogy. Pulverized samples are roasted at 650°C to oxidize any

sulfide and/or carbonaceous material. Samples are then subjected to a series of acid treatments to

eliminate any silica matrix and to dissolve the sample. The Spheron Thiol 1000 is added to the

sample solution, and then with sorbed gold is filtered out, washed, and ignited at 550°C. The

residue is dissolved in aqua regia, evaporated, dissolved in distilled water, transferred to a

volumetric flask, and analyzed by ET AAS.

The limits of detection of gold, based on the 3 σ definition, were 0.5 ng g–1 for 10-g samples

(rocks, sediments, soils) and 0.05 ng mL–1 for 1-L water samples. Precision of determination

expressed by the relative standard deviation varied from 2.9% to 16.4%. The accuracy of the

method is verified by analysis of certified reference materials. The obtained analytical results are in

good agreement with attested values. The developed method was applied for gold determination in

environmental samples affected by the acidification (acid mine drainage which is mainly a product

of pyrite oxidation) from an open quartzite mine in the Šobov region situated NE of the city of

Banská Štiavnica (Slovakia).

References: Medveď, J., Bujdoš, M., Matúš, P., Kubová, J. Anal. Bioanal. Chem., in press.

This work was financially supported by the VEGA Slovak Grant Agency, Grant

No.1/0047/03.

46

P - 13 URINARY STEROID MEASUREMENTS IN SOME ENDOCRINE AND

PSYCHIATRIC DISEASES V. Poór(1), S. Juricskay(1), A. Bufa(1), I. Bíró(1), E. Telegdy(2), T. Tényi(3), Á.Gáti(3), P.Osváth(3),

F. Wilhelm(4)

(1) Institute of Bioanalysis, Faculty of Medicine, University of Pécs 12 Szigeti u., Pécs, Hungary, H-7643, Tel: +36/72-536-438, Fax: +36/72-536-254, [email protected]

(2) Department of Dermatology, Pécs University Medical School, 20 Kodály Zoltán u. Pécs, Hungary H-7624 (3) Department of Psychiatry and Medical Psychology, Faculty of Medicine, University of Pécs, Hungary

(4) Department of Obstetrics and Gynecology, Faculty of Medicine, University of Pécs 17 Édesanyák útja, Pécs, Hungary, H-7624

In 1990 the worldwide accepted Shackleton method, which provides a possibility of

determining the steroid metabolites from urine after solid extraction, enzyme hydrolysis and

methoxim-silyl derivatization with capillary gas chromatography were adopted in our laboratory.

Using three internal standards, programmed temperature from 50 oC to 300 oC, and FID detection,

on ULTRA-1 capillary column, the separation of 28 steroid components is possible. The procedure

is very useful in the diagnosis making of dysfunction or absence of enzymes with an important role

in steroid metabolism, and control of the treatment in patients with these diseases, or in the

diagnosis making in different diseases, such as hirsutismus, polycystas ovarium syndrome (PCOS),

androgen alopecia (AA), pubertas disorders etc.

Besides the proximate clinical application, the method gives a convenient tool to study the

steroid background of these disorders, helping us understand the mechanism of their development.

Not only the concentrations of the steroid components, but also the ratios of certain metabolites

give information on the function of central enzymes in the steroid metabolism.

In the last few years we have examined the steroid profile of patients with hair problems (AA

in women and men, effluvium /E/ in women) and psychiatric problems (major depression /MD/ in

women and men), and osteoporosis (/OP/ in women).

The elevation of certain steroid metabolites in women with AA and E refers to the increased

steroid secretion of not only the adrenal gland, but also of the end organs (human scalp hair

follicles). In all of the 3 examined hair loss disease increased 5α-reductase has been found.

We could observe the alteration of the activity of 11β-hydroxylase enzyme and marked

gender differences in the changes of the steroid metabolism in patients with MD.

In patients with OP the significantly decreased level of certain metabolites points to the role

of testosterone, androstenedione and DHEA in postmenopausal bone loss in women.

Our experiences contribute to the knowledge of the nature and steroid background of some

endocrine and psychiatric diseases.

References 1. F., Lucena-Conde; L, Prat; Anal. Chim. Acta, 1957, 16, 473. 2. T., Hori; M., Sugiyama; S., Himeno; Analyst, 1988, 113, 1639.

