19th International Symposium on Separation Sciences · HIGH PERFORMANCE LIQUID CHROMATOGRAPHY ......

CROATIAN SOCIETY OF CHEMICAL ENGINEERS

SECTION FOR CHROMATOGRAPHY

19th International Symposium on Separation SciencesSeparation Sciences

NEW ACHIEVEMENT IN CHROMATOGRAPHY

BOOK OF ABSTRACTS

25‐28 September 2013Poreč, Croatia

Publisher: Croatian Society of Chemical Engineers, Berislavićeva 6/I, 10000 Zagreb, Croatia

Phone: +385 1 4872 499

Fax: +385 14872490

E-mail: [email protected]

Editors: Šime Ukić and Tomislav Bolanča

A CIP catalogue record for this book is available in the Online Catalogue of the National and

University Library in Zagreb as 854962.

ISBN: 978-953-6470-64-8

The professional and grammatical errors of the abstracts are authors’ responsibility.

International Scientific Committee Organizing Committee

D. Berek*, Bratislava, Slovakia D. Ašperger,Zagreb, Croatia

T. Bolanča* (Chairman), Zagreb, Croatia S. Babić, Zagreb, Croatia

G. Bonn, Insbruck, Austria M. Bojić, Zagreb, Croatia

W. Buchberger*, Linz, Austria T. Bolanča, Zagreb, Croatia

B. Buszewski*, Toruń, Poland S. Malčić, Zagreb, Croatia

V. Coman*, Cluj-Napoca, Romania O. Platiša, Zagreb, Croatia

D. Corradini*, Rome, Italy Z. Šmit, Rijeka, Croatia

M. Gertsiuk*, Kiev, Ukraine L. Štajduhar, Zagreb, Croatia

P. Jandera*, Pardubice, Czech Republic D. Štanfel, Rijeka, Croatia

I. Klebovich*, Budapest, Hungary S. Telen, Zagreb, Croatia

M. Medić-Šarić, Zagreb, Croatia Š. Ukić, Zagreb, Croatia

P. Sandra, Ghent, Belgium

P. Schoenmakers, Amsterdam, Netherlands

N. Šegudović†, Zagreb, Croatia

I. Vovk*, Ljubljana, Slovenia

T. Welsch, Ulm, Germany

*permanent member of CEGSS

Sponsored by (in alphabetic order):

Golden sponsors

AlphaCrom d.o.o. Zagreb Croatia

Instrumentalia Adria d.o.o. Zagreb Croatia

Kemolab d.o.o. Zagreb Croatia

Kobis d.o.o. Trzin Slovenia

Shimadzu d.o.o. Zagreb Croatia

Silver sponsor

Merck d.o.o. Zagreb Croatia

Bronze sponsors

Vita Lab Nova d.o.o. Zagreb Croatia

Primalab d.o.o. Polzela Slovenia

Labomar d.o.o. Zagreb Croatia

Exhibitors (in alphabetic order):

Anas d.o.o. IND - EKO d.o.o. Zagreb Croatia

KEFO d.o.o. Sisak Croatia

Labena d.o.o. Zagreb Croatia

Sartorius Croatia – Libra Elektronik d.o.o. Zaprešić Croatia

PREFACE

Welcome to

19th International Symposium on Separation Sciences

“New Achievements in Chromatography”

Thousands of scientists and engineers have worked on the development of

chromatography over the last several decades. The result is one of the most versatile

techniques that we have in chemical science today. The development is still going on

with thousands of papers and many books being published every year. All this has

been accomplished because there is an understanding of the physico-chemical

principles of the separation and detection process. An expert in chromatography

should understand these principles and implement them into daily practice.

The origin of the International Symposium on Separation Science can be found

in national GC meetings held in Zagreb, Croatia, since 1967. During the years these

meeting have become international initiating foundation of Central European Group

for Separation Science on 1998. The 19th International Symposium on Separation

Science is organized by Croatian Society of Chemical Engineers under the

auspicious of Central European Group of Separation Science and European Society

for Separation Science.

The quality of the ISSS program has always been an imperative and therefore

eminent scientist will join symposium as invited lecturers to share valuable

experiences while representatives of instruments, chemicals and other consumables

production companies will offer practical solutions for particular problem. The topics

prepared for Symposium will cover all aspects of chromatography and other

separation and detection techniques including multidimensionality in separation,

hyphenations in detection, chemometrics and quality assesment, confirming the

cutting edge power of chromatography and separation techniques. The applications

in different areas will be even more emphasized by numerous posters. Special

education session, 14th International Chromatography School, will cover fundaments

of chromatography. Young scientists are especially encouraged to attend the

symposium. Even though the program will be very intensively scheduled, hopefully

there will be some spear moment to enjoy Adriatic coast late summer surroundings of

Poreč.

On behalf of the Organizing Committee it is my honor and privilege to welcome

all the participants on the 19th International Symposium on Separation Scinece, “New

Achivements in Chromatography”.

Prof. dr. Tomislav Bolanča

19th ISSS chairman

New Achievements in Chromatography 19

th International Symposium on Separation Sciences

25-28 September 2013, Poreč, Croatia

CONTENTS




INTERNATIONAL CHROMATOGRAPHY SCHOOL

J. Weiss:

ISOCRATIC VS. GRADIENT ELUTION IN ION CHROMATOGRAPHY ...................... 2

M. Boras:

ULTRAPERFORMANCE CONVERGENCE CHROMATOGRAPHY: EXPANDING

SELECTIVITY FOR THE CHROMATOGRAPHIC LABORATORY ............................... 3

LECTURES

S. Malitsky, I. Rogachev, A. Aharoni, A. Tishbee:

APPLYING THE HIGH EFFICIENCY, AND UNIQUE SELECTIVITY, OF SUPER

CRITICAL FLUID CHROMATOGRAPHY (SFC)-MS, UPLC-MS, AND GC-MS,

IN PLANT METABOLOMICS ............................................................................................. 5

R.M. Sample: INSTRUMENTAL ASSISTANCE IN THE TRANSFER OF METHODS BETWEEN

DIFFERENT HPLC/UHPLC SYSTEMS .............................................................................. 6

I. Klebovich:

BIOMEDICAL AND BIOANALYTICAL ASPECTS OF PRECLINICAL AND

CLINICAL PHARMACOKINETICS AND DRUG METABOLISM RESEARCH ............ 7

B. Buszewski, M. Jaćkowska, M. Grochowicz, B. Gawdzik

NEW GENERATION OF FUNCTIONALISED STATIONARY PHASES FOR ION

CHROMATOGRAPHY (IC) ................................................................................................. 8

N. Avdalović, U. Aich:

GLYCANPAC AXH-1 COLUMNS FOR THE HIGH-RESOLUTION

SEPARATION AND ANALYSIS OF GLYCANS ............................................................... 9

J. Weiss:

HIGH-PRESSURE ION CHROMATOGRAPHY – A NEW PLATFORM FOR HIGH

RESOLUTION OR HIGH THROUGHPUT SEPARATIONS OF IONIC

COMPOUNDS ..................................................................................................................... 10

W. Goessler:

THE ROLE OF LIQUID CHROMATOGRAPHY COUPLED TO ICPMS FOR

ARSENIC SPECIATION ANALYSIS ................................................................................ 11

S. Studzińska, B. Buszewski:

THE STUDY OF OLIGONUCLEOTIDES RETENTION BY

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY .............................................. 12




P. Jandera, M. Staňková, T. Hájek:

NEW MONOLITHIC POLY(METHACRYLATE) COLUMNS FOR REVERSED-

PHASE AND AQUEOUS NORMAL-PHASE SEPARATION OF

LOW-MOLECULAR COMPOUNDS IN 1D AND 2D HPLC ........................................... 13

T. Welsch, U. Hermann:

FLOW RATE CAUSED SELECTIVITY CHANGES OF SERIALLY COUPLED

COLUMNS IN HPLC .......................................................................................................... 14

I. Vovk, B. Simonovska, V. Glavnik, L. Brulc,

A. Albreht, Z. Rodić:

HPLC AND HPTLC OF THE MAJOR DIETARY CAROTENOIDS ............................... 15

F. Cacciola, Paola Donato, M. Beccaria, P. Dugo, A. Cotroneo, L. Mondello:

ADVANCED LIQUID CHROMATOGRAPHY TECHNIQUES FOR THE

SCREENING OF LIPID MOLECULES IN FOOD ............................................................ 16

A. Albreht, I. Vovk, B. Simonovska:

CHROMATOGRAPHY AND MASS SPECTROMETRY AS A TOOL FOR THE

CHARACTERIZATION OF INTERACTIONS BETWEEN SHIKONIN

AND β-LACTOGLOBULIN ............................................................................................... 17

P. Sandra, K. Sandra:

Analytical Aspects of Bringing a Biopharmaceutical to the Market.................................... 18

G.K. Bonn:

NOVEL ADVANCEMENTS IN CHROMATOGRAPHIC APPLICATIONS FOR

THE ENRICHMENT AND SEPARATION OF BIOMOLECULES ................................. 19

D. Corradini, I. Nicoletti:

ROLE OF THE LIQUID PHASE ON THE SEPARATION OF BIOMOLECULES

BY HPLC AND CAPILLARY ELECTROPHORESIS: FUNDAMENTAL AND

PRACTICAL ASPECTS ...................................................................................................... 20

Ž. Debeljak:

ESTABLISHMENT OF THE CORRECT DIAGNOSIS BY CHROMATOGRAPHY

AND MASS SPECTROMETRY ......................................................................................... 21

M. Cindrić:

PEPTIDE DE NOVO SEQUENCE ASSIGNMENT ........................................................... 22

D. Berek:

COMPREHENSIVE MOLECULAR CHARACTERIZATION OF COMPLEX

POLYMER SYSTEMS BY ADVANCED LIQUID

CHROMATOGRAPHY METHODS .................................................................................. 23

W. Buchberger, S. Beissmann, I. Hintersteiner, L. Sternbauer:

SOLVED AND UNSOLVED PROBLEMS IN EVALUATING THE PROPERTIES OF

PLASTIC MATERIALS BY CHROMATOGRAPHIC AND

MASS SPECTROMETRIC METHODS ............................................................................. 25




L.A. Clementi, J.R. Vega, G.R. Meira:

RANDOMLY-BRANCHED POLYMERS BY SIZE EXCLUSION

CHROMATOGRAPHY WITH TRIPLE DETECTION: COMPUTER SIMULATION

STUDY ON BIASES IN DISTRIBUTIONS OF MOLAR MASSES AND

DEGREES OF BRANCHING ............................................................................................. 26

M. Petrović, M. Gorga, R. Lopez-Serna, D. Barceló:

RECENT ADVANCES IN ON-LINE SAMPLE PREPARATION METHODS

COUPLED TO LC-TANDEM MS FOR THE ANALYSIS OF EMERGING

CONTAMINANTS IN ENVIRONMENTAL SAMPLES .................................................. 27

V. Coman, F. Copaciu, L. Copolovici, D. Simedru:

CHROMATOGRAPHIC RESEARCHES ON THE TEXTILE DYES AND THEIR

POLLUTION EFFECTS ON WHEAT PLANTS ............................................................... 28

L. Ciofi, L. Checchini, U. Chiuminatto, E. Coppini, M. Del Bubba:

DETERMINATION OF ALKYLPHENOLS POLYETHOXYLATES (APEOs) AND

ALKYLPHENOLS (APs) IN WASTEWATER AND SURFACE-WATER

THROUGH A UNIQUE HPLC-ESI-MS/MS METHOD ................................................... 29

M. Periša, M. Mitrevski, S. Babić:

IDENTIFICATION OF SULFONAMIDES PHOTODEGRADATION PRODUCTS

IN DIFFERENT WATER MATRICES ............................................................................... 31

M. Gertsiuk, G. Lysychenko:

IDENTIFICATION METHODS OF FORBIDDEN AND OBSOLETE PESTICIDES ..... 32

D. Stipaničev, S. Repec, S. Širac:

SCREENING OF ORGANIC POLLUTANTS IN SURFACE WATER BY

ULTRA HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

COUPLED TO iFUNNEL Q-TOF/MS ................................................................................ 33

T. Kosjek, S. Perko, D. Žigon, E. Heath:

ANALYSIS, OCCURRENCE, DEGRADATION AND TRANSFORMATION

OF FLUOROURACIL IN THE ENVIRONMENT ............................................................. 34

M. Zrnčić, S. Babić, M. Kaštelan-Macan:

DEVELOPMENT OF HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

TANDEM MASS SPECTROMETRY (HPLC – MS/MS) METHOD FOR

DETERMINATION OF DIFFERENT CLASSES OF PHARMACEUTICALS

IN WASTE WATERS ......................................................................................................... 35

M. Concetta Bruzzoniti, R. Maria De Carlo, C. Sarzanini, R. Maina, V. Tumiatti:

COMPARISON OF GAS CHROMATOGRAPHIC METHODS FOR THE

DETERMINATION OF METHANOL AND ETHANOL IN INSULATING

MINERAL OIL AS MARKERS OF CELLULOSE DEGRADATION

IN POWER TRANSFORMER ............................................................................................ 36




P.Q. Tranchida, F.A. Franchina, M. Zoccali, P. Dugo, L. Mondello:

HIGH SENSITIVITY FLOW-MODULATED COMPREHENSIVE TWO-

DIMENSIONAL GAS CHROMATOGRAPHY-MASS SPECTROMETRY .................... 38

O. Platiša, S. Telen:

PROFILE DEFINITION OF AROMATIC HYDROCARBONS IN

CRUDE OIL FRACTIONS BY COMPREHENSIVE TWO-DIMENSIONAL

GAS CHROMATOGRAPHY .............................................................................................. 39

R.A. Salkar:

GC-TIME OF FLIGHT-THERMAL DESORPTION TECHNIQUE FOR THE

DETECTION AND QUANTIFICATION OF VOLATILES IN THE CONTEXT

OF HOME AND PERSONAL CARE PRODUCTS ........................................................... 40

B. Strzemiecka, A. Voelkel, M. Hinz, M. Rogozik:

APPLICATION OF INVERSE GAS CHROMATOGRAPHY IN PHYSICOCHEMICAL

CHARACTERIZATION OF ABRASIVE TOOLS ............................................................ 41

J. Švarc-Gajić:

NEW TRENDS IN THE DEVELOPMENT OF MICROEXTRACTION

TECHNIQUES FOR COUPLING SAMPLE PREPARATION AND

CHROMATOGRAPHIC ANALYSIS ................................................................................. 42

M. Pietrzyńska, A. Voelkel:

MONOLITHIC IN-NEEDLE EXTRACTION (MINE) DEVICE AS

A NEW APPROACH FOR THE DIRECT ANALYSIS OF LIQUID SAMPLE ............... 43

I. Jerković:

PREPARATIVE AND CHROMATOGRAPHIC TECHNIQUES IN CHEMICAL

FINGERPRINTING - SELECTED EXAMPLES ............................................................... 44

R. Martins, C. Maia, J.A. Queiroz, F. Sousa:

AFFINITY-BASED METHOD FOR RNA PURIFICATION PURSUING

mRNA VACCINATION ...................................................................................................... 45

É. Mota, F. Sousa, Â. Sousa, J.A. Queiroz, C. Cruz:

STUDY OF THE INTERACTIONS BETWEEN IMMOBILIZED L-METHIONINE

AND OLIGONUCLEOTIDES BY CHROMATOGRAPHY, NMR

AND SPR ANALYSIS ........................................................................................................ 46

POSTER PRESENTATIONS

I. Rykowska, W. Wasiak:

DETERMINATION OF ESTROGENS IN WATER COLLECTED FROM

SEWAGE PLANTS BY GC-MS USING A NEW STIR BAR COATING

OR SORPTIVE EXTRACTION .......................................................................................... 48




L.S. Sokolova, A.V. Pirogov, O.A. Shpigun:

THE USE OF MICROEMULSIONS FOR THE SAMPLE PRETREATMENT

AND AS THE MOBILE PHASE IN MELC ....................................................................... 49

A.V. Pirogov, L.S. Sokolova, O.A. Shpigun:

DERIVATIZATION OF ANTIBIOTICS IN MICROEMULSION MEDIUM

FOLLOWED BY HPLC DETERMINATION .................................................................... 50

O.I. Shchukina, A.V. Zatirakha, A.D. Smolenkov , O.A. Shpigun:

HYDROPHILIC SPACERS FOR MOVING THE FUNCTIONAL GROUPS

AWAYFROM THE MATRIX OF THE ANION EXCHANGERS IN

ION CHROMATOGRAPHY .............................................................................................. 52

E.B. Pashkova, A.V. Pirogov, L.S. Sokolova:

DETERMINATION OF 2-THIOCYANO-3,5-DINITROPYRIDINE IN OINTMENTS

BY MICROEMULSION LIQUID CHROMATOGRAPHY .............................................. 53

J. Oskonbaeva:

FUNDAMENTALS OF GAS CHROMATOGRAPHY ...................................................... 54

A.A. Kostromskikh, A.V. Pirogov, L.S. Sokolova, O.A. Shpigun:

SAMPLE STACKING AND ON-LINE DERIVATIZATION FOR THE ANALYSIS

OF AMPICILLIN AND AMOXICILLIN BY MICROEMULSION

ELECTROKINETIC CHROMATOGRAPHY .................................................................... 55

D. Bicho, A. Sousa, F. Sousa, J.A. Queiroz, C.T. Tomaz:

DYNAMIC BINDING CAPACITY OF A NON-GRAFTED MONOLITHIC

SUPPORT USING DIFFERENT PLASMIDS .................................................................... 57

Catarina Caramelo-Nunes, Paulo Almeida, João C. Marcos, Cândida T. Tomaz:

DAPP-SEPHAROSE AFFINITY CHROMATOGRAPHY: SUPERCOILED PDNA

PURIFICATION FROM CLARIFIED E. COLI LYSATE SOLUTIONS .......................... 58

T.A. Balotnik, A.D. Smolenkov, R.S. Smirnov, O.A. Shpigun:

DETERMINATION OF THE LOW CONCENTRATIONS OF ROCKET KEROSENE

IN WATER BY GAS CHROMATOGRAPHY-MASS SPECTROMETRY WITH

PRE-MICROEXTRACTION BY N-HEXANE ................................................................... 59

A.A. Bendryshev, B.A. Sarvin, A.V. Pirogov, O.A. Shpigun:

DETERMINATION OF WATER SOLUBLE VITAMINS IN ENERGY DRINKS

AND PHARMACEUTICALS BY HYDROPHILIC INTERACTION LIQUID

CHROMATOGRAPHY ....................................................................................................... 60

L. Markovic, P. Jackson:

HILIC APPLICATION IN PHARMACEUTICAL INDUSTRIES: ROBUSTNESS

AND SPECIFIC SELECTIVITY OF HILIC ....................................................................... 61




M. Szultka, R. Krzeminski, M. Jackowski, B. Buszewski:

IN VITRO CYTOCHROME P450 ACTIVITY: DEVELOPMENT AND

VALIDATION OF LC-MS/MS METHOD FOR THE SIMULTANEOUS

DETERMINATION OF AMOXICILLIN AND ITS METABOLITES .............................. 62

M. Szultka, R. Krzemiński, J. Szeliga, M. Jackowski, B. Buszewski:

APPLICATION OF SPME-LC/MSn FOR METABOLOMICS

DRUG MONITORING IN BIOLOGICAL SAMPLES ...................................................... 63

S. Hasić, M. Kežić, S. Ćavar, A. Goga, A. Čaušević:

ISOLATION AND GC-MS QUANTIFICATION OF ACRYLAMIDE

FROM CEREAL AND POTATO FOODS ......................................................................... 64

A. Goga, M. Kežić, S. Ćavar, S. Hasić, A. Čaušević:

ISOLATION AND GC-MS QUANTIFICATION OF PHTHALATES FROM

CHILDREN`S TOY USING DIFFERENT EXTRACTION TECHNIQUES .................... 65

M.P. Godoy-Caballero, M.I. Acedo-Valenzuela, T. Galeano-Díaz:

DEVELOPMENT OF A REVERSED PHASE DISPERSIVE LIQUID-LIQUID

MICROEXTRACTION FOR THE ANALYSIS OF PHENOLIC COMPOUNDS

IN VOO ................................................................................................................................ 66

M.P. Godoy-Caballero, T. Galeano-Díaz, M.I. Acedo-Valenzuela:

QUANTIFICATION OF PHENOLIC COMPOUNDS IN VOO BY RAPID

RESOLUTION LCDAD-MS PREVIOUS REVERSED PHASE DISPERSIVE

LIQUID-LIQUID MICROEXTRACTION ......................................................................... 67

S. Studzińska, B. Buszewski:

UTILIZATION OF ULTRA HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY FOR THE STUDY OF OLIGONUCLEOTIDES ........................ 68

F. Belal, M.K. Sharaf El-Din, M.I. Eid, R.M. El-Gamal:

MICELLAR HPLC METHOD USING MONOLITHIC COLUMN FOR

THE DETERMINATION OF LINEZOLID AND RIFAMPICIN IN

PHARMACEUTICALS AND BIOLOGICAL FLUIDS ..................................................... 69

E. Dziubakiewicz, K. Hrynkiewicz, M. Walczyk, B. Buszewski:

DETERMINATION OF CHARGE DISTRIBUTION OF BACTERIAL CELL ................ 70

E. Heath, S. Perko, T. Kosjek:

BENZODIAZEPINES IN THE ENVIRONMENT: OCCURRENCE, FATE

AND TRANSFORMATION ............................................................................................... 71

Y.-M. Lee, K.-O. Shin, C.-H. Seo, S.-H. Lee:

SPHINGOSINE1-PHOSPHATELYASE ACTIVITY MEASUREMENT BY

HPLC-FLUORESCENCE DETECTION AFTER

5,5-DIMETHYL CYCLOHEXANEDIONE DERIVATIZATION..................................... 72




A. Šišková, E. Macová, D. Berek:

APPLICATION OF LIQUID CHROMATOGRAPHY UNDER LIMITING

CONDITIONS OF DESORPTION TO SEPARATION OF DIFFICULT

COMPLEX POLYMER SYSTEMS .................................................................................... 73

Ž. Zgorelec, A. Jurišić, M. Mesić, I. Šestak, B. Benko, S. Fabek, B. Novak,

A.-M. Špicnagel:

CHROMATOGRAPHY SEPARATION OF ESSENTIAL IONS IN STEVIA ................. 75

R. Wawrzyniak, W. Wasiak, K. Buczkowska, J. Ostrowska:

CHARACTERISTICS OF VOLATILE COMPOUNDS IN THE LIVERWORT

ANEURA PINGUIS COLLECTED IN THE POLAND....................................................... 76

J. Urban, V. Škeříková:

CAPILLARY LIQUID CHROMATOGRAPHY OF SMALL MOLECULES ON

HYPERCROSSLINKED MONOLITHIC COLUMNS ...................................................... 77

M. Studziński, I. Malinowska, W. Jesionek, H. Malinowski:

MAGNETO-TLC AS A TOOL FOR PLANT EXTRACT ANALYSIS ............................ 78

J. Soukup, P. Jandera:

COMPARISON OF NON-AQUEOUS NORMAL PHASE LIQUID

CHROMATOGRAPHY WITH AQUEOUS NORMAL-PHASE LIQUID

CHROMATOGRAPHY ON HYDROSILATED SILICA-BASED

STATIONARY PHASES .................................................................................................... 79

I. Jerković, M. Obradović, P.M. Kuś, M. Šarolić:

HEADSPACE SOLID-PHASE MICROEXTRACTION OF RARE

CORIANDRUM SATIVUM L. HONEY ............................................................................... 80

P.M. Kuś, I. Jerković, Z. Marijanović, M. Šarolić:

GC-MS PROFILING OF RARE UNIFLORAL PRUNUS CERASUS L.

HONEY HEADSPACE ....................................................................................................... 81

M. Šarolić, M. Gugić, Z. Marijanović, M. Šuste, I. Jerković:

GAS CHROMATOGRAPHIC HEADSPACE FINGERPRINT OF VIRGIN

OLIVE OIL FROM AUTOCHTHONOUS VARIETY MAŠNJAČA .................................. 83

J.F.P. Valente, I.J. Correia, F. Sousa:

p53 SUPERCOILED PLASMID PURIFICATION BY

A L-METHIONINE-AGAROSE MATRIX ........................................................................ 84

L. Maslov, I. Tomaz, M. Medić-Šarić:

DETERMINATION OF POLYPHENOLS AND INDOLE-3-ACETIC ACID

IN WINES BY HPLC-DAD-FLD ....................................................................................... 85




M. Buszewska-Forajta, J. Raczak-Gutknecht, K. Walkowiak, M. Patejko, D. Siluk,

R. Kaliszan:

THE STUDY OF DIFFERENCES IN METABOLOMIC PROFILES OF

GRASSHOPPER’S CHORTHIPPUS SPP. ABDOMINAL SECRETION

OBTAINED FROM THREE SPECIES OF INSECTS ....................................................... 86

J. Raczak-Gutknecht, R. Bujak, R. Gadzała-Kopciuch, M. Buszewska-Forajta,

A. Nowaczyk, E. Daghir, P. Kośliński, B. Buszewski, R. Kaliszan, M.J. Markuszewski:

COCAINE AND BENZOYLECGONINE SELECTIVE EXTRACTION

FROM WATER SAMPLES USING MOLECULARLY IMPRINTED POLYMERS ....... 87

E. Okrągła, G. Gałęzowska, L. Wolska:

HYDROPHILIC INTERACTION CHROMATOGRAPHY OF URINARY COMPOUNDS

TOXIC TOWARD VIBRIO FISCHERI BACTERIA .......................................................... 89

W. Jesionek, I. Choma, B. Majer-Dziedzic:

PLANAR CHROMATOGRAPHY –BIOAUTOGRAPHY AS A TOOL

FOR INVESTIGATION OF BIOLOGICALLY ACTIVE COMPOUNDS

IN PLANT EXTRACTS ...................................................................................................... 90

A. Yumba Mpanga, W. Struck-Lewicka, M. Buszewska-Forajta, D. Szczesny,

M. Markuszewski, M. Roslan, R. Kaliszan, M.J. Markuszewski:

NON-TARGETED METABOLOMIC ANALYSIS OF URINE IN UROGENITAL

TRACT CANCER DISEASES USING LC-MS ................................................................. 91

R. Koeck, M. Fischnaller, R. Bakry, R. Tessadri, G.K. Bonn:

EFFICIENT SEPARATION OF BIOMOLECULES AND SMALL MOLECULES BY

MONOLITHIC POLY (N-VINYLCARBAZOLE-CO-1,4-DIVINYLBENZENE)

CAPILLARY COLUMNS ................................................................................................... 92

S.B. Tóth:

EXAMINATION OF DEGRADATION OF DIFFERENT MYCOTOXIN AND

DEGRADATION PRODUCTS WITH OZONE-ENRICHED MEDIUM

BY HPLC-MS TECHNIQUE .............................................................................................. 93

I. Valentić, M. Buratović, D. Štanfel:

UHPLC STUDY ON THE DEGRADATION PROFILES OF ACTIVE SUBSTANCES

IN THE EYE DROPS SUBJECTED TO HEAT AND FILTRATION

STERILIZATION METHODS ............................................................................................ 94

R. Halko, I. Hukelová:

SEPARATION AND DETERMINATION OF CARBOXYLIC ACIDS IN WINE

AND HUMAN URINE SAMPLES BY ION-EXCLUSION CHROMATOGRAPHY ...... 95

S.B. Tóth:

REDUCTION OF QUANTITY OF MYCOTOXINS IN FOOD PRODUCTS BY

DIFFERENT LEAVENING AGENTS, ANALYSIS OF METABOLITE ......................... 96




A. Moos, A. Bocheńska, R. Wietecha-Posłuszny, P. Kościelniak:

APPLICATION OF MANUAL AND AUTOMATED MICROEXTRACTION BY

PACKED SORBENT FOR ISOLATION OF BENZODIAZEPINES FROM TWO

ALTERNATIVE BIOLOGICAL MATERIALS ................................................................. 98

J. Nowak, M. Ciechomska, M. Woźniakiewicz, P. Kościelniak:

APPLICATION OF DOEHLERT EXPERIMENTAL DESIGN FOR

OPTIMIZATION OF MICROWAVE-ASSISTED EXTRACTION OF

TROPANE ALKALOIDS FROM SOLANACEAE FAMILY PLANTS ............................. 99

M. Szewczyńska, M. Pośniak, E. Dobrzyńska:

THE USE OF HIGH LIQUID PERFORMANCE CHROMATOGRAPHY FOR

THE DETERMINATION OF PAHS IN THE FINE PARTICLE FRACTION

OF DIESEL EXHAUST .................................................................................................... 100

M. Pośniak, E. Dobrzyńska, M. Szewczyńska:

HIGH LIQUID PERFORMANCE CHROMATOGRAPHY AS A METHOD FOR

OCCUPATIONAL EXPOSURE ASSESSMENT OF ACTIVE SUBSTANCES

IN THE PHARMACEUTICAL INDUSTRY .................................................................... 101

J.L. Vera, V.F. Domingues, A. Almeida, J.M. Costa, C. Mansilha, C. Delerue-Matos:

SIMULTANEOUS DETERMINATION OF 29 ENDOCRINE DISRUPTING

PESTICIDES IN SEDIMENTS FROM RIA DE AVEIRO

USING QuEChERS BY GC-MS ....................................................................................... 102

W. Struck-Lewicka, D. Siluk, A. Yumba Mpanga, M. Markuszewski, R. Kaliszan,

M.J. Markuszewski:

TARGETED AND UNTARGETED STUDY OF URINARY METABOLITES

AS POTENTIAL CANCER MARKERS .......................................................................... 103

M. Mavrinac, S. Kamber, D. Štanfel, I. Valentić, K. Mihaljević:

DETERMINATION OF DEXPANTHENOL DEGRADATION PRODUCTS IN

SEMISOLIDS .................................................................................................................... 104

G. Mendaš, B. Tariba, S. Stipičević, M. Dvoršćak, V. Drevenkar:

SIMULTANEOUS DETERMINATION OF PHENYLUREA AND TRIAZINE

HERBICIDES IN RIVER WATER BY SOLID-PHASE EXTRACTION AND LIQUID

CHROMATOGRAPHY ..................................................................................................... 105

H. Rechak, M. Lahouel, A. Hamdi:

PHARMACOKINETICS BEHAVIOUR OF VERAPAMIL AFTER INTRAVENOUS

AND ORAL ADMINISTRATION OF DIAZEPAM IN RATS BY HPLC ...................... 106

N.M Karaseva, V.G. Amelin, A.V. Tretaykov:

COMBINATION QuEChERS, DISPERSIVE LIQUID-LIQUID

MICROEXTRACTION AND HPLC WITH FLUORESCENCE DETECTION

FOR SIMULTANEOUS EXTRACTION AND DETERMINATION OF EIGHT

MYCOTOXINS IN CEREAL AND FEED ....................................................................... 107




Simona Čurmová, Lenka Okenicová, Radoslav Halko:

THE ANALYTICAL USE OF NEOCUPROINE FOR DETERMINATION

OF COPPER IN HUMAN URINE .................................................................................... 108

O. Ferroukhi, S. Bouanani, M. Bensebaa, H. Mebarki:

DEVELOPMENT OF A RAPID QUANTIFICATION METHOD OF

10 PESTICIDES IN WHOLE BLOOD BY

LIQUID CHROMATOGRAPHY /TANDEM MASS SPECTROMETRY LC-MS2 ........ 109

I. Trbojević Akmačić, J. Štambuk, F. Vučković, M. Novokmet, M. Pučić Baković,

G. Lauc:

EVALUATION OF THE UPLC METHOD FOR IgG GLYCAN ANALYSIS

FOR IT'S ROBUSTNESS AND REPEATABILITY ........................................................ 110

N.M. Volkova, A.A. Timofeev, V.G. Amelin, A.V. Tretaykov:

QuEChERS SAMPLE PREPARATION IN SIMULTANEOUS DETERMINATION

OF RESIDUAL AMOUNTS OF ANTIBIOTICS QUINOLONE SERIES

AND CHLORAMPHENICOL IN FOOD BY HPLC-DAD .............................................. 111

K. Sosnowiec, G. Gałęzowska, M. Cieszyńska, L. Wolska:

IDENTIFICATION OF VOCS EMISSION FROM INDOOR MATERIALS AND

THE ATTEMPT OF BIOTESTS IMPLEMENTATION IN TOXICITY

ASSESSMENT OF EMISSION STREAM ....................................................................... 113

M. Miliša Gregurić, Z. Kauzlarić:

COMPARISON OF DIFFERENT CHROMATOGRAPHIC METHODS FOR THE

DETERMINATION OF HYDROCARBON TYPES, BENZENE AND

OXYGENATES IN MOTOR GASOLINE ....................................................................... 114

A. Wronka, I. Malinowska:

A CHROMATOGRAPHIC STUDY OF THE NEW HETERONUCLEAR

COMPLEXES WITH THE SCHIFF BASE AS A MAIN LIGAND ................................ 115

A. Soares, J.A. Queiroz, F. Sousa, Â. Sousa:

PURIFICATION OF HPV16 E6/E7 PLASMID DNA-BASED VACCINE USING

A MODIFIED MONOLITHIC SUPPORT ........................................................................ 116

P. Pereira, A. Sousa, I.J. Correia, A. Figueiras , F. Sousa:

EXPLOITING MULTIPLE INTERACTIONS IN PRE-miR-29 PURIFICATION BY

ARGININE-AFFINITY CHROMATOGRAPHY ............................................................. 117

P. Pereira, A. Sousa, I.J. Correia, A. Figueiras, F. Sousa:

IMPROVED NATIVE PRE-miR-29 PURIFICATION WITH

LYSINE-AFFINITY CHROMATOGRAPHY .................................................................. 118

V. Stankov, H. Farkaš, A. Bognar, B. Marošanović:

DETERMINATION OF AFLATOXIN M1 IN MILK AND MILK PRODUCTS BY

UHPLC-MS/MS ................................................................................................................. 119




K. Sabo, M. Pandurević Todorović, B. Marošanović:

REVIEW OF THE RATIO OF OMEGA-6/OMEGA-3 FATTY ACIDS

IN OILS AND FATS ......................................................................................................... 121

W. Hewelt-Belka, J. Nakonieczna, A. Kot-Wasik, J. Namieśnik:

DEVELOPMENT OF LC-MS-Q-TOF-MS METHOD FOR COMPREHENSIVE

NONTARGETED LIPID PROFILING OF STAPHYLOCOCCUS AUREUS .................. 122

W. Hewelt-Belka, P. Kubica, K. Wilczewska, A. Kot-Wasik, J. Namieśnik:

COMPARISON OF MOBILE PHASE ADDITIVES FOR DETERMINATION

OF AMINO ACIDS IN REVERSED PHASE MODE ...................................................... 123

A. Jakimska, W. Hewelt-Belka, A. Kot-Wasik, J. Namieśnik:

ELUCIDATION OF DEGRADATION PATHWAY OF FUROSEMIDE

BY UPLC-QTOF-MS ........................................................................................................ 124

A. Jakimska, W. Hewelt-Belka, A. Kot-Wasik, J. Namieśnik:

OCCURRENCE OF CAFFEINE IN THE AQUEOUS ENVIRONMENT

BY SPE-HPLC-APCI-MS/MS .......................................................................................... 125

M. Belka, J. Sławiński, T. Bączek

QUANTITATIVE STRUCTURE - RETENTION RELATIONSHIP AS

A SUPPORTIVE TOOL FOR IDENTIFICATION OF CIS- AND TRANS- ISOMERS

IN STABILITY STUDIES OF A SERIES OF NOVEL BENZENSULFONAMIDE

DERIVATIVES – POTENTIAL ANTICANCER COMPOUNDS ................................... 126

R. Góra, M. Hutta, P. Rohárik, N. Bielčíková:

CHARACTERIZATION OF SELECTED ENVIROMACROMOLECULES BY

COMBINATION OF REVERSED-PHASE HIGH-PERFORMANCE LIQUID

CHROMATOGRAPHY AND NARROW-BORE

SIZE-EXCLUSION CHROMATOGRAPHY ................................................................... 128

L. Konieczna, M. Belka, M. Niedźwiecki, T. Bączek:

AMINO ACIDS EVALUATION IN CEREBROSPINAL FLUID IN LEUKEMIA

CHILDREN USING HILIC-ESI-MS METHOD .............................................................. 129

M. Szafarz, A. Zakrzewska, G. Koralewicz, A. Gonciarz, A. Kij, K. Kuś, J. Suraj,

M. Walczak:

DEVELOPMENT OF BIOANALYTICAL LC/MS/MS ASSAY FOR

QUANTIFYING 5-FLUOROURACIL AND ITS TWO METABOLITES

IN HUMAN PLASMA ...................................................................................................... 131

L. Checchini, C. Ancillotti, L. Ciofi, S. Furlanetto, M. Del Bubba:

DIRECT RESOLUTION AND QUANTITATIVE ANALYSIS OF FLURBIPROFEN

ENANTIOMERS USING MICROCRYSTALLINE CELLULOSE TRIACETATE

PLATES: APPLICATIONS TO THE ENANTIOMERIC PURITY CONTROL AND

OPTICAL ISOMER DETERMINATION IN WIDELY CONSUMED DRUGS ............. 132




V. Komorowska, M. Hutta:

ANALYTICAL APPROACH TO VALIDATION OF HPLC METHOD FOR

ANALYSIS AND CHARACTERISATION OF PROTEINS

IN PROTEINACEOUS FOOD SUPPLEMENT ............................................................... 133

K. Kwaśniewska, R. Gadzała-Kopciuch:

SYNTHESIS OF MOLECULAR IMPRINTED POLYMERS AS A FILLING FOR

CHROMATOGRAPHY COLUMNS FOR THE DETERMINATION

OF MYCOTOXIN ............................................................................................................. 134

A. Gonciarz, A. Kij, K. Kuś, J. Suraj, M. Szafarz, A. Zakrzewska, M. Walczak:

INFLUENCE OF DIFFERENT KINDS OF CYCLODEXTRINS ON SEPARATION

OF PRAVASTATIN AND BOVINE SERUM ALBUMIN- APPLICATION

TO PROTEIN BINDING STUDIES BY CAPILLARY ELECTROPHORESIS ............. 135

K. Kuś, A. Gonciarz, A. Kij, J. Suraj, M. Szafarz, A. Zakrzewska, M. Walczak:

OXIDATIVE METABOLISM OF CARVEDILOL BASED ON

ELECTROCHEMICAL SIMULATION COUPLED TO

LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY (EC-LC/MS/MS) ............ 137

A. Kij, A. Gonciarz, K. Kuś, J. Suraj, M. Szafarz, A. Zakrzewska, M. Walczak:

METHOD DEVELOPMENT FOR SEPARATION OF ARACHIDONIC ACID

METABOLITES FROM BIOLOGICAL SAMPLES AND THEIR

QUANTIFICATION USING LC-MS/MS TECHNIQUE ................................................. 138

F.A. Franchina, P.Q. Tranchida, I. Bonaccorsi, L. Schipilliti, P. Dugo,

L. Mondello:

FAST GC COMBINED WITH A HIGH-SPEED TRIPLE QUADRUPOLE MASS

SPECTROMETER FOR THE ANALYSIS OF UNKNOWN AND TARGET CITRUS

ESSENTIAL OIL VOLATILES ........................................................................................ 140

F.A. Franchina, M. Zoccali, P.Q. Tranchida, R. Costa, P. Dugo, L. Mondello:

FAST SPME GC COUPLED WITH HIGH-SPEED TRIPLE QUADRUPOLE MASS

SPECTROMETRY FOR THE UNTARGETED AND TARGETED ANALYSIS

OF TEA SAMPLES ........................................................................................................... 141

M. Mokhtar, J. Soukup, P. Donato, F. Cacciola, P. Dugo, A. Riazi, P. Jandera,

L. Mondello:

DETERMINATION OF BIO-ACTIVE POLYPHENOL COMPONENTS OF

PEPPER FRUIT CAPSICUM ANNUUM L. BY LIQUID CHROMATOGRAPHY

COUPLED TO MASS SPECTROMETRY DETECTION ............................................... 142

I. Jakovljević, G. Pehnec, V. Vađić:

PAH’S CONCENTRATIONS IN PM10, PM2.5 AND PM1 PARTICULATE

FRACTION IN THE AIR .................................................................................................. 143

K. Naumoska, M. Puklavec, A. Albreht, I. Vovk:

NEW TRITERPENOIDS AND PHYTOSTEROLS IN VEGETABLES

IDENTIFIED BY TLC-MS ............................................................................................... 144




K. Tyrpień, C. Dobosz, A. Damasiewicz-Bodzek:

URINARY NICOTINE METABOLITES IN PATIENTS WITH MULTIPLE

SCLEROSIS DETERMINED BY PLANAR CHROMATOGRAPHY ............................ 145

C. Dobosz, K. Tyrpień, M. Szumska:

ASSESSMENT OF EXPOSURE TO PAHs PEOPLE WITH

MULTIPLE SCLEROSIS .................................................................................................. 146

T. Buhač, A. Mornar, M. Sertić, B. Nigović:

DEVELOPMENT OF A NEW LC/DAD/MS/MS METHOD FOR SIMULTANEOUS

QUANTITATION OF 11 ACTIVE INGREDIENTS IN VARIOUS DIETARY

SUPPLEMENT MATERIALS AND FORMULATED PRODUCTS FOR

MENOPAUSAL SYMPTOMS .......................................................................................... 147

M. Sertić, A. Mornar, B. Nigović:

FAST ALLICIN SCREENING IN GARLIC DIETARY SUPPLEMENTS

BY DIRECT INJECTION TANDEM MASS SPECTROMETRY ................................... 148

A.M. Sulej, Ż. Polkowska, J. Namieśnik:

ANALYSIS OF PAHs IN STORMWATERS COLLECTED FROM

AIRPORT AREA ............................................................................................................... 149

A.M. Sulej, Ż. Polkowska, J. Namieśnik: SAMPLE PREPARATION PROCEDURES FOR DETERMINATION

OF ANTI-COROSIVE AGENTS IN AIRPORT RUNOFF WATERS ............................. 150

M. Ruzicka, M. Jirasek, M. Cizkova, F. Teply, D. Koval, V. Kasicka:

PARTIAL-FILLING AFFINITY CAPILLARY ELECTROPHORESIS

IN STUDY OF DNA-INTECALATOR INTERACTIONS .............................................. 151

S. Koprivica, I. Krizman, I. Mikac, I. Senta, S. Terzić, M. Ahel:

EVALUATION OF AN INTEGRATED PROTOCOL FOR NON-TARGET

ANALYSIS OF HYDROPHOBIC AND POLAR CONTAMINANTS IN AQUEOUS

SAMPLES USING GAS CHROMATOGRAPHY/MASS SPECTROMETRY AND

LIQUID CHROMATOGRAPHY/TIME-OF-FLIGHT MASS SPECTROMETRY ........ 153

A. Chrzanowska, A. Poliwoda, P.P. Wieczorek:

MOLECULARLY IMPRINTED SILICA MICROPARTICLES FOR SELECTIVE

SPE CONCENTRATION AND SEPARATION OF CHOSEN PHYTOESTROGENS .. 154

E. Ariburnu, I. Vovk, E. Yesilada:

DEVELOPMENT AND VALIDATION OF AN HPTLC METHOD FOR THE

QUANTIFICATION OF APIGENIN-7-O-GLUCOSIDE IN GERMAN

CHAMOMILE FLOWERS (MATRICARIA RECUTITA L.) AND ITS

APPLICATION FOR DETECTION OF ADULTERANTS ............................................. 155




I. Belamarić, J. Jelić-Balta, M. Brusić:

GAS CHROMATOGRAPHY METHOD FOR DETERMINATION OF SULFUR

COMPOUNDS IN NATURAL GAS ................................................................................ 156

F. Congiu, C. Tuberoso:

EVOLUTION OF AMINO ACIDS AND BIOGENIC AMINEs IN CANNONAU

WINE FERMENTATION PROCESSES .......................................................................... 157

D. Keškić, N. Trkulja:

OIL AND BITUMEN HYDROCARBON DISTRIBUTION FROM WELL A,

SAVA DEPRESSION, CROATIA, ANALYZED BY GAS CHROMATOGRAPHY .... 158

K. Klepac, D. Žgela:

DEVELOPMENT OF RP-HPLC METHOD FOR DETERMINATION OF SIMETHICONE IN ORAL SUSPENSION ...................................................................... 160

P. Žuvela, M. Novak, Š. Ukić, T. Bolanča:

DEVELOPMENT OF GRADIENT RETENTION MODEL IN ION

CHROMATOGRAPHY – PART I: CONVENTIONAL QSRR APPROACH................. 161

A. Vlahović, M. Novak, Š. Ukić, T. Bolanča:


CHROMATOGRAPHY – PART II: ARTIFICIAL INTELIGENCE QSRR

APPROACH ....................................................................................................................... 162

S. Mutka:

DEVELOPMENT OF UPLC METHOD FOR DETERMINATION OF IMPURITIES

IN DRUG PRODUCT USING QUALITY BY DESIGN (QbD) APPROACH ................ 163

D. Ašperger, V. Tišler, D. Mutavdžić Pavlović, S. Babić, A.J.M. Horvat,

M. Kaštelan-Macan:

HPLC-DAD-FLD DETERMINATION OF VETERINARY PHARMACEUTICALS

IN WASTEWATERS OF PHARMACEUTICAL INDUSTRY WITH AND

WITHOUT PRECOLUMN DERIVATIZATION BY FLUORESCAMINE .................... 164

D. Ašperger, S. Prašnički, M. Gavranić, D. Drljača, D. Mutavdžić Pavlović, S. Babić,

I. Mikac, M. Ahel:

SEDIMENT SAMPLE PREPARATION FOR CHROMATOGRAPHIC

DETERMINATION OF VETERINARY PHARMACEUTICALS .................................. 166

I. Vinković Vrček, F. Mlynek, W. Goessler:

NANOSILVER PARTICLES VERSUS IONIC SILVER – CHROMATOGRAPHIC

DIFFERENTIATION AND QUANTIFICATION ............................................................ 168

S. Lock, E. Loge:

THE USE OF MICROFLOW UHPLC AS A WAY TO SOLVENT USAGE IN

PESTICIDE SCREENING OF FOOD SAMPLES BY LC-MS/MS ................................. 169




S. Lock, E. Loge:

Can LCMSMS be used in horse meat detection? ............................................................... 170

S. Lock, E. Loge, C. Hunter, R. van Soest, X. Zhu:

EXPLORING THE SENSITIVITY DIFFERENCES FOR TARGETED PEPTIDE

QUANTIFICATION IN THE LOW FLOW RATE REGIME .......................................... 171

C. Kanakaki, E. Rosenberg, A. Trifonova, J.-H. Han:

GAS CHROMATOGRAPHIC/MASS SPECTROMETRIC TECHNIQUES FOR

THE ANALYSIS OF DECOMPOSITION PRODUCTS AND GASES EMITTED

FROM LITHIUM-ION CELLS UNDER EXTREME CONDITIONS OF USE .............. 172

A. Poliwoda, K. Orłowska, K. Bury, P.P. Wieczorek:

LIQUID MEMBRANE EXTRACTION TECHNIQUES AS A SAMPLE

PRETREATMENT PROCEDURE IN ANALYSIS OF ENDOCRINE

DISRUPTING COMPOUNDS FROM FOOD SAMPLE MATRICES ............................ 173

A.I.C. Gonçalves, L.A. Rocha, J.M. Dias, A. Sousa, L.A. Passarinha:

STUDY OF A ZINC BASED GELLAN GUM CHROMATOGRAPHIC

STATIONARY PHASE USING EXPERIMENTAL DESIGN ........................................ 174

M. Bevardi, J. Bošnir, G. Horvat, S. Serdar, D. Brkić:

DETERMINATION OF BIOGENIC AMINE, HISTAMINE, IN SAMPLES OF

CANNED FISH USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY .... 175

S. Jelušić, A. Galić, L. Barušić, D. Brkić, J. Bošnir:

DETERMINATION OF SPECIFIED POLYCHLORINATED BIPHENYLS

IN PAPER AND BOARD .................................................................................................. 176

O. Ferroukhi, S. Guermouche, M.H. Guermouche, J.P. Bayle:

INFLUENCE OF LATERAL DODECYLOXY GROUPS ON THERMAL AND

ANALYTICAL PROPERTIES OF A NEW HPLC BONDED LIQUID CRYSTAL

STATIONARY PHASE ..................................................................................................... 177




INTERNATIONAL

CHROMATOGRAPHY

SCHOOL




S 1

ISOCRATIC VS. GRADIENT ELUTION IN ION CHROMATOGRAPHY

J. Weiss

Thermo Fisher Scientific GmbH, Am Woertzgarten 10, 65510 Idstein, Germany


Ever since the introductory paper on ion chromatography (IC) by Small et al. in 1975,

the versatile mixture of sodium carbonate and sodium bicarbonate finds widespread

application in traditional anion exchange chromatography, employing suppressed conductivity

detection. There are a number of reasons why this eluent mixture became so popular:

The selectivity is determined solely by the concentration ratio of these two

compounds.