47

3. S., Himeno; T., Ueda; H., Niiya; I., Iwai; T., Hori; Anal. Sci., 1997, 13, 369. 4. I., Kitazumi; Y., Nakashima; S., Himeno; J. Chromatogr. A, 2001, 939, 123-129. 5. Y., Nakashima; T., Goto; I., Kitazumi; S., Himeno; Electrophoresis, 2001, 22, 3377. 6. S., Himeno; N., Ishio; J. Electroanal. Chem., 1998, 451, 203.

48

P - 14 PROTEIN PHOSPHATASE-BASED BIOSENSOR FOR

DETERMINATION OF MICROCYSTINS

Dorota Szydłowska 1, Monica Campàs 2, Jean-Louis Marty 2 and Marek Trojanowicz 1

1)Department of Chemistry, Warsaw University, Pasteura 1, 02-093 Warsaw, Poland [email protected]

2) University of Perpignan, BIOMEM group, Perpignan, France

Microcystins are cyclic heptapeptide compounds produced by cyanobacteria in lakes,

ponds and reservoirs. Due to their hepatotoxicity, the World Health Organisation (WHO) has

proposed a value of 1µg/L microcystin-LR as acceptable level in drinking water. Conventional

detection techniques are based on bioassays and chromatographic methods, the disadvantages being

the non-specificity and low sensitivity for the former, and the non-indication of the toxicity and the

requirement for skilled personnel for the latter. Microcystins are potent inhibitors of

serine/threonine phosphatases, such as protein phosphatase 1 (PP1) and protein phosphatase 2A

(PP2A). This inhibitory power provides the tool for the enzymatic approach.

For this purpose, a protein phosphatase has been produced by molecular engineering and

its performance has been compared to that of commercial PP2A. Three methods have been used to

immobilise the enzyme on the support: entrapment by sol-gel or photocrosslinkable polyvinyl

alcohol bearing styrylpyridium groups (PVA-SbQ), and covalent linking by glutaraldehyde. The

PVA-SbQ immobilisation has provided the highest immobilisation yields and enzyme stability

values, much higher than those of the free enzyme in solution. The carbon screen-printed

electrodes, ELISA plates and membranes have been used as immobilisation supports in order to

develop both the colorimetric and the electrochemical systems.

Colorimetric enzymatic assays have demonstrated the viability of the approach, the protein

phosphatase being able to detect both Microcystin-LR and Microcystin-RR variants with

appropriate detection limits and linear ranges. For the electrochemical sensing, several

phosphorylated substrates have been designed and synthesised. These substrates are

electrochemically active only after their dephosphorylation by protein phosphatase.

49

P - 15

STUDY OF THE LABELING OF HUMAN SERUM TRANSFERRIN USING FITC

T. Konecsni, F. Kilár

Inst. of Bioanalysis, Fac. of Medicine, Univ. of Pécs, Szigeti út 12., 7624 Pécs, Hungary, Tunde.

[email protected] Fluorescently labeled proteins are used in wide range to investigate several biological problems. In

cases when specific proteins are needed and their fluorescent derivatives are not commercially

available, the researcher has to do the labeling. Most of the fluorescent probes react with accessible

primer amines exposed on the protein surface. The distribution and accessibility of these amino

groups differ from protein to protein. However, general protocols for getting optimally stained

conjugate are available. The conditions of labeling should be optimal for keeping the biological

activity of the protein and also effective enough for further procedures. The derivatization depends

on several conditions, e.g., pH of the buffer, concentration of protein and dye, the molar excess of

dye to protein, etc. Human serum transferrin stained with FITC serves as a good model to

investigate the labeling mechanism of amine reactive dyes using different dye to protein ratios

because the derivatized protein can be separated well from the excess of FITC by capillary

electrophoresis without previous purification step. Iron free transferrin samples in the range of

0.125 µM to 0.125 mM were labeled in the presence of 1, 0.1 and 0.01mM FITC. UV, broadband

fluorescent and laser induced fluorescent detection was applied for evaluation. The time

dependence of the derivatization can be determined by CE. Increasing the degree of labeling the

mobility of the labeled protein decreases. The higher is the dye to protein ratio in the reaction

mixture, the more effective is the labeling. measured with LIF detector. An estimation for the dye

to transferrin ratio can be obtained by quantitative evaluation of the labeled protein peak and was

found to be 1-3 FITC per protein molecule using FITC in molar excess of 5 to 120 in the labeling

reaction. Successful staining was possible with a protein concentration of 10 µg/ml = 0.125 µM.