Carbonate/bicarbonate mixtures represent a chemical buffer system which is of

advantage, because sample pH usually differs from eluent pH.

Precise adjustment of eluent pH (Henderson-Hasselbach equation).

A great variety of inorganic and organic anions can be separated with this eluent

combination. As the product of the suppressor reaction, the carbonic acid is only weakly

dissociated, so that the background conductivity is relatively low. However, the residual

conductivity of the suppressor product (carbonic acid) has some undesirable effects on the

performance of such separations. It creates a large void dip which can hamper the integration

of early eluting peaks such as fluoride and acetate. Also, in a background of carbonic acid, the

calibration functions for most species are not linear. Removing the residual carbonic acid

prior to conductivity detection by employing a Carbonate Removal Device (CRD 300)

eliminates most of these limitations, but gradient elution is still impractical with

carbonate/bicarbonate eluents.

The application of pure hydroxide eluents was regarded to be disadvantageous in the

past, although water as the suppressor product produces virtually no background conductance.

However, since hydroxide ions exhibit only a small affinity towards the stationary phase, it is

necessary to work with relatively high concentrations to elute anions with more than one

negative charge. This has an adverse effect on the background conductance because packed

bed suppressors and hollow fiber suppressors used in the past possessed only a limited

suppression capacity. Only since the introduction of high-capacity membrane-based

suppressors it became possible to use hydroxide eluents with concentrations up to 0.1 mol/L

at analytical flow rates up to 1 mL/min. With modern capillary IC, the maximum hydroxide

concentration that can be suppressed could even be increased to 0.2 mol/L due to the small

flow rate of only 10 µL/min. At these high concentrations it is possible to elute even

polyvalent analyte ions. Hydroxide eluents are, therefore, perfectly suited for gradient elution

of anions in combination with suppressed conductivity detection and predominantly used for

this purpose today. However, the concept of electrolytic eluent generation is almost

mandatory for a successful application of gradient elution in anion exchange chromatography.

Based on a number of applications, isocratic and gradient elution techniques in anion

exchange chromatography with suppressed conductivity detection will be compared in this

presentation. Example will be given to illustrate the applicability of both techniques, and the

influence of the selected chromatographic conditions on the retention and calibration

behaviors will be discussed.

2

mailto:[email protected]




S 2

ULTRAPERFORMANCE CONVERGENCE CHROMATOGRAPHY:

EXPANDING SELECTIVITY FOR THE CHROMATOGRAPHIC

LABORATORY

M. Boras

Waters GesmbH, Hietzinger Hauptstraße 145, 1130 Vienna, Austria


UltraPerformance Convergence Chromatography [UPC2] is a holistically designed

chromatographic system that utilizes liquid CO2 as a mobile phase to leverage the

chromatographic principles and selectivity of normal phase chromatography while providing

the ease-of-use of reversed-phase LC.

Built utilizing proven UPLC Technology, the ACQUITY UPC2 System is designed to

enable scientists the ability to address routine and complex separation challenges while

delivering reliability, robustness, sensitivity and throughput never before possible for this

analytical technique.

Unlike normal phase LC, the supercritical CO2 used in convergence chromatography

is miscible with the entire eluotropic series of solvents. In addition, both traditional normal

phase and reversed-phase column chemistries can be utilized. The ability to merge an

exceptional number of column and solvent choices provides a chromatographer significant

range of selectivity combinations to develop separations for a diverse range of compounds. In

this seminar, we will discuss the exceptional increase in available selectivity that convergence

chromatography brings to the chromatographic laboratory.

3





LECTURES




L 1

APPLYING THE HIGH EFFICIENCY, AND UNIQUE SELECTIVITY,

OF SUPER CRITICAL FLUID CHROMATOGRAPHY (SFC)-MS,

UPLC-MS, AND GC-MS, IN PLANT METABOLOMICS

S. Malitsky, I. Rogachev, A. Aharoni, A. Tishbee

The Weizmann Institute of Science, Rehovot, Israel


Plant constant interaction with the environment, yielded a most dynamic and diverse

Metablome profile.

The estimated size of the Metabolome in yeast: approx. 700 compounds, while in

plants: up to 200,000 compounds in a single plant.

Metabolome describes the complement of all metabolites expressed in a cell, tissue or

organism during its lifetime

Evolutionary processes directed towards enhancing plant fitness most probably

stimulated formation of new structures like, fruit flavor and aroma compounds, flower and

fruit pigments, "sun screen" metabolites such as the flavonols, and sulfur-containing defense

compounds (i.e. glucosinolates). Metabolite composition offers a powerful tool for decoding

gene function and regulatory processes.

Multiple modes of analysis are needed to enable identification as well as quantitation

to get a better understanding of these thousands of Metabolites.

Supercritical fluid chromatography (SFC), use carbon dioxide (CO2) as a primary

mobile phase. Its supercritical gas-like properties result in higher diffusion and mass transfer

rates, and its polarity can be controlled with a cosolvent.

The new modern SFC instrument utilizing sub micron particle columns, have high

efficiency short analysis time and reproducibility, As a result, SFC- Q-TOF-MS, proved to be

a very useful tool analyzing metabolites with a wide polarity range, including the thermally

labile ones.

SFC- Q-TOF-MS; UPLC and GC-MS is applied to analyze identify and quantify

carotenoids, chlorophyls, and brassinosteroids in plant tissue.

Brassinosteroids are polyhydroxylated steroids. with a wide spectrum of physiological

effects, including promotion of cell elongation and division, enhancement of tracheary

element differentiation, retardation of Shedding of flowers and leaves and fruit, enhancement

of gravitropic-induced bending, promotion of ethylene biosynthesis, and enhancement of

stress resistance.

5





L 2

INSTRUMENTAL ASSISTANCE IN THE TRANSFER OF METHODS

BETWEEN DIFFERENT HPLC/UHPLC SYSTEMS

R.M. Sample

Agilent Technologies Ltd., Lakeside, Cheadle Royal Business Park,

Stockport, Cheshire, SK8 3GR, UK


With the continual development of HPLC/UHPLC systems there is a wider range of

fundamental performance characteristics available in the market than ever before. This

presents an increasing challenge to regulated laboratories that are required to run methods on

different systems as properties such as different delay volumes can change the resulting

separation. Careful calculation of equivalent method parameters or the physical alteration of

delay volumes can often mitigate these differences but this is not always enough. In

particular, the different mixing characteristics of pumps can affect the shape of a gradient

profile and this can only be measured experimentally. This presentation examines these issues

and considers different approaches using hardware, software or method modifications to

ensure accurate method transfer.

6





L 3

BIOMEDICAL AND BIOANALYTICAL ASPECTS OF PRECLINICAL

AND CLINICAL PHARMACOKINETICS AND

DRUG METABOLISM RESEARCH

I. Klebovich

Semmelweis University, Department of Pharmaceutics

H-1092 Budapest, Hőgyes Endre St. 7., Hungary


The present lecture gives a comprehensive overview about the preclinical and clinical

pharmacokinetic and metabolism studies carried out on five different species which are

essential for the marketing authorization in accordance with the requirements of health

authorities.

The extreme sensitivity (atg/ml, pg/ml, fg/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 GC, HPLC bioanalytical methods of conventional

detections.

The different types of radioactive detection enables high selectivity (only the 3H-,

14C- labeled compounds and their metabolites are detectable) with extremely good sensitivity

and resolution. The different imaging techniques (in vitro: DAR, Phosphor Imaging

Technology, MALDI Imaging, in vivo: animal PET, MRI, CT) will be demonstrated in

different phases of preclinical research.

The presentation intends to give an overview of the process and up-to-date

bioanalytical tools of the in vitro and in vivo drug metabolism. Several examples illustrate the

possibilities of the quick fingerprint radio-bioanalytical examination of drug candidate in the

comparison of species. In silico and in vitro metabolism studies bring important decisional

elements for the selection of the best candidate(s) entering clinical development and

represent valuable tools to optimize future clinical studies. The minor and subminor

metabolites can be separated and detected by a novel on-line hyphenated technique method

OPLC-DAD-RD-MS/MS.

The pharmacokinetic and metabolism information of different species (mouse, rat,

dog, rabbit and human), contributing to registration, are also summarized.

7





L 4

NEW GENERATION OF FUNCTIONALISED STATIONARY PHASES

FOR ION CHROMATOGRAPHY (IC)

B. Buszewski1, M. Jaćkowska

1, M. Grochowicz

2, B. Gawdzik

2

1Chair of Environmental Chemistry & Bioanalytics, Faculty of Chemistry

Nicolas Copernicus University 7 Gagarin, 87 – 100 Toruń 2Department of Polimer Chemistry, Faculty of Chemistry

Maria Curie-Skłodowska University, 33 Gliniana, 20-614 Lublin


Ion chromatography (IC) is one of the most widely used analytical techniques for the

determination of ionic compounds. Recently, the number of ion-exchange materials used in

the IC has increased enormously. Effective ion chromatography requires the packings, which

are made of very small particles with a narrow range of changes. As a packing material there

may be used a wide range of various organic and inorganic materials, where have the

presence on their surface functional groups capable for ion exchange is a common feature. In

the case of stationary phases, for the separation of anions as functional groups are used

quaternary ammonium groups which exhibit a good selectivity for the separation of inorganic

and organic anions.

This paper presents the synthesis and properties of multilayer polymeric stationary

phases with dendrimerics structure. This material has a quaternary ammonium groups as an

ion exchange centers and can be used for the determination of anions by IC. Stationary phases

were obtained as a result of chemical modification of the support surface of the organic

polymer of 1,4-di (2-hydroxy-3-metakryloyloksypropoksy)-phenol (1,4-DMH-TRIM) using

the two monomers: 1,4-butanodiolodiglicydylowego ether (BDDE) and methylamine (MA).

Obtained packings were undergone a complete physico-chemical characterization by

porosimetry, elemental analysis (CHN), microscopy (SEM, AFM), 13 C CP-MAS NMR,

ATR-FTIR, DSC and chromatography.

As a result of a multi-stage, controlled synthesis there was obtained a series of

stationary phases with a different number of layers (3, 7, 11 and 15) associated with the

carrier. The materials have been used to separate a mixture of seven anions: F-, Cl

-, NO

2-, Br

-,

NO3-

, SO42-

and HPO42-

. As eluents were applied: NaOH, Na2CO3, NaHCO3 and mixtures

thereof. The obtained column packings are characterized by a high selectivity in the

chromatographic assays.

8





L 5

GLYCANPAC AXH-1 COLUMNS FOR THE HIGH-RESOLUTION

SEPARATION AND ANALYSIS OF GLYCANS

N. Avdalović, U. Aich

ThermoFisher Scientific, Sunnyvale, CA, USA


The GlycanPac™ AXH-1 column is a high-performance, silica-based HPLC column

for simultaneous separation of glycans by charge, size, and polarity. It is designed for high-

resolution and high-throughput analysis with unique selectivity for biologically important

glycans, either labeled or native, by LC-fluorescence and LC-MS methods.

The GlycanPac AXH-1 column can be used for qualitative, quantitative, and structural

characterization of uncharged and charged glycans present in biological molecules (e.g.,

proteins).

9





L 6

HIGH-PRESSURE ION CHROMATOGRAPHY – A NEW PLATFORM

FOR HIGH RESOLUTION OR HIGH THROUGHPUT SEPARATIONS

OF IONIC COMPOUNDS

J. Weiss

Thermo Fisher Scientific GmbH, Am Woertzgarten 10, 65510 Idstein, Germany


One of the most topical subjects in conventional HPLC is the increase of sample

throughput without sacrificing resolution by utilizing UHPLC techniques. This is typically

achieved by packing separator columns of shorter length and smaller internal diameter with

separation materials of smaller particle sizes. However, even at optimal flow rates the

resulting back pressure often exceeds the pressure tolerance of traditional HPLC hardware.

Therefore, we currently witness the development of HPLC instruments with significantly

improved back pressure tolerance well above 80 MPa.

Since ion chromatography is part of liquid chromatography, it is not surprising that a

similar solution for IC is demanded as well. The fundamental difference in instrument design,

however, is the fact that the fluidic pathways in ion chromatography instruments are made of

metal-free components with a significantly lower pressure tolerance which excludes the use of

particle sizes of around 2 µm (or smaller) typically employed in UHPLC separations. While

particle sizes of common ion-exchange materials used in analytical IC are typically around

8.5 µm, so-called fast ion-exchange columns do exist, featuring 5 µm particle sizes in smaller

column formats (150 mm × 3 mm i. d.). Thus, the analysis times for anion and cation profiles

could be decreased by 50% in comparison with conventional ion exchangers. But even under

these conditions, typical anion or cation profiles are characterized by a run time of around

eight minutes.

One possibility for further decreasing analysis times in IC is a flow rate increase

beyond the van Deemter optimum, which goes along with a loss of resolution due the

relatively large particle size of the ion-exchange material. Thus, this approach is only feasible

for samples with a simple analyte composition and little or no matrix contamination.

Doubling the linear velocity of the mobile phase through the separator column cuts the

analysis time in half, while keeping the back pressure of the separator column well below the

maximum pressure tolerance of the system.

The latest development in ion chromatography hardware design is the expanded

pressure tolerance of electrolytic eluent generation in capillary and analytical IC systems up to

34.5 MPa (5000 psi). This allows the use of higher linear velocities of the mobile phase in

conventional ion exchangers or the use of separator columns packed with a resin of smaller

particle size (4 µm). On the other hand, it also facilitates high resolution separations of

complex samples through the use of longer conventional or 4 µm separator columns with

standard length.

Besides the two major detection techniques for ion chromatography (conductivity and

amperometry), a new type of detection mode based on charge measurements will be presented

showing increased sensitivity and linear calibration behavior for weakly dissociated anions

and cations.

10





L 7

THE ROLE OF LIQUID CHROMATOGRAPHY COUPLED TO ICPMS

FOR ARSENIC SPECIATION ANALYSIS

W. Goessler

Institute for Chemistry – Analytical Chemistry, University Graz

Universitätsplatz 1, 8010 Graz, Austria


Already hundred years ago British scientists have found high arsenic concentrations in

marine fish and algal samples and speculated that the arsenic cannot be present as arsenic

trioxide, the well-known poison. It took more than 50 years until the mystery of arsenic in

marine samples was solved by Australian scientists. They identified arsenobetaine as

dominating arsenic compound in marine animals and arsenoribosides as major compounds in

marine algae. For the identification of these arsenicals they had to extract them from large

amounts of samples, purify the extracts and identify the compounds with NMR and X-ray

crystallography.

As soon as these compounds were identified people developed chromatographic

methods to separate the arsenic compounds and used atomic absorption as element-selective

detectors. With flame atomic absorption the detection limits were not good enough. For

graphite furnace atomic absorption the chromatographic flow rates were too low to obtain

good detection limits. Since its introduction around 25 years ago inductively coupled plasma

mass spectrometry (ICPMS) became a valuable detector for arsenic speciation analysis. The

ICP is certainly the most efficient ionization source and therefore ideal for quantification of

the separated arsenic compounds. Depending on the pH the arsenic compounds can be present

as cations, neutrals and anions. Therefore, ion chromatography is often employed for the

separation of the arsenicals.

The presentation discusses the role of liquid chromatography coupled to ICPMS in

arsenic speciation analysis and highlights the importance of this combination for investigating

the cycling of arsenic in our environment.

11





L 8

THE STUDY OF OLIGONUCLEOTIDES RETENTION BY HIGH

PERFORMANCE LIQUID CHROMATOGRAPHY

S. Studzińska, B. Buszewski

Chair of Environmental Chemistry and Bioanalytics, Faculty of Chemistry,

Nicolaus Copernicus University, 7 Gagarin St., PL- 87-100 Toruń, Poland


The field of synthetic oligonucleotides is developing quickly since these compounds

are finding many applications in experimental medicine (antisense drugs), molecular biology

and biochemistry. Their large-scale synthesis may be characterized by a high efficiency,

however it affords products that may contain closely related impurities. For this reason

additional purification step is required very often. Nowadays two separation techniques are

utilized for the separation of oligonucleotides: electrophoresis and high-performance liquid

chromatography (HPLC). HPLC has a potential to be used very widely in the study of

oligonucleotides. Numerous HPLC columns for the separation of these compounds are

commercially available, however most of them are conventional octadecyl ones. Therefore

other packing materials should be used. Moreover, due to the polyanionic character of

oligonucleotides, three modes of HPLC may be used for their chromatographic analysis,

namely ion-exchange, mixed mode and ion-pair reversed-phase chromatography (IP RP

HPLC). IP RP HPLC is used mainly for the oligonucleotide purification. The separation

mechanism is mixed-mode, since it comprises reversed-phase and anion exchange selectivity.

The principal aim of our work was to investigate the retention of oligonucleotides on

various stationary phases (octadecyl, alkylamide, cholesterol, alkyl-phosphate) for liquid

chromatography. IP RP HPLC has been used to separate modified and unmodified

oligonucleotides. Different ion-pair reagents were tested. Several oligonucleotides differing at

various base positions in sequences of 20 bases was studied. The dynamic nature of IP RP

HPLC mechanism allows for selectivity changing based on charge as well as sequence.

Acknowledgements

The authors are grateful to Foundation for Polish Science for Parent-Bridge

programme (POMOST/2011-3/9), cofinanced from European Union, Regional Development

Fund.

12





L 9

NEW MONOLITHIC POLY(METHACRYLATE) COLUMNS FOR

REVERSED-PHASE AND AQUEOUS NORMAL-PHASE SEPARATION

OF LOW-MOLECULAR COMPOUNDS IN 1D AND 2D HPLC

P. Jandera, M. Staňková, T. Hájek

Department of Analytical Chemistry, Faculty of Chemical Technology, University of

Pardubice, Studentská 573, CZ-53210 Pardubice, Czech Republic


Generally, it is difficult to achieve good efficiency for low-molecular compounds on

organic polymer monolithic columns. We investigated the effects of the polarity and size of

functional and cross-linking monomers used in the polymerization mixture on the separation

properties of capillary- and micro-HPLC monolithic columns prepared by in-situ

polymerization in fused silica capillaries. By careful optimization of polymerization

conditions, we prepared new types of stable poly(methacrylate) micro-columns with inner

diameters 0.1 - 0.5 mm for the HPLC separation in the HILIC and RP separation mode,

providing the efficiencies of up to 80 000 theoretical plates/m for low-molecular compounds.

The columns showed excellent permeability and very good batch-to-batch reproducibility.

The zwitterionic (poly)methacrylate micro-columns prepared by in-situ polymerization using

cross-linkers with larger polar molecules can be used alternatively either in the reversed-

phase, or in the aqueous normal-phase (HILIC) mode. The columns show also size-exclusion

and chiral selectivity for some types of compounds. The new 0.5 mm i.d. zwitterionic

columns show excellent performance, outperforming similar particulate zwitterionic columns,

in two-dimensional LCxLC of phenolic compounds and flavonoids, when used in the first

dimension, coupled on-line with various "fast" monolithic or superficially porous second-

dimension columns. The monolithic columns can be used in the first dimension in alternating

RP and HILIC application modes, just by applying different mobile phase gradients. This

approach thus implements certain features of three-dimensional separations.

Acknowledgement

This work was supported by the Grant Agency of the Czech Republic under project

P206/12/0398.

References

1. P.Jandera, M. Staňková, V. Škeříková, J. Urban, J. Chromatogr. A, 1274 (2013) 97.

2. M. Staňková, P. Jandera, J. Urban, V. Škeříková, J. Chromatogr. A, 1289 (2013) 47.

3. P. Jandera, T. Hájek, M. Staňková, K. Vyňuchalová, P. Česla, J. Chromatogr. A, 1268

(2012) 91.