The limit of detection for highly derivatized samples was 10 ng/ml = 0.125 nM protein.

50

P - 16

APPLICATION OF PORPHYRINS FOR PRECONCENTRATION OF VANADIUM USING SOLID-PHASE EXTRACTION

Krystyna Pyrzyńska and Tomasz Wierzbicki

Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland

[email protected]

Determination of vanadium is of great importance in biological, geochemical and

environmental studies. However, its low concentration in most natural samples and high content of

matrix components very often requires the application of preconcentration and/or techniques prior

to the detection. Recently, great attention has been devoted to application of solid-phase extraction

for the enrichment processes due to simple equipment involved and the possibility to applied on-

line sample pretreatment. Development of the new ion-exchange resin for metal preconcentration is

still interesting research purpose. The sorbents with specific functional groups for their selectivity

are preferred for this purpose.

In this study porphyrins, naturally occurred macrocyclic compounds, were used for

modification of commercially available sorbents in order to obtain a higher selectivity and better

kinetics ion-exchange reaction. Tetra(4-carboxyphenylo)porphyrin (TCPP) was examined as ligand

for metal complexes, which were retained on anionic polymeric resin IRA 904 and silicagel. For

comparison of results two set of experiments were carried out with sorption of metals on solid

phase modified with TCPP and sorption of previously prepared V-TCPP complexes on non-

modified sorbent.

51

P - 17

CHEMICAL COMPOSITION OF ANCIENT RESIN OF ALEPPO PINE (PINUS HALEPENSIS): A PRELIMINARY REPORT

B. Zlateva, I. Kuleff, R. Djingova

Department of Analytical Chemistry, Faculty of Chemistry, University of Sofia 1, J. Bouchier str., Sofia 1164, Bulgaria

The resin of Aleppo pine (Pinus Halepensis) was an important source of preservation material for

ancient Greece. Adding resin to wine is a process that dates back to antiquity when either pitch,

pine resin or a combination of plaster and resin was used to make impermeable clay vases

(amphorae, pitoses) in which wine was transported vie sea. Ancient Greeks observed that pine

resin not only helped seal the amphorae from moisture, but also helped to preserve the wine within.

In this way was born the technology that nowadays is used for production of the famous Greek

wine “Retsina”.

We used the chance of this archaeological find to analyze the ancient resin with the aim to proof if

ancient Greek people used resin from the same source – Aleppo pine (Pinus Halepensis) as it is

today by preparing of “Retsina”.

In this study using IR and NMR spectroscopy for determination of chemical compounds of ancient

and modern resin samples some preliminary results are presented.

The ancient resin sample was found in ceramic vessel (bottom of amphorae) in Sveshtari-the

former capital of the Thracians state in North-Eastern Bulgaria. The find is dated 4th – 3th century

BC while the modern resin samples were collected of Aleppo pine trees, which are grown in Attica

Region, near Athens (Greece).

The obtained results indicated that chemical composition of ancient and modern resin samples are

very similar. The differences between samples are not so significant and probably they are due to

oxidation processes to the ancient samples, what we will try to proof in additional investigations

using gas-chromatography, HPLC, etc.

Although we investigate one sample of ancient resin, the interpretation of analytical data indicates

that ancient Greeks are used the same resin for preservation of wine as today for preparing of

“Retsina”. Our result confirm the trade contacts of the Thracian population with ancient Greek

towns for supplying of wine, nevertheless that it live far a way from the Black Sea coast.

Acknowledgements The authors are indebted to Dr. Diana Gergova (Archaeological Institute, Bulgarian Academy of Sciences,

Sofia) for kindly supplying the archaeological materials for this investigation.

52

P - 18

CANDIDA ANTARCTICA LIPASE A IN THE DYNAMIC RESOLUTION OF NOVEL FURYLBENZOTIAZOL –BASED CYANOHYDRIN

ACETATES

Csaba Paizsa, Cornelia Majdika, Monica Toşaa, Paula Moldovana, Liisa T. Kanervab and Florin Dan Irimiea*,

aDepartment of Biochemistry and Biochemical Engineering, Babeş-Bolyai University, Arany János 11, RO-3400 Cluj-Napoca, Romania; [email protected]

b Laboratory of Synthetic Drug Chemistry and Department of Chemistry, University of Turku, Lemminkäisenkatu 2, FIN-20520 Turku, Finland

A series of novel (R)-furylbenzotiazol-based cyanohydrin acetates were prepared over 90% isolated

chemical yields from the corresponding furancarbaldehydes. The one-pot method combined a basic

resin to produce hydrogen cyanide from acetone cyanohydrin, an equilibrium between the

formation and decomposition of furylbenzotiazol-based cyanohydrins and further the unique

enantioselectivity of Candida antarctica lipase A, allowing the acylation of a (R)-cyanohydrins in

the presence of vinyl acetate in anhydrous acetonitrile.

O

N

S

R2

R1

OH

N

O

N

S

R2

R1

O

N

OO

N

S

R2

R1

OH

N +

Acetone cyanohydrin

Amberlite OH-

Amberlite OH-

CAL-AVinyl acetate

3a-d rac-4a-d

(S)-4a-d (R)-2a-d

O

N

SO

R2

R11

2

34

51'

2'3'

3a'4'5'6'

7'7a'

One pot synthesis of (R)-(+)-2a-d by dynamic resolution . Tetrahedron: Asymetry, 2003, 14, 619-627

53

P - 19

DETERMINATION OF SULFITE IN WINE BY ZONE ELECTROPHORESIS WITH ON-LINE ISOTACHOPHORESIS SAMPLE

PRETREATMENT ON A CHIP

M. Masár*1, M. Danková1, E. Ölvecká1, A. Stachurová1, D. Kaniansky1, B. Stanislawski2

1Department of Analytical Chemistry, Faculty of Natural Sciences, Comenius University, Mlynská

Dolina CH-2, SK-84215 Bratislava, Slovakia 2Merck KGaA, Frankfurter Straβe 250, D-64293 Darmstadt, Germany

54

P - 20

PEPTIDE MAPPING BY CAPILLARY ZONE ELECTROPHORESIS ON-LINE COUPLED WITH CAPILLARY ISOTACHOPHORESIS

M.Danková*, S. Strašík, J. Jánošková, J. Pivovarská, D. Kaniansky


55

P - 21 CAPILLARY ISOTACHOPHORESIS WITH DIODE-ARRAY DETECTION

AND CHEMOMETRY DATA PROCESSING

S. Strašík*, M. Danková, M. Molnárová, M. Lučaníková, D. Kaniansky


56

P - 22 SEPARATION OF PROTEINS BY ZONE ELECTROPHORESIS ON-LINE

COUPLED WITH ISOTACHOPHORESIS ON A COLUMN-COUPLING CHIP WITH CONDUCTIVITY DETECTION

E. Ölvecká*1, B. Pollák1, D. Kaniansky1, B. Stanislawski2

1Department of Analytical Chemistry, Faculty of Natural Sciences, Comenius University, Mlynská

Dolina CH-2, SK-84215 Bratislava, Slovakia 2Merck KGaA, Frankfurter Straβe 250, D-64293 Darmstadt, Germany

57

P - 23 COMPUTER ASSISTED CHOICE OF DISCRETE SPACERS FOR

ISOTACHOPHORESIS SEPARATIONS

V. Vaváková*, J. Marák, D. Kaniansky

Department of Analytical Chemistry, Faculty of Natural Sciences, Comenius University, Mlynská Dolina CH-2, SK-842 15 Bratislava, Slovakia

58

P - 24 SYNTHESIS OF NEW FERULOYLAMINO ACID DERIVATIVES

Maja Spasova1, Galja Ivanova3, Tamara Pajpanova1,2, Tsenka Milkova1,3

1 Department of Chemistry, South-West University “Neofit Rilsky”, Blagoevgrad, Bulgaria, 2 Institute of Molecular Biology, Bulgarian Academy of Sciences, Bulgaria,

3 Institute of Organic Chemistry with Center of Phytochemistry, Bulgarian Academy of Sciences, Bulgaria

In the present work we present the synthesis of feruloylamino acid amides. These

compounds could be considered as potentially antioxidants.

Coupling of different amino acid esters and unprotected hydroxy group of ferulic acid using

DIC/HOBt method provided corresponding amides.

teaching and learning “analytical and · pdf file“atomic absorption spectometry...

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