13





L 10

FLOW RATE CAUSED SELECTIVITY CHANGES OF SERIALLY

COUPLED COLUMNS IN HPLC

T. Welsch, U. Hermann

Ulm University, Department of Analytical and Bioanalytical Chemistry,

Albert-Einstein-Allee 11, 89069 Ulm, Germany


Admittedly, selectivity plays a crucial role in the successful separation of analytes in

all chromatographic methods but it is especially important in liquid chromatography because

of the limited plate numbers of HPLC columns. In liquid chromatography selectivity is

mainly determined by the stationary phase, the type and composition of the mobile phase and

- long time underestimated - on temperature.

In case of serially coupled columns having a different retention characteristic

respectively, the total selectivity is additionally influenced by the local mobile phase

velocities and the lengths and phase ratios of the individual columns. These influences can

advantageously be used to optimize the selectivity for pairs of analytes. The individual mobile

phase velocities in the columns involved can be adjusted by adding or draining a mobile phase

flow between the coupled columns via a T-piece.

We reported first time flow rate driven selectivity changes in HPLC by adding a

secondary mobile phase flow at the mid-point between the two columns [1]. Further examples

for this type of selectivity tuning in HPLC in contrast to true two-dimensional HPLC were

presented in several papers [1-3]. Albeit the technique is so simple, it found not much

acceptance in practice. Maybe one reason was the then lack of commercially available

columns with alternative selectivities. This has changed over the course of the last five years.

We report in this contribution on our latest results of flow rate controlled selectivity

changes of different serially coupled column systems by variation of a split flow between the

columns. The theory behind will be briefly discussed and technical details and applications

examples will be presented.

References

1. T. Welsch, U. Dornberger, D. Lerche, J. High Resol. Chromatogr. 16 (1993) 18

2. J. Dungelová, J.Lehotay, J.Krupcik, J. Cizmárik, T. Welsch, D.W. Armstrong,

J. Chromatogr. Sci. 42 (2004) 135

3. A.P. Köhne, U. Dornberger, T. Welsch, Chromatographia 48 (1998) 9

14





L 11

HPLC AND HPTLC OF THE MAJOR DIETARY CAROTENOIDS

I. Vovk1,2

, B. Simonovska1, V. Glavnik

1,2, L.

Brulc

1, A. Albreht

1,2, Z. Rodić

1

1National Institute of Chemistry, Laboratory for Food Chemistry, Hajdrihova 19,

SI-1000 Ljubljana, Slovenia 2EN-FIST Centre of Excellence, Dunajska 156, SI-1000 Ljubljana, Slovenia


Carotenoids β-carotene, α-carotene, β-crpytoxanthin, lycopene, lutein, zeaxanthin, and

astaxanthin are part of the everyday human diet and show different health protective effects

(e.g. preventing vitamin A deficiency, antioxidant and immune-enhancing activity, cancer

prevention). Apart from those extracted from natural sources, synthetically produced

carotenoids became a big world business: projection is 1.3 billion $ till 2017.

Carotenoids are not stable compounds, especially if exposed to oxygen, heat, light and

acids and require special care during the analysis. Nowadays, HPLC methods coupled to PDA

and MS detector prevail for their separation and quantification in a wide range of

concentrations in samples as plasma, vegetables, fruits and food supplements. However, TLC

sometimes offers additional useful information about the analytes and represents an

alternative and complementary technique. Different layers and numerous development

solvents were applied for the TLC separations of carotenoids in the past. Besides the limited

separation capacity, stability of carotenoids on the TLC plate represented the main drawback

compared to HPLC.

The aim of our work was to develop TLC and HPLC chromatographic methods for the

analysis of the major dietary carotenoids in plants, foods and food supplements after

optimised extraction or saponification. A substantial improvement of stability of carotenoids

on the RP C18 HPTLC plates enabled confirmation of identity by in situ visible spectra and

TLC-MS, as well as densitometric quantitation in ng range. Triethylammonium acetate buffer

(pH 7) introduced to the mobile phases in the HPLC separations of carotenoids performed on

C30 and additionally C18 core-shell columns resulted in enhanced peak areas and lower RSD

of peak areas. The advantage of using triethylammonium acetate buffer instead of

triethylamine in mobile phases is avoiding peak tailing and high back pressure. Besides the

prevailing all-trans compounds a number of geometric isomers were separated and identified

by visible spectra.

15





L 12

ADVANCED LIQUID CHROMATOGRAPHY TECHNIQUES FOR THE

SCREENING OF LIPID MOLECULES IN FOOD

F. Cacciola1,2

, Paola Donato3,4

, M. Beccaria4, P. Dugo

4,3, A. Cotroneo

4, L. Mondello

4,3

1Dipartimento di Scienze dell'Ambiente, della Sicurezza, del Territorio, degli Alimenti e della

Salute,University of Messina, viale F. Stagno d'Alcontres 31, 98166 Messina, Italy 2Chromaleont s.r.l. A start-up of the University of Messina, c/o Dipartimento di Scienze del

Farmaco e dei Prodotti della Salute, University of Messina, viale Annunziata,

98168 Messina, Italy 3Centro Integrato di Ricerca (C.I.R.), University Campus Bio-Medico of Rome, Via Álvaro del

Portillo, 21, 00128 Roma, Italy 4Dipartimento di Scienze del Farmaco e dei Prodotti per la Salute, University of Messina,

viale Annunziata, 98168 Messina, Italy


Lipids are one of the major constituents of foods and are important in our diet for

several reasons. Firstly, they are a major source of energy providing essential lipid nutrients;

also, in many foods the lipid component plays a major role in determining the overall physical

characteristics, such as flavor, texture, mouthfeel and appearance.

From an analytical standpoint, liquid chromatography (LC) is the most widely applied

technique to the analysis of food lipids. As far as detection is concerned, as lipids typically

lack a chromophore for the required light absorption, mass spectrometry (MS) is normally

used for a full characterization of the sample.

However, when dealing with very complex samples, the resolving power of liquid

chromatography is often not satisfactory thus requiring more sophisticated analytical

techniques. This contribution highlights how very high separation efficiency could be

achieved by using serially coupled columns or two-dimensional comprehensive liquid

chromatography (LC×LC) techniques. In the former case, the possibility to serially connect

octadecylsilica columns, under ultra high pressure conditions (UHPLC), turned out to be a

powerful tool for unravelling the complexity of many lipid real-world samples. However, a

further separation improvement was achieved through the employment of the LC×LC

technique, where two columns with complementary selectivity, connected by means of high-

pressure switching valves, are usually employed. The enhanced separation power also

rendered the interpretation of MS data fairly easier and more reliable due to the attainment of

high-quality spectra for completely resolved lipid molecules.

Acknowlegments

The Project was funded by the Italian Ministry for the University and Research

(MIUR) within the Relevant National Interest Projects (PRIN) prot. 2009ZHMKA5

“Advanced analytical and chemometric techniques applied to assessment of triacylglycerol

and linear esters profile in edible oils as a tool for purity assessment and geographical

traceability”.

16





L 13

CHROMATOGRAPHY AND MASS SPECTROMETRY AS A TOOL

FOR THE CHARACTERIZATION OF INTERACTIONS BETWEEN

SHIKONIN AND Β-LACTOGLOBULIN

A. Albreht1,2

, I. Vovk1,2

, B. Simonovska1

1National Institute of Chemistry, Laboratory for Food Chemistry, Hajdrihova 19, SI-1000

Ljubljana, Slovenia 2EN-FIST Centre of Excellence, Dunajska 156, SI-1000 Ljubljana, Slovenia


Shikonin and its ester derivatives (shikonins) are well known secondary metabolites

which are most often found in the epidermis of the roots of many Boraginaceous plants. These

naphthoquinones are natural red pigments with a wide array of beneficial effects on human

health. Recent findings show that, apart from having anti-inflammatory, antioxidant,

antitumor, wound healing, antithrombotic, antibacterial, and antifungal properties, these

compounds could also be utilized in prevention of diabetes and obesity. Oral intake of

shikonins is at the moment somewhat limited due to their lipophilic nature and, consequently,

their poor solubility in aqueous media.

In our previous study we increased the water solubility of shikonin up to 181-fold by

adding β-lactoglobulin to the aqueous solution. β-Lactoglobulin is one of the major water

soluble whey proteins, is generally recognized as safe, and can act as a carrier for various

small lipophilic molecules; as such, it stabilizes these ligands and increases their water

solubility. Our preliminary results from the interaction characterization studies showed that

some shikonin molecules bind to the protein via non-covalent interactions on the β-

lactoglobulin surface, and at least one pigment molecule might react with the protein and form

a covalent bond.

Here we report how chromatography and mass spectrometry can be utilized to

elucidate if possible covalent reaction between shikonin and β-lactoglobulin really takes

place. Thin-layer chromatography and high-performance liquid chromatography in

combination with mass spectrometry were used in the course of this study. Shikonin-β-

lactoglobulin product was characterized either under protein denaturing conditions, or after

tryptic digest of the product, or after protection of particular amino acid residues prior product

formation, etc. A conclusion, from all of the obtained results, can be drawn that at least one

shikonin molecule indeed covalently binds to the protein via Cys121

.

17





L 14

ANALYTICAL ASPECTS OF BRINGING A BIOPHARMACEUTICAL

TO THE MARKET

P. Sandra, K. Sandra

Research Institute for Chromatography, President Kennedypark 26,

B-8500 Kortrijk, Belgium.


Today the pharmaceutical market is shaped with a diverse set of molecules ranging

from small synthetic molecules up to large recombinant proteins produced in living

organisms. Synthetic small molecules are still dominating the pharmaceutical market but it is

expected that within the current decade, more than 50 % of the new drug approvals will be

large biomolecules. Analytical challenges are different when one is confronted with small or

large molecule therapeutics.

In the current contribution the challenges for the analysis of bio-therapeutics will be

highlighted and solutions to tackle these challenges will be proposed and discussed.

Throughout the presentation, the recent successful introduction of a biopharmaceutical

accompanied by US and EU submission for approval, will be used to illustrate the different

analytical aspects.

18





L 15

NOVEL ADVANCEMENTS IN CHROMATOGRAPHIC

APPLICATIONS FOR THE ENRICHMENT AND SEPARATION OF

BIOMOLECULES

G.K. Bonn

Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University

Innsbruck, Innrain 80-82, 6020 Innsbruck, Austria


Separation of biological samples has become increasingly important and new

advancements in chromatography have boosted progress in many bioanalytical applications.

Considerable progress has been made in the development of highly efficient stationary phases

for sample preparation and separation. In this talk, novel enrichment and desalting methods

based on organic monoliths with incorporated nanoparticles in shape of pipette tips for

desalting and phosphopeptide recovery will be presented. In another approach precipitation of

phosphorylated proteins and peptides is shown by trivalent lanthanide ions for further

investigations by MALDI and LC-MS. Major focus is placed on the development of novel

innovative analytical techniques using high performance single- and multi-dimensional

separation methods including µ-HPLC and capillary electrophoresis CE coupled to mass

spectrometry (MS). New polymeric capillary monoliths, especially tailored for miniaturized

HPLC offer a highly efficient unique separation platform for applications in metabolomics

and proteomics. The monolithic structure reduces the diffusion path and provides high

permeability, resulting in excellent separation efficiency. Operation at high and very low

flow-rates, stability at high pH, and low column pressure drop can yield flexibility and speed

for many HPLC separations. All of the presented monolithic capillaries have been

successfully designed for the application of high- and low molecular weight compounds,

showing that organic monoliths are serious alternatives toward silica-based stationary phases.

Separation efficiencies of the presented capillaries are outstanding, as they have no inter-

particular void volume and retaining frits, which results in a reduced resistance to mass

transfer as a result of convective flow. Characterization of stationary phases is carried out by

high performance spectroscopic methods including near- and mid-infrared spectroscopy.

19





L 16

ROLE OF THE LIQUID PHASE ON THE SEPARATION OF

BIOMOLECULES BY HPLC AND CAPILLARY ELECTROPHORESIS:

FUNDAMENTAL AND PRACTICAL ASPECTS

D. Corradini, I. Nicoletti

Institute of Chemical Methodologies, National Research Council,

Area della Ricerca di Roma 1

I-00015 Montelibretti, Rome, Italy


Most chromatographic and electrophoretic methods used for the analytical separation

of biomolecules are performed in aqueous solutions whose composition might affect to

different extent selectivity, resolution and efficiency. This aspect is particularly relevant for a

significant number of biomolecules bearing different concomitant functionalities, consisting

of ionisable and/or hydrogen-bonding groups, hydrophobic regions, and hydrophilic moieties.

Such multifunctional molecules may interact to different extents with the various components

of the surrounding aqueous solution and with either the stationary phase or the capillary wall,

in chromatography and in capillary electrophoresis, respectively. This communication

discusses the results of our recent studies conducted to investigate a variety of factors that

influence both electrophoretic and chromatographic behaviour of biomolecules of particular

interest in plant physiology and food chemistry. The presentation evaluates the influence of

the composition of either the electrolyte solution (BGE) or the mobile phase on the selective

separation of representative plant secondary metabolites in capillary zone electrophoresis

(CZE) and in reversed phase liquid chromatography (RP-HPLC), respectively. Appropriate

selection of the composition of either the BGE or the mobile phase involves the evaluation of

the equilibrium in solution that might take place between the analytes and the components of

such solutions. The result is the possibility of tailoring selectivity and efficiency of the

considered separation systems by incorporating suitable buffering agents and additives into

the BGE or the mobile phase, respectively. Practical applications of these approaches to the

analysis of phenolic compounds in samples extracted from a variety of plant tissues and food

of vegetable origin are then discussed.

20





L 17

ESTABLISHMENT OF THE CORRECT DIAGNOSIS BY

CHROMATOGRAPHY AND MASS SPECTROMETRY

Ž. Debeljak

Department of Clinical Laboratory Diagnostics, Clinical Hospital Center „Osijek“,

31000 Osijek, Croatia

E-mail [email protected]

During the last few decades chromatographic and mass spectrometry techniques

entered clinical diagnostics field. In some instances they replaced exisitng immunoassays or

spectrofotometric methods but they also enabled introduction of completely new analytes i.e.

diagnostic parameters in clinical practice. Thanks to these techniques even the new clinical

entities have been recognized.

Almost all types of chromatography and mass spectrometry have been applied in

clinical diagnostics. But, HPLC, GCMS and LCMSMS play the most prominent roles in

routine use. The main advantages of these analytical techniques over other diagnostic

approaches are their sensitivity, selectivity and the substance identification capability.

Submicromolar concentrations of different analytes can be easily measured by these

approaches. Other techniques already available in clinical laboratories are rarely so sensitive.

Numerous interferences present in biological samples are easily recognized and their

influence can be eliminated by these techniques. Therefore, different chromatographic and

mass spectrometry methods have become reference methods for various diagnostic

parameters.

Still, these techniques are not widely used in routine. The main limitations are

demanding sample preparation and a limited robustness. These properties disable full

analytical process automation needed to fulfill ever increasing demands for short turn around

times. In instances when high accuracy is not an issue other techniques fill in. But therapeutic

drug monitoring, toxicology and forensic pathology, congenital metabolic diseases and

endocrinological disorders are clinical fields that take full advantage of chromatography and

mass spectrometry. It is expected of these techniques to take a more prominent role in protein

diagnostics. Clinical utility, current status and future clinical prospects of chromatography and

mass spectrometry will be reviewed through paradigmatic case studies.

21





L 18

PEPTIDE DE NOVO SEQUENCE ASSIGNMENT

M. Cindrić

Centre for Proteomics and Mass Spectrometry, Division Of Molecular Medicine,

„Ruđer Bošković“ Institute, Planinska 1, Zagreb, Croatia


A novel method for peptide amino acid sequence determination upgraded with

genome fingerprint scanning (GFS) was designed to investigate gene expression of the

organisms which proteome is still not well known. Unknown proteomes can be used as

models in these experiments (e.g. congenital diseases, cancer cells). The method links

proteomic data, consisting of determined tryptic peptides amino acid sequences obtained by

negative and positive ion mode MALDI tandem mass spectrometry (MS/MS) matched against

sequenced genome of the observed organism and conformed by mass spectrometry (MS) data

of elucidated peptide mass fingerprints sequences derived from the annotated genome [1]. The

idea that lies behind this new technology is enhanced de novo sequencing of unknown peptide

amino acid sequences in negative (enabled by mild overnight derivatization by carrier that

contains two negatively charged groups without observed side reactions or peptide

degradation) and positive ion mode of the same peptide used as quality control (MS/MS of

derivatized or underivatized precursor ions).

The method can be compared to DIGE (Differential Gel Electrophoresis) [2] when

fluorescence tag would be compared to negatively charged N-terminal tag in negative ion

mode mass spectrometry. The enhancement of negatively charged ions’ ionizability (similar

to fluorescence effect in spectroscopy) and dissociation in negative ion mode ensures data

confidence without ion adduction or in-source decay interferences characteristic for positive

ion mode peptide mixture analysis. This technology enables unambiguous determination of at

least five to six amino acids and often more in a row from a MS/MS experiment only in one

ion mode, but it can be combined as b-ion series from N-terminus (negative ion mode) and

vice versa y-ion series from C-terminus (positive ion mode), even though derivatized ions are

“invisible” as precursor ions in positive ion mode.

References

1. Giddings M.C. DDT: TARGETS, Genome fingerprint scanning for protein

identification and gene finding, 2004, 3, S56-S62

2. Friedman, D.B. et al. Proteomics, Proteome analysis of human colon cancer by two-

dimensional difference gel electrophoresis and mass spectrometry, 2004, 4, 793–811

22





L 19

COMPREHENSIVE MOLECULAR CHARACTERIZATION OF

COMPLEX POLYMER SYSTEMS BY ADVANCED LIQUID

CHROMATOGRAPHY METHODS

D. Berek

Polymer Institute, Slovak Academy of Sciences, 845 41 Bratislava, Slovakia


Molecular characteristics of macromolecules that co-determine the end-use properties of

resulting polymeric materials are molar mass, chemical structure (composition) and physical

architecture. All molecular characteristics of synthetic polymers exhibit certain dispersity

(distribution). Complex polymers possess several dispersities in their molecular characteristics.

Typical examples are various copolymers, functional, and stereoregular polymers, as well as

blends of linear homopolymers. A mixture of a complex polymer with other macromolecular

substance(s) of distinct nature is called complex polymer system. Comprehensive molecular

characterization of complex polymer systems represents a challenging task. Molar mass

averages and dispersities of synthetic polymers are most often determined by size exclusion

chromatography, SEC. As known, SEC separates macromolecules according to their size in

solution, which depends not only on molar mass but also on other molecular characteristics of

polymers to be characterized. Therefore, SEC cannot directly produce exact, quantitative data

on the molar mass averages and dispersities of complex polymers and complex polymer

systems. Due to its limited both selectivity of separation and sensitivity of detection, SEC can

hardly discriminate polymer species of different nature that possess similar size in solution and

identify minor macromolecular constituents (<1%) in complex polymer systems even if their

molar masses differ significantly. To solve these tasks, the exclusion, entropy controlled

separation mechanism of SEC is to be combined with one or several interaction, enthalpy

driven separation mechanisms. The resulting procedures are designated coupled methods of

polymer liquid chromatography such as eluent gradient liquid chromatography, EG LC,

temperature gradient interaction liquid chromatography, TGIC, liquid chromatography under

critical conditions of enthalpic interactions, LC CC and liquid chromatography under limiting

conditions of desorption, LC LCD. The principle, advantages and limitations of LC CC and LC

LCD methods will be elucidated and compared in the contribution. The coupling of separation

mechanisms within one single HPLC column is often insufficient for assessment of complex

polymers, and especially for separation and molecular characterization of complex polymer

systems. It is necessary to combine two different HPLC procedures executed in two

independent chromatographic systems. Such combination is denoted two-dimensional polymer

liquid chromatography, for short 2D-LC or also LCxLC. Principles and limitations of the

existing 2D-LC procedures will be outlined. A novel approach will be presented, namely a

combination of LC LCD and SEC, called sequenced two-dimensional polymer liquid

chromatography, S2D-LC. The procedure includes fast separation of multicomponent complex

polymer systems with help of LC LCD into defined fractions while the latter are in their

entirety online forwarded into the SEC column(s). In this way, constituents of complex polymer

systems can be comprehensively characterized as to their amount and molar mass average and

dispersity. Minor macromolecular admixtures well below one percent can be analyzed with

help of S2D-LC. Typical examples of sequenced two-dimensional separations will be

23





presented, the separation and molecular characterization of parent homopolymers in block

copolymers being the important application.

Acknowledgement This work was supported with the Slovak Grant Agencies VEGA (Project 2/0001/12)

and APVV (Project 0109/10).

24




L 20

SOLVED AND UNSOLVED PROBLEMS IN EVALUATING THE

PROPERTIES OF PLASTIC MATERIALS BY CHROMATOGRAPHIC

AND MASS SPECTROMETRIC METHODS

W. Buchberger, S. Beissmann, I. Hintersteiner, L. Sternbauer

Johannes-Kepler-University, Analytical Chemistry, Altenbergerstrasse 69,

A-4040 Linz, Austria


Plastic materials made from synthetic polymers often provide substantial advantages

in comparison to traditional materials. However, these materials are also prone to

degradation by sunlight and oxygen. Therefore, the addition of different stabilizers is

essential. Rapid screening methods for such stabilizers are of significant importance in order

to evaluate the suitability of materials of unknown origin for certain application areas, to

clarify reasons for failure of materials, or for comparison of materials from different

suppliers.

Qualitative analysis of solid polymeric materials without sample preparation turned

out to be possible by advanced mass spectrometric approaches like direct analysis in real

time (DART). Unfortunately, not all of the different chemical classes of stabilizers are suited

for this technique. As an alternative, extracts of plastic materials were analyzed by flow

injection / tandem mass spectrometry with electrospray ionization. The selection of

appropriate transitions from precursor to product ions allowed simultaneous

semiquantitative screening of 36 stabilizers without interferences. Nevertheless, quantitation

of contents of stabilizers still required chromatographic techniques, preferably HPLC.

Besides traditional reversed phase columns, new phases stable under alkaline pH conditions

were investigated for the class of hindered amine light stabilizers that had up to now been

difficult to separate.

Finally, the applicability of GC was systematically investigated. Stabilizers may have

molecular weights up to 1000 which at first sight makes the use of this technique

questionable. Even so a careful optimization of injection conditions revealed that high

temperature GC is suited for a wide range of these analytes.

The bottleneck may still be the sample preparation step for real polymer samples.

Microwave-assisted extraction is one of the most promising pretreatment steps that can easily

be combined with chromatographic instrumentation and provides a high potential for

automated analysis.

25





L 21

RANDOMLY-BRANCHED POLYMERS BY SIZE EXCLUSION

CHROMATOGRAPHY WITH TRIPLE DETECTION: COMPUTER

SIMULATION STUDY ON BIASES IN DISTRIBUTIONS OF MOLAR

MASSES AND DEGREES OF BRANCHING

L.A. Clementi1,2

, J.R. Vega1,2

, G.R. Meira1

1INTEC (CONICET and Univ. Nac. del Litoral), Guemes 3450, Santa Fe (3000), Argentina.

2FRSF –UTN. Santa Fe, Argentina.


This article theoretically evaluates the biases introduced into the distributions of molar

masses (MMD) and number of long chain branches per molecule (LCBD), when randomly-

branched polymers are analyzed by size exclusion chromatography (SEC) with light

scattering and specific viscosity detectors. The MMDs of fractions containing from 0 to up to

75 tetrafunctional branch units per molecule were calculated with the Stockmayer equation

(1); and the calibrations of the branched classes were estimated from an imposed calibration

for the linear class combined with appropriate contraction factors. An ideal SEC model was

simulated that assumes perfect fractionation by hydrodynamic volume, except for a minor

mixing in the detector cells. Under these ideal conditions, the MMD exhibits a negligible bias;

with a maximum local dispersity of 1.0035 at the high molar masses. These results are in

accord with previous investigations (2, 3), but differ quantitatively from the observations of

Ref. (4). The MMD still remains essentially unbiased when band broadening is introduced in

the column, while maintaining perfect and noise-free measurements. However, rather poor

MMD estimates are obtained when even minor (zero-mean random) noises are added onto the

chromatograms. Only gross estimates of the LCBD are possible. This is due to theoretical

uncertainties, to errors in the required physico-chemical parameters, and to a fast propagation

of errors caused by the highly nonlinear nature of the problem. The work was carried out

under the auspices of the IUPAC project: “Data Treatment in SEC and Other Techniques of

Polymer Characterization. Correction for Band Broadening and Other Sources of Error”,

Chair G. Meira, http://www.iupac.org/web/ins/2009-019-2-400.

References

1. Stockmayer, W.H. (1943). J. Chem. Phys., 11(2), 45-55.

2. Jackson, C. (1994). J. Chromatogr. A, 662, 1-12.

3. Netopilík, M. (2012). J. Chromatogr. A, 1260, 97-101.

4. M. Gaborieau, J. Nicolas, M. Save, B. Charleux, J-P. Vairon, R.G. Gilbert, P.

Castignolles (2008). J. Chromatogr. A, 1190, 215-223.

26





L 22

RECENT ADVANCES IN ON-LINE SAMPLE PREPARATION

METHODS COUPLED TO LC-TANDEM MS FOR THE ANALYSIS OF

EMERGING CONTAMINANTS IN ENVIRONMENTAL SAMPLES

M. Petrović1,2

, M. Gorga3, R. Lopez-Serna

3, D. Barceló

2,3

1Catalan Institute for Water Research (ICRA), Scientific and Technological Park of the

University of Girona, Emili Grahit,101, E- 17003 Girona, Spain

E-mail: [email protected] 2Catalan Institution for Research and Advanced Studies (ICREA),

Passeig Lluís Companys, 23, E-08010 Barcelona, Spain 3Dept. of Environmental Chemistry, IDAEA-CSIC, Jordi Girona 18-26,

E-08034 Barcelona, Spain

The common problem in the analysis of environmental samples is that the extract

obtained by exhaustive extraction techniques typically contains a large number of matrix

components, which may co-elute with the analytes and disturb the quantitative analysis.

Generally, time and labor consuming sample pretreatments, aimed at the reduction of the

matrix content and the enrichment of the target compounds, are applied. Such tedious

extraction and clean-up protocols often constitute the bottleneck of the analytical method

since they account for more than 75% of analysis time. The growing number of samples to be

analyzed in laboratories carrying out monitoring studies requires employment of high-

throughput and fully automated analytical techniques. Because of these reasons, great effort is

going into the development of cost-effective sample handling techniques characterized by the

efficiency and simplicity of operations and devices.

Over the past several years, there has been an increase in the use of automated

instruments that integrate extraction, purification and detection step in the analysis of

emerging contaminants in environmental and wastewater samples and biosolids. Several

generic approaches have been developed for on-line sample extraction coupled to LC-MS

using different extraction supports or sorbents such as disposable SPE cartridges, large-size

particles or monolithic materials or dual LC column systems.

This presentation gives an overview of recent trend in the trace level determination of

two groups of emerging contaminants, pharmaceutical residues and their metabolites and

endocrine disrupting compounds (EDCs) in water and sediment by on-line sample preparation

methods coupled to LC-tandem MS systems. Several practical examples of analysis of multi-

class pharmaceuticals and EDCs in wastewaters using EQuanTM

Direct Injection Technology

and Turboflow systems will be presented and discussed.

27





L 23

CHROMATOGRAPHIC RESEARCHES ON THE TEXTILE DYES AND

THEIR POLLUTION EFFECTS ON WHEAT PLANTS

V. Coman1, F. Copaciu

1, L. Copolovici

2, D. Simedru

3

1Babeş-Bolyai University, “Raluca Ripan” Institute for Research in Chemistry,

30 Fântânele Street, 400294 Cluj-Napoca, Romania 2Institute of Technical and Natural Sciences Research-Development of “Aurel Vlaicu”

University, 2 Elena Drăgoi Street, 310330 Arad, Romania 3INCDO-INOE 2000, Research Institute for Analytical Instrumentation ICIA,

67 Donath Street, 400293 Cluj-Napoca, Romania.


Textile industry is a common and important sector in the world being a high consumer

of water. The residue accumulation in water in amounts exceeding the natural power of

transformation and integration of the environmental factors conducts to the appearance of

some imbalances of natural life.

In this paper are presented some modern and performance chromatographic methods

for the identification and quantification of six textile dyes (Optilan Blue and Lanasyn Blue -

anthraquinone dyes, and Lanasyn Red, Nylosan Red, Nylosan Dark Brown and Lanasyn Dark

Brown - azo dyes), and their use in the monitoring of these dyes from wastewater samples.

All performed methods were validated.

Five textile dyes (Lanasyn Blue, Nylosan Dark Brown, Nylosan Red, Lanasyn Dark

Brown, and Lanasyn Red) were studied by high performance thin layer chromatography

(HPTLC) using Alugram RP-18W/UV254 plates with the n-butanol-ethyl acetate-ammonium

hydroxide mobile phase. The HPTLC method was used to monitor these dyes in the effluent

wastewaters from a textile factory, effluents which are discharged into the sewerage network.

Five textile dyes (Nylosan Red, Optilan Blue, Lanasyn Red, Lanasyn Dark Brown, and

Nylosan Dark Brown) were studied by liquid chromatography coupled with mass

spectrometer detector (LC-ESI/MS-MS). These dyes were monitored in the influent and

effluent wastewaters from a wastewater treatment plant which potentially contain textile dyes.

The target textile dyes from the wastewater samples were isolated on Strata cartridges

by solid phase extraction (SPE).

The eco-toxicological influence of these dyes on wheat plants (Triticum aestivum L.)

was also studied. These effects were evaluated on the foliage photosynthesis, and on the

secondary metabolites (photosynthetic pigments, emissions of lipoxygenase pathway

products, and emissions of monoterpenes) in wheat.

28





L 24

DETERMINATION OF ALKYLPHENOLS POLYETHOXYLATES

(APEOS) AND ALKYLPHENOLS (APS) IN WASTEWATER AND

SURFACE-WATER THROUGH A UNIQUE

HPLC-ESI-MS/MS METHOD

L. Ciofi1, L. Checchini

1, U. Chiuminatto

2, E. Coppini

3, M. Del Bubba

1

1Department of Chemistry, Via della Lastruccia 3 – 50019 Sesto Fiorentino, Florence, Italy

E-mail: [email protected] 2ABSciex, Viale Lombardia, 218, 20047 Brugherio, Monza Brianza, Italy

3GIDA S.p.A., Via Baciacavallo, 36 – 59100 Prato, Italy

APEOS with an average ethoxylate number between 3 and 10, have been widely used

in many industrial applications, as non-ionic surfactants or antioxidants [1]. The presence of

APEOs and their biodegradation derivatives have been frequently reported in literature for

different fresh water ecosystems [2,3]. Most persistent APEOs degradation metabolites are

APEOs oligomers with 1-3 ethoxylate groups and alkylphenols (APs), which represent the

last degradation stage of the ethoxylate chain. Moreover, short-chain alkylphenoxy

carboxylates (APECs) have been also found as a result of APEO degradation [4]. Several

studies have shown that APEOs, APECs and APs exhibit estrogenicity; because of these

evidences, the European Community, by means of the Directive 2003/53/EC and subsequent

regulations, has restricted the commercialization and the use of some of these compounds.

Nevertheless, these restrictions do not cover the use of APEOs with an alkyl chain length

different from nine carbon atoms, such as polyethoxylated octylphenols (OPEOs) and the

commercialization or use of pretreated goods imported from non-European countries.

Accordingly, in some recent studies concerning the emission of nonylphenols (NPs) and their

ethoxylate derivatives (NPEOs) from goods and products, textile and leather manufacturing

have been identified as an important source of NPs to wastewater treatment plants (WWTP)

[5]. Thus, this topic is still of current concern and high-throughput analytical methods are

necessary for monitoring campaigns with the aim of evaluating the presence of these

pollutants in the environment.

Several methods, mainly based on HPLC-MS/MS, have been recently reported in

literature [6-8]. However, since APs and APEOs are characterized by different ionization

mechanism, the above-mentioned authors proposed two different runs for their determination.

Only Jahnke et al. in 2004 [9] optimized a single LC run for their simultaneous determination,

but it involved a time-consuming sample preparation and a questionable chromatographic

analysis. In the present study a deep look into the ionization key-parameters has been given,

optimizing the concentration of the mobile phase modifier, as well as the chromatographic

separation and the scheduled MS/MS experiments, allowing for developing a unique

highthroughput SPE-HPLC-MS/MS method for the determination of both APs and APEOs in

surface water and wastewater at ppt levels.

References

1. OSPAR Commission, 2009

2. G.G. Ying, B. Williams, R. Kookana. Environ. Int. 28 (2002) 215

29





3. A. Soares, B. Guieysse, B. Jefferson, E. Cartmell, J.N. Lester. Environ. Int. 34 (2008)

1033

4. M. Ahel, W. Giger, M. Koch. Water Res. 28 (1994) 1131

5. N. Månsson, L. Sörme, C. Wahlberg, B. Bergbäck. Water Air Soil Pollut. 8 (2008)

445

6. J.E. Loyo-Rosales, C. P. Rice, A. Torrents. Chemosphere 68 (2007) 2118

7. R. Loos, G. Hanke, G. Umlauf, S.J. Eisenreich. Chemosphere 66 (2007) 690

8. T. Vega-Morales, Z. Sosa-Ferrera, J.J. Santana-Rodríguez. J. Hazard. Mater. 183

(2010) 701

9. A. Jahnke, J. Gandrass, W. Ruck. J. Chromatogr. A 1035 (2004) 115

30




L 25

IDENTIFICATION OF SULFONAMIDES PHOTODEGRADATION

PRODUCTS IN DIFFERENT WATER MATRICES

M. Periša, M. Mitrevski, S. Babić

Department of Analytical Chemistry, Faculty of Chemical Engineering and Technology,

University of Zagreb, Marulićev trg 19, Zagreb, Croatia


Today it is well established that the source, presence and the fate of pharmaceutical

active compounds in the environment is of concern. This area of research has progressively

received more attention as many pharmaceuticals in surface water becomes evident. Once

released into the aquatic environment, pharmaceuticals may undergo different degradation

processes. Photodegradation, for example, might be an important elimination process for

light-sensitive pharmaceuticals, such as antibiotics. It takes place mainly in surface water,

while it may not occur when the compounds are present in turbid water, if the creek, river or

lake is shadowed by trees, or if the compounds are in soil, sewage, and sewage pipes since

they have low light exposure. The effectiveness of photodegradation depends on light

intensity and frequency, pH and hardness of the water, and type of matrix and location, and it

also varies with season and latitude.

Sulfonamides represent one of the commonly used families of antibiotics in veterinary

medicine, although they were frequently applied as human medicines to treat many kinds of

infection. Since they have aromatic rings, heteroatoms, and other functional chromophore

groups that can absorb solar radiation, photodegradation can be promising process for their

elimination.

To investigate the appearance of photodegradation products, three sulphonamides

(sulfadiazine, sulfamethazine, sulfamethoxazole) were irradiated under simulated sunlight in

three different water matrices (MilliQ water, natural water and synthetic wastewater similar

by composition to wastewater of pharmaceutical industry). Irradiated solutions were analyzed

by LC-MS/MS, which was performed with an Agilent 6410 triple-quadrupole mass

spectrometer coupled with an Agilent Series 1200 HPLC system. Except the structures of

sulfonamides photodegradation products which were suggested on the basis of mass spectra,

photodegradation kinetics of selected sulfonamides was also investigated.

Acknowledgement

This work has been supported by the Croatian Ministry of Science, Education and

Sports Project: 125-1253008-1350 „Advanced analytical methods for pharmaceuticals

determination in the environment“

31





L 26

IDENTIFICATION METHODS OF FORBIDDEN AND OBSOLETE

PESTICIDES

M. Gertsiuk, G. Lysychenko

The Institute of Environmental Geochemistry of National Academy of Sciences of Ukraine,

34а Palladin ave. Kyiv-142, 03680, Ukraine


In Ukraine and other CIS countries since the days of the former Soviet Union were

tens of thousands of tons of forbidden and obsolete pesticides .In most cases, they are stored

in unsuitable conditions in damaged packaging, without labels, often in these warehouses is

missing the real owner and documentation about pesticides. . These objects represent a

significant threat to the environment and health of people living in the area. In order to utilize

of these pesticides, or to change the packaging for further temporary storage, they should be

identified. For this purpose are used the methods of gas chromatography-mass spectrometry.

However, under these conditions, sample preparation and used equipment is often too

complex and time consuming. Elemental analysis in combination with chromatographic

methods using allows simplifying identification. Identification pesticides scheme is

considered.

32





L 27

SCREENING OF ORGANIC POLLUTANTS IN SURFACE WATER BY

ULTRA HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

COUPLED TO iFUNNEL Q-TOF/MS

D. Stipaničev1, S. Repec

1, S. Širac

2

1Hrvatske vode, Central Water Management Laboratory, Ulica Grada Vukovara 220,

10000 Zagreb, Croatia

E-mail: [email protected], [email protected] 2Hrvatske vode, Department of Development, Ulica Grada Vukovara 220,

10000 Zagreb, Croatia


The main goal of the European Union Water Framework Directive (EU WFD) is to

achieve compliance with a list of priority hazardous substances and in the ecological quality

in water bodies to identify all other chemicals that could cause deterioration. A large number

of new pesticides, as well as emerging pollutants (human and veterinary pharmaceuticals,

personal care products, steroids, explosives) are released into the environment. In the context

of the WFD there is a need to prioritize chemicals within river basins for reduction, for

monitoring or for improving scientific knowledge in order to meet European requirements and

to improve quality of waters. The challenging task for environmental researches is screening

of surface waters because different organic substances occurring in surface waters are difficult

to characterize by chemical analyses since these complex mixtures occurs at a very low

concentrations and requires both a specific analytical methods and instruments for

identification. There is a growing demand for high resolution instruments in screening

methods, which are able to identify unexpected compounds and also to quantify known

contaminants.

New generation of UHPLC-Q-TOF/MS with main characteristics like high resolution

(>10000 at full width half maximum, FWHM) accurate mass (mass accuracy <1 ppm) and

satisfactory sensitivity in full-acquisition mode for the rapid screening and quantification of

multi-class organic pollutants in water, with little sample manipulation open new multiple

possibilities for challenging environmental analysis. Full-acquisition MS data and

characteristic MS/MS scanning obtained by Q-TOF/MS provide valuable qualitative

information, served by advanced software tools which facilitates safe identification of

different compounds in surface water samples. For screening of non-target organic pollutants

in this work direct injection of large volume surface water samples on ultra high performance

liquid chromatography (UHPLC) coupled to time-of-flight mass spectrometry (Q-TOF/MS)

was used. This approach will be detailed and illustrated with surface water examples.

33







L 28

ANALYSIS, OCCURRENCE, DEGRADATION AND

TRANSFORMATION OF FLUOROURACIL IN THE ENVIRONMENT

T. Kosjek, S. Perko, D. Žigon, E. Heath

Jožef Stefan Institute, Ljubljana, Slovenia


5-Fluorouracil (5-FU) is a fluorinated pyrimidine analogue important in the treatment of

cancer whose fate in the environment is yet to be fully addressed. Due to its high polarity 5-FU

requires challenging sample preparation and therefore we thoroughly investigated different

solid phase extraction mechanisms (ion pair, ion exchange, reversed phase), sorbents and

derivatisation agents to enable trace-level analysis of 5-FU based on GC-MS/MS in natural and

wastewaters. While ion pair and ion exchange retention mechanisms were shown inappropriate

for complex environmental matrices, the reversed phase sorbent Isolute ENV+ gave the best

extraction efficiencies (53% and 93% for wastewaters and surface waters, respectivelly).

Further, alkylation was rejected in favour of silylation with MTBSTFA. The achieved LOQ for

waste and surface waters were 1.6 ng/L and 0.54 ng/L, respectively. The method was used to

analyse samples of hospital, wastewater treatment plant influent and effluent and surface

waters. 5-FU was quantified in four out of the twelve samples of oncological ward wastewaters

and municipal wastewater treatment plant influents in concentrations from 4.7 to 92 ng/L. This

work is also the first to study the environmental transformation of 5-FU and its prodrug

capecitabine (CAP). Their removal and transformation was simulated using a series of

biodegradation and photodegradation experiments, where 5-FU proved more degradable in

comparison to CAP. Transformation of 5-FU and CAP was studied by using ultra-performance

liquid chromatography coupled to a quadrupole time-of-flight mass spectrometry (UPLC-

qTOF). Overall, six transformation products for 5-FU and ten for CAP are proposed; 13 of

these are to our knowledge published for the first time.

34


http://en.wikipedia.org/wiki/Cancer




L 29

DEVELOPMENT OF HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY TANDEM MASS SPECTROMETRY (HPLC –

MS/MS) METHOD FOR DETERMINATION OF DIFFERENT CLASSES

OF PHARMACEUTICALS IN WASTE WATERS

M. Zrnčić, S. Babić, M. Kaštelan-Macan


University of Zagreb, Marulićev trg 20, Zagreb, Croatia


The presence of pharmaceuticals in the environment has been a great concern among

scientists in the last few decades. They have been classified as emerging contaminants and

their presence should be investigated in the environment. There are many ways

pharmaceuticals reach the environment but the most significant one is via wastewater

treatment plants (WWTP), because some of the pharmaceuticals are not eliminated

completely during the treatment. Majority of those end up in the water systems and can reach

drinking water sources. Once present in the environment they can have negative effects on

organisms and influence the equilibrium of ecosystems. Their possible impact on human

health also cannot be neglected. This environmental issue is still present and there is a

constant need to develop new analytical methods for the determination and quantification of

pharmaceuticals in the environment.

The aim of this study was to develop a method based on solid phase extraction (SPE)

followed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-

ESI-MS/MS) for simultaneous analysis of 22 pharmaceuticals. The choice of analytes covered

six different groups of pharmaceuticals: macrolides, fluoroquinolones, tetracyclines,

sulfonamides, anthelmintics and anesthetics. After the MS parameters were optimized, the

optimal chromatographic conditions were obtained using phenyl column. The result of sample

preparation step optimization was sorbent Oasis HLB, 60 mg with acetonitrile as eluent.

Environmental samples are very complex and the matrix can affect the efficiency of

ionization process in the ESI. In this study different approaches to compensate matrix effects

were investigated in order to obtain more accurate results.

After the optimization the method was validated and applied to the analysis of real

waste water samples.

35





L 30

COMPARISON OF GAS CHROMATOGRAPHIC METHODS FOR THE

DETERMINATION OF METHANOL AND ETHANOL IN INSULATING

MINERAL OIL AS MARKERS OF CELLULOSE DEGRADATION IN

POWER TRANSFORMER

M. Concetta Bruzzoniti1, R. Maria De Carlo

1, C. Sarzanini

1, R. Maina

2, V. Tumiatti

2

1University of Torino, Department of Chemistry, via P. Giuria 5, 10125 Torino, Italy

2Sea Marconi Technologies, Via Ungheria 20, 10093 Collegno, Torino, Italy


The presence of methanol and ethanol in transformer oils has been recently correlated

to the degradation of the solid insulation in power transformer, i.e. Kraft paper. The

evaluation and the quantification of their presence have been proposed as a useful tool to

evaluate the thermal and mechanical degradation of cellulose. Despite the advantages to use

methanol and ethanol as cellulose degradation indicators compared to the currently used

markers (carbon oxides: CO and CO2 and 2-furaldehyde), their sensitive and accurate

determination is hampered by the complexity of oil matrix.

In this work, we optimized and compared the performance of two head-space gas

chromatographic methods using different detectors (flame ionization and mass spectrometry)

to determine methanol and ethanol in mineral insulating oil samples. Linearity of both

methods was verified in the range 20-3000 ng g-1

.

As for the head-space gas chromatographic method with flame ionization detection

(HS-GC-FID), the detection limits (calculated by multiplying the Student’s t-value by the

standard deviation of the replicates at a concentration close to the estimated detection limit)

were 12 ng g−1

for methanol and 27 ng g-1

for ethanol, whereas for HS-GC-MS method, the

detection limits were 1.3 ng g−1

and 3.1 ng g-1

, respectively.

The method was validated evaluating repeatability for both analytes at different

concentration levels with both the methods in real samples of transformer oils. For HS-GC-

FID the relative standard deviation (RSD) was 4 % for 79 ng g-1

methanol and 6% for

50 ng g-1

ethanol. For HS-GC-MS, RSD was 12 % for 3 ng g-1

methanol and 7% for 14 ng g-1

ethanol. The precisions of the methods were compared by a F-test which assessed that no

significant difference in precision between HS-GC-FID and HS-GC-MS is present.

The accuracy of the methods was validated under a proficiency test within Cigré JWG

A2/D1.46.

The applicability of both the methods to the direct analysis of trace methanol and

ethanol in oil from field transformer samples was successfully demonstrated. Twenty-one in-

service oils were analyzed and the results obtained were compared by a one-sided paired t-

test, confirming that the two methods are not statistically different.

For each of the samples considered, 2-furaldehyde and CO2 were also measured and

the values obtained compared with those of methanol and ethanol.

Correlations of methanol and ethanol content in sampled oils to the years of their

activity are provided.

Both the methods developed allow reliable identification of methanol and ethanol as

new markers of the level of thermal degradation of cellulose. Despite the slightly better

detection limits achieved by HS-GC-MS, the HS-GC-FID method is characterized by less

36





expensive instrumentation within every laboratory reach and hence can represent a more user-

friendly tool for solid insulation degradation surveillance of power transformers.

37




L 31

HIGH SENSITIVITY FLOW-MODULATED COMPREHENSIVE TWO-

DIMENSIONAL GAS CHROMATOGRAPHY-MASS SPECTROMETRY

P.Q. Tranchida1, F.A. Franchina

1, M. Zoccali

1, P. Dugo

1,2, L. Mondello

1,2

1Dipartimento di Scienze del Farmaco e dei Prodotti per la Salute - University of Messina,

Viale Annunziata, 98168 Messina, Italy 2Centro Integrato di Ricerca (C.I.R.), University Campus Bio-Medico, Via Álvaro del

Portillo, 21, 00128 Roma, Italy

E-mail: [email protected], [email protected], [email protected],

[email protected], [email protected]

One of the major limitations of current-day flow-modulated comprehensive two-

dimensional gas chromatography (FM GC×GC) is the generation of high gas flows (e.g., 20

mL/min) in the second analytical dimension. Even though such high flows are necessary to

efficiently flush the content of the modulator onto the second dimension, they also greatly

restrict the employment of mass spectrometry (MS). One way to enable the use of MS

systems, in FM applications, is to divert a substiantial part of the second-dimension flow to

waste. It is obvious that such a choice has a negative impact on sensitivity.

The present contribution is focused on the development of high sensitivity methods

using flow-modulated comprehensive two-dimensional gas chromatography-mass

spectrometry. Specifically, an FM GC×GC-MS approach was developed in which the flows

necessary to efficiently flush the modulator were greatly reduced. Consequently, there was no

need to divert flow to waste. The efficiency of the set-up is demonstrated on real-world

samples.

Acknowlegments

The Project was funded by the “Italian Ministry for the University and Research

(MIUR)” within the National Operative Project “Hi-Life Health Products from the industry of

foods”. Project ID: PON01_01499.

38









L 32

PROFILE DEFINITION OF AROMATIC HYDROCARBONS IN CRUDE

OIL FRACTIONS BY COMPREHENSIVE TWO-DIMENSIONAL GAS

CHROMATOGRAPHY

O. Platiša, S. Telen

Refineries and Marketing Development Sector, INA d.d., Lovinčićeva 4,

Croatia-10002 Zagreb

E-mail: [email protected], [email protected]

High price and availability of crude oils on the market push refineries to focus on

every processed and sold molecule. Understanding of composition of crude oil and its fraction

is essential for molecular based refinery management. Therefore, compositional knowledge

enables the refinery to predict the problems and conduct the processes more efficiently.

Crude oil is a mixture of hydrocarbon molecules which are organic compounds that

may include from one to sixty carbon atoms in more than 10.000 chemical structures. The

properties of hydrocarbons depend on the number and arrangement of the carbon and

hydrogen atoms in the molecules. The refining processes use chemicals, catalysts, heat and

pressure to separate and combine the basic types of hydrocarbon molecules naturally found in

crude oil into groups of similar molecules. The refining processes also rearrange their

structures and bonding patterns into different hydrocarbon molecules and compounds.

Therefore it is the type of hydrocarbons (paraffins, naphthenes, aromatics) that is significant

in refinery processes.

Aromatics are hydrocarbon molecules which contain at least one benzene ring. Their

importance in refinery processes is in high energy of the aromatic molecules and their high

density which is important for finished products. Profile of aromatics along the distillation

curve could therefore significantly improve the understanding of refinery processes.

Comprehensive two-dimensional gas chromatography (GC×GC) is a powerful

multidimensional GC technique that combines two independent separations to provide

detailed sample characterization. GC×GC subjects entire sample to orthogonal separation

within two chromatographic columns of different polarity connected together in series and

separated by modulator. First column acts in term of conventional gas chromatography and

the second one in term of fast chromatography. GC×GC principle of separation gives the

opportunity to separate the entire sample on the basis of two different chemical or physical

properties and therefore gets much more information about the entire sample than any other

conventional separation technique.

This study demonstrates contribution of GC×GC in understanding the refinery

processes and properties of finished products.

39






L 33

GC-TIME OF FLIGHT-THERMAL DESORPTION TECHNIQUE FOR

THE DETECTION AND QUANTIFICATION OF VOLATILES IN THE

CONTEXT OF HOME AND PERSONAL CARE PRODUCTS

R.A. Salkar

Unilever Research and Development, Quarry Road East, Bebington, Wirral,

Merseyside, CH633JW, United Kingdom


Hyphenated chromatographic techniques are being increasingly used for the detection

of volatiles for a number of reasons ranging from higher sensitivity, flexibility of sampling

and that of sample introduction.

The current talk discussed the use of GC-Time of Flight-thermal desorption technique

for the analysis of volatiles relevant to the Home and Personal care business. Novel sample

introduction and analysis techniques have been applied for a number of applications ranging

from real time monitoring of volatiles to low level detection of analytes in complex sample

matrices.

40





L 34

APPLICATION OF INVERSE GAS CHROMATOGRAPHY IN

PHYSICOCHEMICAL CHARACTERIZATION OF ABRASIVE TOOLS

B. Strzemiecka, A. Voelkel, M. Hinz, M. Rogozik

Poznań University of Technology, Institute of Chemical Technology and Engineering, pl. M.

Skłodowskiej-Curie 2, 60-965 Poznań, Poland


The production of the abrasive materials includes the following stages: covering of

abrasive materials by wetting agent, addition of the filler with the binding material, mixing of

the components, stabilization of the composition and the hardening. The most important

processes influencing the properties of the final product are: the coverage of the abrasive

materials, mixing and proper hardening. The homogeneity of the mixture of semi-product

increases the quality of the final product. The effectiveness of the hardening process depends

also on the applied temperature programme.

Although the determination of wettability of abrasive materials is relatively simple but

the controlling of the hardening process is much more complicated. Therefore, we present the

procedure for the determination of the degree of hardening (DH) of the resins applied in the

abrasive articles by using Inverse Gas Chromatography (IGC). IGC is a version of gas

chromatography dedicated to the investigation of various materials being placed in the

chromatographic column and playing a role of the stationary phase. In this case the column

filling consisted of semi-products containing resin, various fillers and abrasive grains. Degree

of hardening was estimated after different steps of heating procedure, i.e. at different

temperatures and after different times of heating. The influence of the content of fillers on the

DH values was investigated. IGC results were verified by using FTIR and extraction method

in Soxhlet apparatus. Results showed that IGC is simple and suitable method for qualitative

estimation of the degree of hardening in grinding tools.

This work was supported by PBS1/A1/7/2012 The National Centre for Research and

Development project.

41





L 35

NEW TRENDS IN THE DEVELOPMENT OF MICROEXTRACTION

TECHNIQUES FOR COUPLING SAMPLE PREPARATION AND

CHROMATOGRAPHIC ANALYSIS

J. Švarc-Gajić

Faculty of Technology, University of Novi Sad, Bulevar cara Lazara 1,

21 000 Novi Sad, Serbia


Development in food, environmental and pharmaceutical analysis is marked with

expressed tendency to minimize analytical instruments and to integrate several analytical

steps in one. Commercial analytical instruments that couple sample preparation and analytical

steps, reducing overall measurement uncertainty, offer such alternative. Such approach allows

automatization which is important in great sample inputs. By applying modern sample

preparation techniques, relying on the use of analyte-, class- of interferences-specific

sorbents, samples can be directly subjected to chromatographic analysis, both liquid and gas,

as well as to capillary electrophoresis. Principal focus in the development of preparation

techniques for samples of complex matrices relies on the enhancement of sorbent chemistry,

geometry and softwares used in computerized steps, as well as in the improvement of

interfaces.

Sorbent materials, like class-specific sorbents, such as restricted-access materials and

immunosorbents for selective isolation of analytes, are rapidly developing, improving their

selectivity and capacity. Low-specificity sorbents are used for general application, whereas

high surface area porous polymers and graphitized carbon sorbents are used mostly for

environmental samples.

Recent research is focusing on the design of interfaces for coupling microextraction

techniques (SPME) with chromatographic. Fiber design microextraction devices can be

coupled to HPLC analysis via desorption chamber and a six-port injection valve, whereas in-

tube sorbents are more easily integrated. Sorptive fibers showed to be convenient for

implementation in autosamplers for GC analyses, where the major challenge was to

incorporate agitation and temperature control. Portable SPME systems allow rapid and cost-

effective investigation in the field even of complex organic samples.

42





L 36

MONOLITHIC IN-NEEDLE EXTRACTION (MINE) DEVICE AS A

NEW APPROACH FOR THE DIRECT ANALYSIS OF

LIQUID SAMPLES

M. Pietrzyńska, A. Voelkel

Poznań University of Technology, Institute of Chemical Technology and Engineering,

pl. M. Skłodowskiej-Curie 2, 60-965 Poznań, Poland


Combination of extraction and chromatographic techniques opens NEW possibilities

in sample preparation area. Macroporous poly(styrene-divinylbenzene) (PS-DVB) monoliths

with different proportion of monomers were prepared by in situ polymerization in stainless

steel needles [1]. MINE devices were used in the preparation of a series of test water samples

for chromatographic analysis. Taking into account possible large flow resistance of

monolithic sorbent layer it was necessary to examine the permeability of the in-needle device.

So far in-needle technique was relatively seldom used for direct separation of analytes

from liquid samples and most often was combined with head-space (HS) or purge and trap

(P&T) techniques [2]. This limited application is associated with a high flow resistance

produced by a sorbent layer [3]. New proposal - the application of monolithic filling in the in-

needle device should prevent changes occurring in the sorbent layer and increase the

efficiency of this sample preparation tool. MINE devices with nine monolithic fillings were

characterized by acceptable hydrodynamic characteristics. Scanning electron microscope

images (SEM) were also obtained.

The extraction of phenolic compounds from water samples was carried out by

pumping liquid samples through the MINE device. Breakthrough volume and the sorption

efficiency of prepared monolithic in-needle extraction devices were determined

experimentally. There results indicate a high efficiency of examined systems. The achieved

recovery was higher than 90%.

References

1. M.Pietrzynska, A. Voelkel, K. Bielicka-Daszkiewicz, Anal. Chim. Acta,

DOI: 10.1016/j.aca.2013.03.022

2. H.L.Lord, W. Hang, J. Pawliszyn, Anal. Chim. Acta 677 (2010) 3-18

3. M.Pietrzynska, A. Voelkel, K. Héberger, K. Bielicka-Daszkiewicz, M. Kaczmarek,

Anal. Chim. Acta, 751 (2012) 182–188

43





L 37

PREPARATIVE AND CHROMATOGRAPHIC TECHNIQUES IN

CHEMICAL FINGERPRINTING - SELECTED EXAMPLES

I. Jerković

Faculty of Chemistry and Technology, University of Split, N. Tesle 10/V, 21000 Split, Croatia


Following its widespread application in the quality control of medicinal plants and

foods, chemical fingerprinting has recently gained increased attention tracing product back to

its source, and occasionally fingerprinting to identify a specific source. Mass spectrometry

(MS) in combination with gas chromatography (GC) or liquid chromatography (LC) is most

often used with the capacity to detect overall chemical components of the sample (untargeted

analysis), but also can be focused to specific compounds (targeted analysis). Except

ubiquitous preparative methods (distillations and extractions) prior to GC or LC, several new

methods have been developed in our laboratory (e.g. hydrodistillation-adsorption or co-

distillation with superheated pentane vapour). Formation of artifacts (mainly heat generated)

and co-isolation of accompanying compounds have been major disadvantages of the

preparative methods application. As it is known, the volatile organic compounds (VOCs)

profile is one of the most typical features of a plant and food product, for both organoleptic

quality and authenticity. Selected examples of GC and/or HPLC fingerprints are further

presented.

VOCs (essential oils) have been proved particularly helpful in assessing

chemotaxonomic relationships of several genera in Lamiaceae family. Different essential oil

chemotypes have been found (e.g. oregano thymol/carvacrol chemotype or wormwood (Z)-

epoxyocimene with β-thujone and (Z)-epoxyocimene with chrysantenyl acetate chemotypes).

The use of headspace solid-phase microextraction (HS-SPME) and ultrasonic solvent

extraction (USE) provided different complementary chromatographic fingerprints for

comprehensive screening of the honey. Norisoprenoids, terpenes, benzene derivatives and

others have been proposed as the quality markers for authenticity of the honey floral origin.

Subsequently, some specific-marker compounds have been suggested, e.g., methyl

anthranilate for Citrus spp. honeys, coumarin and vomifoliol for Prunus mahaleb L. honey,

methyl syringate for Asphodelus microcarpus Salzm. et Viv. honey, 3,4-dihydro-3-oxoedulan

for Centaurea cyanus L. honey and others. Kinurenic acid and salicylic acid are useful to

mark Salix. spp. honeydew honey by direct HPLC-DAD. The relationship between (Z,E)- and

(E,E)-abscissic acid isomers abundance (determined by HPLC-DAD) was 1 : 2 (Polish willow

honey), instead of 1 : 3 in Croatian willow honey.

The presence of typical markers of Sardinian unifloral honeys represents a powerful

chemical fingerprint to connect a honey-based Sardinian product abbamele with the territory

of production. The compared GC–MS and HPLC-DAD fingerprints proved to be useful to

obtain information about the use of specific honeys in the production (A. microcarpus Salzm.

et Viv. or Arbutus unedo L.) and to verify citrus addition.

44





L 38

AFFINITY-BASED METHOD FOR RNA PURIFICATION PURSUING

mRNA VACCINATION

R. Martins, C. Maia, J.A. Queiroz, F. Sousa

CICS-UBI – Health Sciences Research Centre, University of Beira Interior, Av. Infante D.

Henrique, 6200-506 Covilhã, Portugal


Coding messenger RNA (mRNA) is emerging as a particularly attractive option in the

development of new approaches for the treatment of cancer or infection diseases focusing on

immunotherapies. mRNA shows several advantages as a vaccine including feasibility,

applicability, safeness, and effectiveness when it comes to the generation of immune

responses. Cervical cancer is the second most common cancer in women worldwide. Human

papillomavirus (HPV) is the primary etiologic agent of cervical cancer. Two HPV16 proteins,

E6 and E7, are consistently expressed in tumor cells. Most therapeutic vaccines target one or

both of these proteins. Due to the increasing success of clinical application of mRNA-based immunotherapies the

development of new tools to improve RNA manufacture process is of great significance. Of

particular importance is the purification process of RNA molecules since rigorous quality criteria

recommended by international regulatory authorities should be fulfilled. Therefore, new

chromatographic strategies for RNA purification were considered, exploiting affinity interactions

between amino acids and nucleic acids. The potential of using affinity chromatography with

histidine or arginine as ligands has been recently demonstrated to selectively isolate and monitor

different RNA species [1-4]. In the present study, a single arginine-affinity chromatography step

was employed for the purification of chemically synthetized mRNA encoding HPV16 E6 and E7

proteins. The chromatographic experiments were developed by optimizing the sodium chloride

gradient steps to purify the RNA transcript from impurities resultant from the transcription

process, such as template, enzymes, nucleotides, salts or buffer. The quality assessment of RNAs

preparations revealed a high recovery yield, high integrity and purity. Moreover, cell transfection

experiments were also conducted in order to evaluate the applicability of the newly purified RNA

transcripts. Overall, these results reveal that this affinity technique can be a promising alternative

for RNA purification, obtaining a reproducible and appropriate RNA quality that can contribute to

the development of new gene therapy strategies employing RNA molecules.

References: 1. Martins, R., Queiroz, J. A., Sousa, F., J. Mol. Recognit. 2010, 23 (6):519-524;

2. Martins, R., Queiroz, J. A., Sousa, F., Biomed. Chromatogr. 2012, 26 (7):781-788;

3. Martins, R., Maia, C. J., Queiroz, J. A., F. Sousa, J. Sep. Sci., 2012, 35 (22):3217-

3226;

4. Martins, R., Queiroz, J. A., F. Sousa, Anal. Bioanal. Chem., 2013, submitted.

Acknowledges: This work was supported by FCT, the Portuguese Foundation for Science and

Technology (PTDC/EBB-BIO/114320/2009 and PEst-C/SAU/UI0709/2011 COMPETE).

Rita Martins also acknowledges a fellowship (SFRH/BD/ 64100/2009) from FCT.

45





L 39

STUDY OF THE INTERACTIONS BETWEEN IMMOBILIZED L-

METHIONINE AND OLIGONUCLEOTIDES BY

CHROMATOGRAPHY, NMR AND SPR ANALYSIS

É. Mota, F. Sousa, Â. Sousa, J.A. Queiroz, C. Cruz

CICS-UBI – Centro de Investigação em Ciências da Saúde,

Universidade da Beira Interior, 6200-506, Covilhã, Portugal


Amino acid-based affinity chromatography appears as a promising approach to purify

supercoiled plasmid DNA since it combines the selectivity of naturally occurring interaction

with the simplicity of a single small ligand. Therefore, the present study explores the effect of

oligonucleotides composition on the mechanism of retention to L-methionine support by

chromatography and saturation transfer difference (STD)-nuclear magnetic resonance (NMR)

techniques. Surface plasmon resonance (SPR) experiments are also performed to evaluate the

binding equilibrium of the oligonucleotides and L-methionine immobilized on the amine

surface. The STD-NMR results show that thymine led to more contacts with the support.

These results are according with binding profiles obtained by chromatography. For synthetic

polynucleotides, polyT had the highest retention time, followed by polyC, polyA and polyG.

In general, the larger homo-oligonucleotides are more retained to the L-methionine support.

All experiments were performed using 1.5 M (NH4)2SO4, indicating the involvement of

hydrophobic interactions. For hetero-oligonucleotides, AAATTT had the highest retention

time followed by CCCAAA and CCCTTT with similar retention time, showing that

oligonucleotides with adenine have the highest retention, and the presence of cytosine reduces

the retention by the L-methionine chromatographic support. From SPR-biosensor, the

equilibrium dissociation constants (KD) ranged from 3.34×10-4

M to 5.52×10-3

M; generally,

cytidine is the base that shows highest affinity, followed by thymine and adenine, while the

lowest affinity is found for guanine. The results show higher affinity with an increase in the

number of nucleotides (bases and backbone). The order of KD values for hetero-

oligonucleotides is CCCTTT > CCCAAA AAATTT > GGGTTT, showing that

oligonucleotides with cytidine have the highest affinity and the presence of guanine reduces

the affinity with the L-methionine immobilized. Combining STD-NMR and SPR biosensor

techniques allied with chromatography was successfully used to screen amino acid affinity

support-nucleotide interactions, providing further insights into supercoiled plasmid DNA

purification for therapeutic applications.

Keywords: chromatographic support, nuclear magnetic resonance, surface plasmon

resonance, L-methionine immobilized, oligonucleotides.

Acknowledgements

This work was supported by FCT, the Portuguese Foundation for Science and

Technology (PTDC/EBB-BIO/114320/2009) and by PEst-C/SAU/UI0709/2011 COMPETE.

E. Mota also acknowledges a fellowship (CENTRO-07-ST24-FEDER-002014) from

“Programa Operacional Regional do Centro 2007-2013 QREN” (“Mais Centro” program).

46





POSTER

PRESENTATIONS




P 1

DETERMINATION OF ESTROGENS IN WATER COLLECTED FROM

SEWAGE PLANTS BY GC-MS USING A NEW STIR BAR COATING

FOR SORPTIVE EXTRACTION

I. Rykowska, W. Wasiak

Faculty of Chemistry, Adam Mickiewicz University, Umultowska 89b,

610-614 Poznań, Poland


The SBSE technique has been applied successfully to trace analysis in environmental,

biomedical and food applications, with a possibility to obtain extremely low detection limits.

Polydimethylsiloxane (PDMS) is a typical coating material in the commercial stir bars.

Due to the apolar character of PDMS, which is the only commercialized coating for SBSE,

stir bar sorptive extraction has been mainly applied to extract non-polar and weakly polar

compounds, and failed in the extraction of strongly polar compounds unless they have been

previously derivatised.

The objective of the presented work is to prepare a novel stir-bar coating based on

modified silica gel with ketoimine groups and its application followed by gas chromatography

and mass spectrometry for the determination of estrogens in two water samples (fresh and

cleaned effluent) collected from sewage plants.

Estrogens were selected as test analytes to investigate the extraction efficiency of the

new stir bar. Based on sample solutions containing these compounds, some research was

taken to optimize conditions for the quantitative determination of these compounds using gas

chromatography.

48






P 2

THE USE OF MICROEMULSIONS FOR THE SAMPLE

PRETREATMENT AND AS THE MOBILE PHASE IN MELC

L.S. Sokolova, A.V. Pirogov, O.A. Shpigun

Lomonosov Moscow State University, Moscow 119991, Russia


Microemulsions are liquid colloidal systems spontaneously formed upon mixing two

fluids with limited solubility (eg, water and oil) with adding a surfactant and in some cases

co-surfactant. In the microemulsions (ME) of the direct type (oil-in-water), the size of the oil

droplets ranges from 10 to 200 nm. As the ME are chemically and structurally very different

from the ordinary widely used organic solvents, they have been used not only in the

petrochemical industry, but also in analytical chemistry. Microemulsions are used in the

modern analytical methods of analysis: capillary electrophoresis and chromatography. The

same microemulsions can be used as a "reactor" for the reaction of derivatization and for the

sample pretreatment. The oil-in-water microemulsions can extract hydrophilic components

from the objects with a high content of oil and carbohydrates.

The aim of this paper is to demonstrate the possibility of using ME in the sample

pretreatment of both medicines and food. Spreads were chose as the food samples, in which

the content of preservatives and sweeteners was determined. The most common synthetic

preservatives are benzoic and sorbic acid as well as theirs salts. Some preservatives can

accumulate in the human body and exhibit the properties of carcinogen, while large

concentrations can cause the allergic reactions. Among the artificial sweeteners the most

popular ones are aspartame, acesulfame potassium, saccharin and cyclamate. The content of

these substances in food is strictly regulated, because these sweeteners are not assimilated in

by the organism, they don’t possess food value, and their excessive consumption cause a large

number of side effects.

Among the pharmaceutical substances we chose Hexestrol, the oil drug for the

injection. It’s a synthetic hormone with hydrophilic properties, used in the medicine. The

main feature of the complex fat-containing objects analysis is the necessity of the target

component extraction with a suitable solvent often followed by the laborious step of the

extract purification.

ME which was used for the sample pretreatment contain the following substances -

surfactant: heptane: n-butanol, 3:0.8:8, mass. %. Sodium dodecyl sulfate (SDS), and PEG-40

hydrogenated castor oil (Eumulgin HRE 40) were used as the surfactants. Chromatographic

determination was performed by means of the traditional RP-HPLC and microemulsion liquid

chromatography (MELC) with UV-detection.

MELC method is suitable for the determination of sorbic and benzoic acids in spreads,

because it has the low limits of detection of 0.06 and 0.02 mg·L-1

, respectively, and a high

degree of recovery (95%). The use of microemulsions as the extracting solvents can reduce

the sample pretreatment procedure to three operations: the dissolution of the sample in the

ultrasonic bath, centrifugation and dilution with the microemulsion of the same composition

in case of high concentrations.

The described method of the sample pretreatment is applicable to the analysis of both

food products and medicines.

49





P 3

DERIVATIZATION OF ANTIBIOTICS IN MICROEMULSION

MEDIUM FOLLOWED BY HPLC DETERMINATION

A.V. Pirogov, L.S. Sokolova, O.A. Shpigun



Ampicillin is an penicillinic antibiotics. It is used for the treatment of the certain

infections caused by bacteria, for example, pneumonia, bronchitis and some infections of ears,

lungs, skin and urinary tract.

Microemulsions (ME) are highly dispersive systems formed by two insoluble liquids.

The diameter of the dispersed phase droplets ranges generally from 10 to 200 nm. Due to the

small size of the droplets, ME are stable and usually transparent as opposed to the

conventional emulsions. ME are widely applied in analytical chemistry and about 10 years

ago they were used as mobile phases in HPLC. This method is called microemulsion liquid

chromatography.

ME are chemically, structurally and functionally different from the traditional

solvents, which are used as media for the derivatization. Application of ME in this case may

affect the rate of the reaction and the yield of the product. The aim of this work was to

determine the degree of the influence ME on the derivatization of ampicillin with 2,3-

naphthalene dialdehyde. The components of the microemulsion were 3% surfactant (sodium

dodecyl sulfate), 0.8% heptane and 8% n-butanol in water phase (% w/v), pH 9.

Chromatographic determination of the pure components and the reaction products was carried

out by means of traditional RP-HPLC and MELC in gradient mode with UV-detection.

Derivatization of ampicillin was carried out with four-fold molar excess of the

derivatizing agent (2,3-naphthalene dialdehyde) in water-MeCN and microemulsion media.

The yield of the reaction product (the derivative of ampicillin) was calculated by the decrease

of the agent in the reaction. In the first case, the reaction proceeds under tough conditions,

which temperature control at 60 ° C for 1 hour. And when ME were used as a "reactor" for the

derivatization, the target product was formed at room temperature in 20 minutes. The yield of

the product under the described conditions in both cases was 98%. These results were

validated by using two chromatographic methods, HPLC and MELC, and by decreasing the

concentrations of the initial substances and by using two different chromatographic columns.

Figure 1. Dependence of the yield of the derivative in

microemulsion and water-MeCN media.

0

50

100

150

0 20 40 60 80

ME (25 °C)

t, min

R, %

50





Fig. 1. shows that the yield of the reaction depends on the medium, in which the

reaction proceeds. The use of microemulsions as a medium, for the derivatization of

ampicillin with 2,3-naphthalene dialdehyde can significantly speed up the kinetics (hundreds

times) and allows one to conduct it at the room temperature.

51




P 4

HYDROPHILIC SPACERS FOR MOVING THE FUNCTIONAL

GROUPS AWAYFROM THE MATRIX OF THE ANION EXCHANGERS

IN ION CHROMATOGRAPHY

O.I. Shchukina, A.V. Zatirakha, A.D. Smolenkov , O.A. Shpigun

Lomonosov Moscow State University, Analytical chemistry department


The main problem with using anion exchangers based on polystyrene-divinylbenzene

is the tailing of the peaks of polarizable anions, such as nitrate or bromide, caused by the

specific interactionsofthese anions witharomatic rings of the polymeric matrix. There are two

approaches for reducing such interactions: spatial moving of the functional groups away from

the matrix andincreasing thestationary phase hydrophilicity.

In the present work the method of the synthesis providing both spatial moving of the

functional groups and hydrophilizationof the matrix surface due to the introduction of the

hydrophilic spacers is proposed. Theapproach is based onthe introduction amino groups to the

anion exchanger’s structure, bythe reactions of acylation and the following

reductiveamination with further modification of the ammonium groups.

The anion exchangers with trimethylammonium functional group moved away from

the matrix by hydrophilic spacers were obtained.In comparison with the anion exchangers

based on the same matrixcontaining the functional groups onthe surface, new anion

exchangers were characterized by higher selectivity and efficiency. Theseparation of seven

inorganic anions in 10-15 minutes waspossible.The maximum efficienciesfor nitrate and

bromide were 45000 TP/m and 50000 TP/m, respectively.

The proposed method of synthesis allows one to investigate the influence of the

furtherhydrophilization of anion exchangersby introducing the hydrophilic radicals to

thequarternary nitrogen atom on thechromatographic properties.For these purposes the

modification of the functional group withoxiraneswas carried out. Epichlorohydrin can also

be used for the further increase of the hydrophilic spacer’slength byalternatingthe alkylation

and amination stages.Such anion exchangerswere highly selectiveand they allow one to carry

out the separation of eight inorganic anions withthe efficiency fornitrate being 18000 TP/m,

and the efficiency for phosphate being 62000 TP/m.

52





P 5

DETERMINATION OF 2-THIOCYANO-3,5-DINITROPYRIDINE IN

OINTMENTS BY MICROEMULSION LIQUID CHROMATOGRAPHY

E.B. Pashkova1, A.V. Pirogov

2, L.S. Sokolova

2

1Pharmaceutical Company «Bion», Obninsk, Kaluga region, 249032, Russia

2Lomonosov Moscow State University, Moscow 119991, Russia


Microemulsion-based HPLC (MELC) is a recent development, offering reduced

sample preparation times for complex samples and generic separation conditions applicable to

a wide range of solutes. Oil-in-Water microemulsions offer alternative partitioning

mechanism while using an ordinary stationary phase of the RP-HPLC column.

Microemulsions have great solubilizing power for both water-insoluble and -soluble

compounds. They, therefore, offer the ability to directly solubilize hydrophobic samples and

matrices, such as creams and ointments, without lengthy pre-extraction steps.

2-thiocyano-3,5-dinitropyridine is a novel pharmaceutical substance with strong

antimicrobial properties. Pharmaceutical preparations, based on this compound, were recently

suggested and clinically tested. Thus, development of methods of the determination of the

active component and control of the related substances is an actual analytical task.

A novel technique for the determination of 2-thiocyano-3,5-dinitropyridine assay,

based on the microemulsion extraction with the following separation on a reversed-phase

column using oil-in-water microemulsion as an eluent was developed. The whole procedure

including sample pretreatment takes about 20 minutes.

The established method was subjected to method validation, and required validation

parameters were defined. Robustness testing, an important part of method validation, was

performed as well. The validated MELC method was found to be suitable for the

simultaneous determination of 2-thiocyano-3,5-dinitropyridine assay and its impurities in

pharmaceuticals.

The intra-day and inter-day precisions (in term of % coefficient of variation) were less

than 2.85 % and, respectively. The influence of the composition of the microemulsion system

was also studied and the method was found to be robust with respect to some changes of the

microemulsion components.

53





P 6

FUNDAMENTALS OF GAS CHROMATOGRAPHY

J. Oskonbaeva

Kyrgyz-Turkish Manas University, Engineering Faculty


Chromatography is a science of study that involves the separation of molecules in a

mixture based on differences in their structure and/or composition. It is one of the most

common physical methods performed in laboratories to separate the most complex mixtures

containing complex molecules. The procedure consists of two parts viz. the mobile phase and

the stationery phase. The mobile phase is the mixture to be separated dissolved in a solvent

and the stationery phase is the layer of a certain material in its solid form through which the

mobile phase passes through and disintegrates into separate components at various stages.

Adsorptive materials are used in the stationery phase.

Gas chromatography is one of the most widely used techniques for analyzing

hydrocarbon mixtures. Some of the advantages of chromatography are the range of

measurement (from ppm levels up to 100%), the detection of a wide range of components,

and the repeatability of the measurements. Chromatography is used in the laboratory, in

permanently installed online systems, and in the field with portable systems. No matter the

location, style, or brand, all gas chromatographs are composed of the same functional

components: the sample handling system, the chromatograph oven, and the controller

electronics.

Gas chromatography is the way in science to many great solutions.

54





P 7

SAMPLE STACKING AND ON-LINE DERIVATIZATION FOR THE

ANALYSIS OF AMPICILLIN AND AMOXICILLIN BY

MICROEMULSION ELECTROKINETIC CHROMATOGRAPHY

A.A. Kostromskikh, A.V. Pirogov, L.S. Sokolova, O.A. Shpigun



Microemulsion electrokinetic chromatography (MEEKC) is an electrodriven

separation technique which uses microemulsion buffer as a background electrolyte. In recent

years MEEKC has gained considerable attention and widespread application because of its

usefulness for determining the substances, which are different in their electrophoretic

mobilities, and offers the possibility of highly efficient separation of both charged and neutral

solutes covering a wide range of water solubilities.

Microemulsion can be used as a reaction medium for providing many chemical

reactions due to its unique composition. In our work it was shown that derivatization of

ampicillin (AMP) and amoxicillin (AMO) with naphthalene-2,3-dicarboxaldehyde (NDA) is

accelerated greatly in microemulsion, compared with the standard derivatization procedure,

no heating being required. This permits the reaction to proceed in the capillary in on-line

mode. A novel method using MEEKC combining the concentration technique called reversed

electrode polarity stacking mode (REPSM) with subsequent on-line derivatization was

developed for the quantitative determination of antibiotics. The effect of each individual

component within the microemulsions, i.e. the oil phase, the surfactant, the co-surfactant and

the buffer concentration on the resolution of the analytes was systematically studied.

Figure 1. Electropherogram of the NDA derivatives of AMP and AMO in a concentration 1

mg/L. Separation conditions: 3.3% (w/w) SDS, 0.8% (w/w) heptane, 8.0% (w/w) 1-butanol

and 87.9% (w/w) 10 mmol/L borate buffer (pH 9.2); fused-silica capillary 48,5 cm×50 µm

(detection window at 40 cm), 40oC, 230 nm. During the analysis gradient of voltage was used.

Sample injection: flushing the capillary for 1 min with the analyte, injection NDA from outlet

vial (-25 mbar for 10 sec), then applying -5 kV until 95% of original current reached.

55





As we can see in Fig.1, the baseline separation of the antibiotics derivatives was

achieved and the limits of detection were 150 µg/l for AMO and 110 µg/l for AMP. The

method developed allowed us to decrease the detection limits by about 200-fold, to reduce the

analysis and preparation time considerably, to diminish the consumption of the expensive

reagents and to avoid high temperatures during the derivatization procedure.

56




P 8

DYNAMIC BINDING CAPACITY OF A NON-GRAFTED

MONOLITHIC SUPPORT USING DIFFERENT PLASMIDS

D. Bicho1, A. Sousa

1, F. Sousa

1, J.A. Queiroz

1,2, C.T. Tomaz

1,2

1CICS-UBI – Health Sciences Research Centre, University of Beira Interior,

Av. Infante D. Henrique, 6200-506 Covilhã, Portugal 2DepartmentofChemistry, Universityof Beira Interior, Rua Marquês d’Ávila e Bolama,

6201-001 Covilhã, Portugal


In recent years, gene therapy and DNA vaccination are becoming suitable alternatives

for the treatment and prevention of severe pathologies such as infectious diseases or cancer.

Researchers have made efforts to improve downstream purification methods that yield large

quantities of highly pure supercoiled (sc) pDNA required to therapeutic applications.

Monoliths have been used successfully for purification approaches due to itshigh binding

capacity and the excellent transfer mass properties. In this work we describe the effect of

parameters such as plasmid size, conformation, salt concentration and flow-rate on the

dynamic binding capacity (DBC) of a non-grafted carbonyldiimidazole (CDI) monolithic

diskin order to prove the potential utility of this monolith. The results showed that it was

possible to have a selective separation of the sc isoformsof the differentplasmids. Higher size

plasmidsneeded a lower (NH4)2SO4 concentration to bind to the monolith possibly due to the

greater number of interactions occurring between these plasmids and the monolith, which

allows a stronger binding. However, DBC experiments for plasmids with different sizes

revealed that the monolith capacity was higher for small plasmids. Additionally, it was

verified that the increase of salt concentration from 2.5 M to 3 M of (NH4)2SO4 promoted an

increment in the capacity of the monolithic support to bind pDNA. Finally, two different flow

rates were tested. Low flow also increased the DBC due to the high contact time between the

binding sites of the monolith and the pDNA molecules. The present study confirms the

possibility of using this monolithic support to purify sc isoform of different size plasmids,

providing a valuable tool for large-scale purification.

Keywords: chromatography, dynamic binding capacity, monoliths, plasmid DNA,

purification.

Acknowledgements

Diana Bicho and Ângela Sousa acknowledge the doctoral and post-doctoral grants

from FCT (SFRH/BD/82196/2011 and SFRH/BPD/79106/2011, respectively). This work was

supported by FCT, the Portuguese Foundation for Science and Technology (PTDC/EBB-

BIO/114320/2009) and PEst-C/SAU/UI0709/2011 COMPETE. The authors acknowledge to

BIA Separations for the monolithic support and to John Blenis and Linzhao Cheng for the

pcDNA3-myc-FLNa S2152A and pEGIP Addgene plasmids.

57






P 9

DAPP-SEPHAROSE AFFINITY CHROMATOGRAPHY:

SUPERCOILED PDNA PURIFICATION FROM CLARIFIED E. COLI

LYSATE SOLUTIONS

Catarina Caramelo-Nunes1,2

, Paulo Almeida2, João C. Marcos

3, Cândida T. Tomaz

1,2

1CICS-UBI – Health Sciences Research Centre, University of Beira Interior,

Av. Infante D. Henrique, 6200-506 Covilhã, Portugal. 2Department of Chemistry, University of Beira Interior, Rua Marquês d’Ávila e Bolama,

6201-001 Covilhã, Portugal 3Center of Chemistry, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal


Affinity chromatography has been established as an important technique for pDNA

purification. It relies on a strong but reversible interaction between the target molecule and the

immobilized ligand. Therefore, choosing the right ligand is a crucial step for the development

of a successful purification system. This work describes the successful application of the

intercalator 3,8 - diamino - 6 - phenylphenanthridine (DAPP) as a chromatographic affinity

ligand for the specific separation and purification of supercoiled plasmid DNA (pDNA) from

clarified E. Coli lysates . DAPP was coupled onto an epoxy-activated Sepharose matrix and

the obtained DAPP-Sepharose support was tested for the purification of supercoiled pDNA of

two plasmid molecules with different sizes (pVAX1-LacZ and pCAMBIA-1303, with 6.05

Kbp and 12.361 Kbp, respectively). Total retention of all lysate components was achieved

without any added salt to the eluent buffer. Elution of impurities was accomplished by adding

to the buffer solution 0.22M of NaCl for pVAX1-LacZ or 0.3M of NaCl for pCAMBIA-130.

Supercoiled pDNA was eluted simply by increasing NaCl concentration to 0.55M for both

plasmid solutions. The recovery yield for pCAMBIA-1303 was 65% and for pVAX1-LacZ

was 94%, with all host impurity levels in accordance with the requirements established by the

regulatory agencies.

Acknowledgments This work was supported by FCT, the Portuguese Foundation for Science and

Technology PTDC/QUI-QUI/100896/2008 and FCT-COMPETE PEst-C/SAU/UI0709/2011.

C. Caramelo-Nunes acknowledges a fellowship (SFRH/BD/64918/2009) from FCT.

58





P 10

DETERMINATION OF THE LOW CONCENTRATIONS OF ROCKET

KEROSENE IN WATER BY GAS CHROMATOGRAPHY-MASS

SPECTROMETRY WITH PRE-MICROEXTRACTION BY N-HEXANE

T.A. Balotnik, A.D. Smolenkov, R.S. Smirnov, O.A. Shpigun



Rocket kerosene enters the environment from the space vehicle launching when rocket

tanks are landing and the residues of fuel spill to the landscapes. The maximum permissible

concentration (MPC) of rocket kerosene in water was established in Russia as 0.01 mg/L.

Only the method of gas chromatography (GC) allows identification the of petroleum products

type. To determine rocket kerosene at such low level of concentration the GC was combined

with the extraction method.

For determining kerosene in water, 1 L of water sample was extracted by 8 mL of

n-hexane in the presence of potassium chloride (20 g/L) for 30 minutes with vigorous stirring

on a magnetic stirrer. After the separation of the phases 1 mL of the extract was dried over

anhydrous sodium sulfate and added to the internal standard solution. 1 µL aliquot of the

extract was injected into a gas chromatograph. 1,4-Bromo-fluorobenzene was used for

assessing the extraction degree of the fuel components as the surrogate standard,

naphthalene-d8 − as an internal standard for construction the calibration plot.

The technique of identifying the petroleum products type was developed. The method

was based on the presence of specific markers compounds, and also on the ratio of their peak

areas.

The selected ion monitoring mode of MSD (m/z 67, 136, 174) was applied for the

determination of rocket kerosene. The ratio at all peaks areas (m/z 67) to the area of the

internal standard (m/z 136) was used as an analytical signal.

With the using of microextraction the obtained pre-concentration factor was 125. The

detection limit was 0.0025 mg/L, the linearity of the calibration curve was found in a range of

0.005-0.05 mg/L. The correctness of the proposed approach was confirmed by the analysis of

the spiked samples.

59





P 11

DETERMINATION OF WATER SOLUBLE VITAMINS

IN ENERGY DRINKS AND PHARMACEUTICALS

BY HYDROPHILIC INTERACTION LIQUID CHROMATOGRAPHY

A.A. Bendryshev, B.A. Sarvin, A.V. Pirogov, O.A. Shpigun

Moscow State University, Chemistry department, GSP-2,

Lenin’s Hills, 119991, Moscow, Russia


The problem of monitoring vitamin content keeps its relevance due to the constant

increase in the number of fortified foods, dietary supplements, and pharmaceuticals. The most

widespread methods for the determination of water-soluble vitamins are based on RPHPLC

separation techniques. However, in some cases, methods based on this technique do not

ensure the separation of target vitamins from matrix components, especially for vitamins with

low retention times. Away to solve this problem is hydrophilic interaction liquid

chromatography (HILIC). This technique is based on the mechanisms of retention other than

those in RP HPLC. This provides different separation selectivity for vitamins and matrix

compounds. The aim of our study was to develop a method for the determination of water-

soluble vitamins using a HILIC technique and test it on real samples.

Effects of the mobile phase composition, pH, buffer solution concentration, organic

modifiers on the retention and separation of water soluble-vitamins in the HILIC mode were

studied. Various profiles of gradient elution were tested. Chromatographic conditions for the

separation and determination of thiamin, riboflavin, nicotinamide, nicotinic acid, pyridoxine,

folic acid, and cyanocobalamin were selected. The total analysis time is 22 min.

Sample preparation procedures for HILIC determination of water-soluble vitamins in

pharmaceutical and energy drinks were developed.

Proposed procedures were used in the analysis of pharmaceuticals and energy drinks.

Obtained vitamin concentrations are in good agreement with results by traditional RP HPLC

and manufacturer’s information.

60





P 12

HILIC APPLICATION IN PHARMACEUTICAL INDUSTRIES:

ROBUSTNESS AND SPECIFIC SELECTIVITY OF HILIC

L. Markovic, P. Jackson

GlaxoSmithKline Research & Development Limited, Gunnels Wood Road, Stevenage,

SG1 2NY, United Kingdom

E-mail: [email protected]; [email protected]

This poster details an investigation of Hydrophilic interaction liquid chromatography

(HILIC) conditions and stationary phase chemistry on the retention behaviour of the test

mixture containing acids, bases and neutral compounds. Analyses are performed on silica and

zwitterionic polar stationary phases with opposite arrangement of functional groups, in order

to observe their selectivity. Using a Design of Experiment (DoE) and model term ranking, the

work demonstrates that the variety of columns available and the differing influence of method

parameters (e.g. buffer concentration, pH and temperature) on the different columns make the

HILIC separation mode attractive for a wide range of separations.

Robustness analyses have been performed using an HILIC method for analysis of an

Active Pharmaceutical Ingredient (API) and its related impurities on a Silica column. DoE

statistical approach has been used during robustness testing with reduced 2-level fractional

factorial design having 12 runs overall. It is observed that HILIC method is robust, even

though complex mixed mode mechanism is known to operate. The water percentage in mobile

phase was the most significant method parameter which impacted resolution but was readily

addressed by procedural controls in the method.

An example of an HILIC-ESI-MS Limit Test Method which has been developed for

the determination of a polar azetidine impurity in a non polar API is given. HILIC

chromatography is very well suited to this type of analysis in terms of mobile phase

compatability, and retention of the polar analytes.

61






P 13

IN VITRO CYTOCHROME P450 ACTIVITY: DEVELOPMENT AND

VALIDATION OF LC-MS/MS METHOD FOR THE SIMULTANEOUS

DETERMINATION OF AMOXICILLIN AND ITS METABOLITES

M. Szultka1, R. Krzeminski

2, M. Jackowski

2, B. Buszewski

1

1Department of Environmental Chemistry and Bioanalytics, Faculty of Chemistry,

Nicolaus Copernicus University, Gagarin 7 Street, 87-100 Torun, Poland 2Department of General, Gastroenterological and Oncological Surgery,

Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University,

Joseph Street 53-59, 85-067 Bydgoszcz, Poland


Metabolism studies of new chemical entities are part of the early stages of the

discovery and development process for new compounds, potential drugs. The methods of

determining metabolic stability in in vitro conditions requires a development of sensitive,

specific and to some degree universal chromatographic method for detecting a set of

structurally related compounds and their metabolites. Because of the ever-rising costs of this

process and necessity of screening an increasing number of biologically active compounds,

high-throughput screening (HTP) methods were created. One of such applications includes

the use of the substrate cocktail in the microsomal incubations for the simultaneous evaluation

of the cytochrome P450 activities.

Compounds mixture consisted of probe substrates metabolized by the major

isoenzymes was incubated with liver microsomes. Metabolic stability test was performed with

the use of microsomes preparation in the presence of NADPH to study possible first phase

metabolic biotransformation catalyzed mostly by cytochrome P450 enzymes. The specific

metabolites were quantified by LC-MS/MS method with gemifloxacin as an internal standard.

The aim of this work was to developed and validate a simple method for the quantification of

specific metabolites, which can be used in metabolic HTP screening analysis. The analytical

system consisted of HPLC 1100 and Triple Quad mass spectrometer equipped with

electrospray ion source ESI. MS analysis were performed using a Mass Hunter Quantum

system in positive and negative multiple reaction monitoring (MRM) modes. Analytical assay

accuracy and precision values were within the limits set by the FDA guidelines. The presented

method was successfully applied to evaluations of CYP450 in vitro activity – cytochrome

P450 inhibition assays.

Acknowledgement This work was supported by National Science Center (Cracow, Poland), no.

2011/01/N/ST4/03178.

62





P 14

APPLICATION OF SPME-LC/MSN FOR METABOLOMICS DRUG

MONITORING IN BIOLOGICAL SAMPLES

M. Szultka1, R. Krzemiński

2, J. Szeliga

2, M. Jackowski

2, B. Buszewski

1

1Department of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus

Copernicus University, Gagarin 7, 87-100 Torun, Poland 2Department of General, Gastroenterological and Oncological Surgery,

Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University,

Joseph Street 53-59, 85-067 Bydgoszcz, Poland


Determination of biologically active compounds from various matrices, including

environmental and biological samples is a serious problem in a modern analytical chemistry.

The most relevant matrices to be analyzed for this purpose are plasma or blood, due to

providing a good correlation between their concentration and pharmacological effects. One of

the major tools in the pharmacokinetic studies (PK) is the combination of high-performance

liquid chromatography and mass spectrometry (LC/MSn). The main aim of this investigation

was to apply a fast and sensitive extraction technique using electrochemically prepared a new

polymeric coatings as sorbents for solid phase microextraction (SPME). SPME blood

sampling technique was evaluated in human PK studies. Polypyrrole (PPy), polythiophene

(PTh) and poly(3-alikilothiopenes) SPME coatings were used and evaluated their ability to

extract selected antibiotic drugs. Mass spectrometric parameters were optimized for target

compound in positive ion mode over the m/z 100-500 range. Quantitation was done using

multiple reaction monitoring (MRM) mode to monitor precursor ion at [M+H]+ to product ion

transition of m/z 366 → 349 for amoxicillin (AMOX), 332 → 288 for ciprofloxacin (CIP),

478 → 322 for gentamycin (GEN), and 172 → 128 for metronidazole (MET). Validation data

for accuracy and precision for intra- and inter-day were good and satisfied FDA’s guidance:

CV between 0.24% and 11.66% and accuracy between 93.8% and 108.7% for all compounds.

The presentation intends to give an overview of the process and bioanalytical tools of

the in vitro and in vivo drug metabolism. A new model of Heart-and-Lung Machine provides

a potential application to study the metabolism and pharmacokinetics as well of biologically

active compounds from different medical classes. It demonstrates the potential of in vitro tool

for chromatographic based metabolomics drug monitoring in the biomedical application.

Moreover, the obtain data are in good correlation with that from pseudo-in vivo study using

clinical samples from patients receiving therapeutic dosages of drugs. Developed method can

be used for the quantitative analysis of selected biologically active compounds, and provide a

potential application to study the metabolism and pharmacokinetics of other drugs from

different medical classes from the biological matrices. The results demonstrate the potential of

in vivo SPME as a useful sample preparation tool for chromatographic based metabolomics

drug monitoring in the biomedical application from patients receiving therapeutic dosages.

Acknowledgement

This work was supported by National Science Center (Cracow, Poland), no.

2011/01/N/ST4/03178.

63





P 15

ISOLATION AND GC-MS QUANTIFICATION OF ACRYLAMIDE

FROM CEREAL AND POTATO FOODS

S. Hasić1, M. Kežić

2, S. Ćavar

1,3, A. Goga

1, A. Čaušević

2

1University of Sarajevo, Faculty of Science, Department of Chemistry, Zmaja od Bosne 33-35,

71000 Sarajevo, Bosnia and Herzegovina

E-mail: [email protected]; [email protected] 2Institute for Public Health FB&H, Department of Hygiene and Environmental Health,

Maršala Tita 9, 71000 Sarajevo, Bosnia and Herzegovina

E-mail: [email protected] 3Palacky University Olomouc, Faculty of Science, Centre of the Region Haná for

Biotechnological and Agricultural Research, Šlechtitelů 586/11,

78371 Olomouc, Czech Republic


Neo-formed contaminants (NFCs) are compounds forming during heating or

preservation processes and exhibiting possible harmful effects to humans. Baking, toasting,

frying, roasting, sterilization result in desired and undesired effects due to various chemical

reactions being Maillard reaction (MR), caramelization and lipid oxidation the most

prominent. Recently, one acrylamide, as neo-formed contaminant has gained much interest

because of its high toxicological potential and its wide occurrence in foods. Acrylamide is

considered as probably or potentially carcinogenic to humans or might be metabolized by

humans to potentially carcinogenic compounds. Its formation in foods has been an intensive

area of research throughout the world.

This study has been mainly focused on determining acrylamide levels in different kind

of cereal and potato foods. The preparation of samples involved grinding a 20 g of sample in a

blender, then mixing with 100-150 mL of water. Ultrasonic extraction was performed within

30 minutes, followed by filtration using Büchner funnel. The samples were derivatized

through using bromination reagent. The sample was kept overnight, and the excess bromine

was decomposed by adding sodium thiosulfate (1 M) as drops until the yellow color

disappeared, and solution was extracted with ethyl acetate. Organic fractions were dried and

evaporated with a rotary evaporator to 1-5 mL.

Calibration curve was prepared using 2,3-dibromopropenamide in the range of

concentration from 5.0 to 80.0 μg/mL. Cereal food products showed levels of acrylamide in

the range from 0.001 ± 0.000 μg/g to 1.745 ± 0.029 μg/g, while potato foodstuff revealed

lower levels of acrylamide in the range from 0.061 ± 0.003 μg/g to 1.070 ± 0.245 μg/g.

64








P 16

ISOLATION AND GC-MS QUANTIFICATION OF PHTHALATES

FROM CHILDREN`S TOY USING DIFFERENT EXTRACTION

TECHNIQUES

A. Goga1, M. Kežić

2, S. Ćavar

1,3, S. Hasić

1, A. Čaušević

2

1University of Sarajevo, Faculty of Science, Department of Chemistry, Zmaja od Bosne 33-35,

71000 Sarajevo, Bosnia and Herzegovina

E-mail: [email protected], [email protected] 2Institute for Public Health FB&H, Department of Hygiene and Environmental Health,

Maršala Tita 9, 71000 Sarajevo, Bosnia and Herzegovina

E-mail: [email protected] 3Palacky University Olomouc, Faculty of Science, Centre of the Region Haná for

Biotechnological and Agricultural Research, Šlechtitelů 586/11,

78371 Olomouc, Czech Republic


Phthalates are dialkyl or alkyl aryl esters of phthalic acid which are used as

plasticizers to make materials more flexible. Due to this property, they are found in almost

every product, such as food, water, food packaging and cosmetics, children's toys,

pharmaceutical products, glue, textiles, building materials, detergents, medical devices, paints

and coatings, electronics, etc. In the last decade, the interest in examination of phthalates

increased solely due to their effects on humans. They are found to be hormone disruptors that

interfere with the balance of hormones in living organisms, causing increase feminization of

male population. There is also evidence of their carcinogenicity (benign tumor of the breast

and testicular cancer), and that they cause various malformations of the testicular tissue.

This work presents the investigation of phthalate content in children's toy in the

dependence of extraction method performed, such as headspace, Soxhlet, ultrasonic, reflux,

hydrodistillation, and cold-mixing extraction. n-Hexane was used as solvent in all techniques.

All extractions last for six hours with sampling every two hours. The phthalate contents were

measured on GC-MS technique. Calibration curves were made using six phthalate standards

that are the most dibutyl phthalate (DBP), butylbenzyl phthalate (BBP), di-n-octyl phthalate

(DNOP), di(2-ethylhexyl) phthalate (DEHP), diizononyl phthalate (DINP) and diizodecyl

phthalate (DIDP). Five phthalates were found with following percentages: DBP to 0.028 ±

0.001 %, DEHP to 0.009 ± 0.000 %, DINP to 0.005 ± 0.000%; DNOP and DIDP were found

in the traces.

As shown, the content of each phthalate depends on extraction technique employed.

Presented results suggest ultrasonic extraction as desirable technique for isolation of these

substances. Furthermore, the results showed that DBP is most common phthalate, which is

consistent with the literature. It is also proven that all identified phthalates found are in the

legal limits, i.e. maximum 0.1% of the total mass of chosen toy.

65








P 17

DEVELOPMENT OF A REVERSED PHASE DISPERSIVE LIQUID-

LIQUID MICROEXTRACTION

FOR THE ANALYSIS OF PHENOLIC COMPOUNDS IN VOO

M.P. Godoy-Caballero, M.I. Acedo-Valenzuela, T. Galeano-Díaz

Analytical Chemistry Department, University of Extremadura, Badajoz, Spain


This communication presents the results of the development of a reversed phase

dispersive liquid-liquid microextraction procedure (RP-DLLME) for the analysis of phenolic

compounds in virgin olive oil (VOO). Phenolic compounds are part of the minor fraction of

compounds in VOO, which constitutes approximately 2 % by weight of total. The interest in

the study and analysis of them is related to the fact that they act as natural antioxidants and

may contribute to the prevention of human disease. In addition, they also contribute to the

sensory properties of VOO as well as to its stability [1]. Different procedures to isolate the

polar phenolic fraction of the VOO have been employed, being mainly used the LLE using

methanol:water mixtures and the SPE using Diol cartridges. However, these procedures are

generally expensive, time and organic toxic solvents consuming and also an intensive labor is

required. In this sense, we propose a simple and fast miniaturized extraction and

preconcentration procedure, based in the dispersive liquid-liquid microextraction (DLLME),

but directly applied onto a non polar sample (VOO). From preliminary experiments a solvent

system composed of 1,4-dioxane (disperser solvent) and ethanol:water (extracting solvent) is

selected. Response surface methodology has been applied by means of a central composite

design (CCD) for the optimization of the variables affecting the extraction procedure

searching for the best recovery. The included variables in the CCD were the total volume of

disperser and extracting solvents, their phase relation (defined as the disperser solvent

volume/extracting solvent volume) and the proportion of ethanol in the hydroalcoholic

mixture used as extracting solvent. The optima results were achieved employing a mixture

composed by 1 mL of 1,4-dioxane as disperser solvent, 150 μL of ethanol:water 60:40 v/v as

extracting solvent and 2 g of VOO. Finally the procedure was satisfactory validated using the

well established SPE with Diol cartridges.

Acknowledgements

María del Pilar Godoy-Caballero is grateful to the Ministerio de Educación of Spain

for a FPU grant (Orden EDU/3083/2009, de 6 de noviembre, BOE nº 277, de 17/11/09,

reference number AP2009-0750). The authors are grateful to the Ministerio de Ciencia e

Innovación of Spain (Project CTQ2011-25388) cofinanced by the European FEDER funds.

Funding from the Junta de Extremadura and European FEDER Funds (Consolidation Project

of Research Group FQM003, Project GR1003) is also acknowledged.

References

1. A. Carrasco-Pancorbo, L. Cerretani, A. Bendini, A. Segura-Carretero, T. Gallina-

Toschi, A. Fernández-Gutiérrez, “Analytical determination of polyphenols in olive

oils” J. Sep. Sci. 28 (2005) 837-858.

66





P 18

QUANTIFICATION OF PHENOLIC COMPOUNDS IN VOO BY RAPID

RESOLUTION LCDAD-MS PREVIOUS REVERSED PHASE

DISPERSIVE LIQUID-LIQUID MICROEXTRACTION

M.P. Godoy-Caballero, T. Galeano-Díaz, M.I. Acedo-Valenzuela

Analytical Chemistry Department, University of Extremadura, Badajoz, Spain


Phenolic compounds are receiving considerable attention, fundamentally due to its

antioxidant activity, strongly related to cancer prevention, inflammatory disorders and

cardiovascular diseases. In addition, phenolic compounds and their strong natural antioxidant

activity contribute to the stability of VOO against oxidation and influence in its organoleptic

characteristics and nutritional qualities [1]. The determination of phenolic compounds in

virgin olive oil (VOO) using a reversed phase dispersive liquid–liquid microextraction

(RPDLLME) previous to the rapid resolution liquid chromatography–diode array and mass

spectrometry detection (RRLC-DAD–MS) have been performed. A rapid resolution Zorbax

Eclipse XDB-C18 column (4.6 mm × 50 mm, 1.8 μm particle size) has been employed and

eighteen phenolic compounds belonging to different families have been identified and

quantified spending a total time of 26 and 13 min with UV-visible and MS detection,

respectively. The validation of the methods was performed through the establishment of the

external standard calibration curves and the analytical figures of merit. Limits of detection

ranging from 40 to 400 ng·mL-1

and 9 to 200 ng·mL-1

were achieved using UV-visible and

MS detection, respectively. The main advantages of the MS detector (higher sensitivity and

lower analysis time) have been shown. However, since MS detector is not always available, it

has been showed that DAD is also a useful detection mode, which in addition shows larger

linearity ranges. Finally, the quantification of the phenolic compounds in VOO from different

olive varieties (Cornicabra, Morisca, Hojiblanca, Picual, Manzanilla Cacereña and

Arbequina in two different ripe stages) was successfully carried out by means of the standard

addition method.

Acknowledgements

María del Pilar Godoy-Caballero is grateful to the Ministerio de Educación of Spain

for a FPU grant (Orden EDU/3083/2009, de 6 de noviembre, BOE nº 277, de 17/11/09,

reference number AP2009-0750). The authors are grateful to the Ministerio de Ciencia e

Innovación of Spain (Project CTQ2011-25388) cofinanced by the European FEDER funds.

Funding from the Junta de Extremadura and European FEDER Funds (Consolidation Project

of Research Group FQM003, Project GR1003) is also acknowledged.

References

1. A. Carrasco-Pancorbo, L. Cerretani, A. Bendini, A. Segura-Carretero, T. Gallina-

Toschi, A. Fernández-Gutiérrez, “Analytical determination of polyphenols in olive

oils” J. Sep. Sci. 28 (2005) 837-858.

67





P 19

UTILIZATION OF ULTRA HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY FOR THE STUDY OF OLIGONUCLEOTIDES

S. Studzińska, B. Buszewski

Chair of Environmental Chemistry and Bioanalytics, Faculty of Chemistry,

Nicolaus Copernicus University, 7 Gagarin St., PL- 87-100 Toruń, Poland


Quantitative and qualitative analysis of oligonucleotides is of great importance in the

field of new drugs development. Synthetic modified and unmodified nucleosides are tested as

a novel medicines for the treatment of various diseases (e.g. cancer, diabetes, Amyotrophic

lateral sclerosis, Duchenne muscular dystrophy, asthma, arthritis). Chromatographic

techniques may be successfully used for the separation and determination of oligonucleotides.

Ion exchange (IEC) and ion pair reversed chromatography (IP RP HPLC) are the most widely

used for this purpose. Both techniques have advantages and disadvantages connected with the

required resolution or selectivity. Resolution of the oligonucleotides may be improved by the

application of Ultra High Performance Liquid Chromatography (UHPLC). Due to the wide

avability of stationary phases with small particle size, UHPLC is a great alternative to IEC

and IP RP HPLC. So far very few papers concern the utilization of UHPLC for the studies of

oligonucleotides.

The main aim of the study was to separate the mixture of 20mer oligonucleotides by

UHPLC with three different stationary phases (octadecyl, octyl and phenyl). The type of

mobile phase was also the goal of the study. Various ion-pairing reagents as well as buffers

were studied to choose the most suitable for the resolution of oligonucleotides.

Acknowledgement Financial support was provided by the National Science Center (Cracow, Poland)

under grant No. 2011/01/D/ST4/04142.

68





P 20

MICELLAR HPLC METHOD USING MONOLITHIC COLUMN FOR

THE DETERMINATION OF LINEZOLID AND RIFAMPICIN IN

PHARMACEUTICALS AND BIOLOGICAL FLUIDS

F. Belal, M.K. Sharaf El-Din, M.I. Eid, R.M. El-Gamal

Department of Analytical Chemistry, Faculty of Pharmacy,

University of Mansoura, 35516, Mansoura, Egypt


A Simple and rapid micellar liquid chromatographic method was developed and

validated for the simultaneous determination linezolid (LNZ) and rifampicin (RIF). The

analysis was achieved using a 50 mm x 4.6 mm i.d, Chromolith® SpeedROD RP-18

endcapped column as a stationary phase. A mobile phase consisting of a mixture of 0.15 M

sodium dodecyl sulphate (SDS), 0.3% triethylamine (TEA), 10% n-propanol in 0.02 M

orthophosphoric acid of pH 6.0 was pumped at a flow rate of 1 mL/min, with ultraviolet

detection at 254 nm. Lamotrigine (LTG) was used as an internal standard (IS). The method

showed good linearity over the concentration ranges of 2.0 – 40.0 g/mL and 6.0 – 120.0

g/mL with limits of detection of 0.65, 1.40 g/mL and limits of quantification of 1.96, 4.24

g/mL for LNZ and RIF, respectively. The suggested method was successfully applied for the

analysis of the studied drugs in their different dosage forms. The method was further extended

to the determination of the studied drugs in spiked human plasma without prior extraction, the

mean percentage recoveries in spiked and human plasma (n = 4) were 99.94 ±1.39 and 99.35

± 4.15 for LNZ and RIF, respectively. Moreover the method was also extended to the

determination of RIF in spiked human urine. Statistical evaluation and comparison of the data

obtained by the proposed and comparison methods revealed good accuracy and precision of

the proposed method.

Keywords: determination, micellar liquid chromatography, monolithic column linezolid,

rifampicin, lamotrigine, dosage forms

69





P 21

DETERMINATION OF CHARGE DISTRIBUTION OF

BACTERIAL CELL

E. Dziubakiewicz, K. Hrynkiewicz, M. Walczyk, B. Buszewski

1Chair of Environmental Chemistry and Bioanalytics, Faculty of Chemistry

Nicolaus Copernicus University, Gagarina 7, 87-100 Toruń, Poland 2Department of Microbiology, Institute of General and Molecular Biology,

Nicolaus Copernicus University, Gagarina 9, 87-100 Toruń, Poland 3Chair of Materials Chemistry, Adsorption and Catalysis, Faculty of Chemistry

Nicolaus Copernicus University, Gagarina 7, 87-100 Torun, Poland


Capillary zone electrophoresis is used in rapid separations and identification of

microorganisms so it gives the potential to use it in bioscience research, medical diagnosis

and profiling of some diseases. However, during electrophoretic separations aggregation

and/or adhesion of bacterial cells may be observed which are serious disadvantages of the

method.

Each bacteria species has a complex and characteristic cell wall composition.

Macromolecules which are present in the cell wall and membranes of bacteria, for example:

proteins, phospholipids, teichoic acid, teichuronic acid and lipopolysaccharides provide a

unique biochemical fingerprint. Consequently, the charged cell wall groups determine the

spontaneous formation of the electrical double layer. The properties of the EDL affect the

behavior of biocolloid including cell-to-cell and cell-to-capillary surface interactions. Electric

properties of bacterial cells are a function of the type and concentration of specific functional

groups on cell surface, the electrolyte composition, the pH value and the ionic strength of the

electrolyte.

This work presents a basic knowledge about interactions of bacterial cells including

negative effect of bacterial adhesion and aggregation on the basis on electrophoretic behavior

of different strains of Gram-positive (B. subtilis, B. cereus, S. aureus(1), M. luteus, S. lutea)

and Gram-negative (E. coli) bacteria. Physicochemical properties of bacterial cell wall

surfaces were investigated by the combination of electrochemical (zeta potential,

potentiometric titration) and spectroscopic techniques (FTIR) allow to obtain concentration

and protonation/deprotonation of the specific functional groups on the microorganism cell

surface.

The results obtained are helpful in obtaining accurate information about the potential

structure of the electrical double layer the examined bacteria cell and allow understanding the

electrophoretic separation of living organisms.

Acknowledgements

This work was supported by the National Science Centre (Narodowe Centrum Nauki,

abbr. NCN (Poland) Grant No. N N204 369040 as well as by the European Social Found, the

Polish National Budget and the budget of the Kujawsko-Pomorskie Region as part of the

“Krok w przyszłość IV” 2012-2013 (Step into the Future) Sectoral Operational Programme –

Human Resources.

70





P 22

BENZODIAZEPINES IN THE ENVIRONMENT: OCCURRENCE, FATE

AND TRANSFORMATION

E. Heath1,2

, S. Perko1, T. Kosjek

1

1Jožef Stefan Institute, Department of Environmental Sciences, Ljubljana, Slovenia

2International Postgraduate School Jožef Stefan, Ljubljana, Slovenia


Benzodiazepine derivatives are prescribed in large quantities globally and are

potentially new emerging environmental contaminants. Unfortunately, a dearth of data exists

concerning occurrence, persistence and fate in the environment. This paper redresses this by

reviewing existing literature, assessing the occurrence of selected benzodiazepine anxiolytics

(diazepam, oxazepam and bromazepam) in wastewater influent and effluent and surface water

from Slovenia, evaluating their removal during water treatment and identifying the

transformation products formed during water treatment. Their occurrence was monitored in

hospital effluent, river water and in wastewater treatment plant influent and effluent. The

study reveals the presence of benzodiazepine derivatives in all samples with the highest

amounts in hospital effluents: 111 ng L−1, 158 ng L−1 and 72 ng L−1 for diazepam,

bromazepam and oxazepam, respectively. Removal efficiencies with respect to biological

treatment of diazepam were 16–18% (oxic), 18–32% (anoxic → oxic), 53–76% (oxic →

anoxic) and 83% (oxic → anoxic → oxic → anoxic cascade bioreactors), while the removal

oxazepam was 20–24% under anoxic conditions. Coupled biological and photochemical

treatment followed by the adsorption to activated carbon resulted in a removal efficiency of

99.99%. Results reveal the recalcitrant nature of benzodiazepine derivatives and suggest that

only combinational treatment is sufficient to remove them. In addition, eight novel diazepam

and four novel oxazepam transformation products are reported.

71

http://www.sciencedirect.com/science/article/pii/S0043135411006646

http://www.sciencedirect.com/science/article/pii/S0043135411006646





P 23

SPHINGOSINE1-PHOSPHATELYASE ACTIVITY MEASUREMENT

BY HPLC-FLUORESCENCE DETECTION AFTER 5,5-DIMETHYL

CYCLOHEXANEDIONE DERIVATIZATION

Y.-M. Lee, K.-O. Shin, C.-H. Seo, S.-H. Lee

College of Pharmacy and MRC, Chungbuk National University, Chongju 361-763, Korea


Sphingosine-1-phosphate (S1P) is a sphingolipid signaling molecule crucial for cell

survival and proliferation. S1P lyase (S1PL) is the only known enzyme that irreversibly

degrades sphingoid base-1-phosphates to phosphoethanolamine and the corresponding fatty

aldehydes. S1PL-mediated degradation of S1P results in the formation of (2E)-hexadecenal,

whereas hexadecanal is the product of dihydrosphingosine-1-phosphate (DHS1P) degradation.

We have devised an assay using a commercially available C17 DHS1P substrate. This

substrate degrades pentadecanal and phosphoethanolamine. We have developed a simple,

highly sensitive protocol for pentadecanal quantitation as a 5,5-dimethyl cyclohexanedione

(5,5-dimethyl CHD) derivative by high performance liquid chromatography fluorescence

detector (HPLC-FLD). We optimized derivative reaction as reaction time and reaction

temperature and 5,5-dimethyl CHD, acetic acid, ammonium acetate concentration. The

reaction is linear over 20 min and total protein concentrations of 10-50 µg. in this method

SPL levels as low as 4 pmol/mg/min were readily detected. The SPL-catalyzed reaction is

linear over a 30 min time period and yields a Km of 2.68 μM for C17 DHS1P.

To confirm our new methods, we assay the SPL level of different cell lines in F9-0,

normal cell and F9-2 cell, S1PL knock down and F9-4 cell, S1PL overexpression.

Furthermore, we treated F9-4 cells with different SPL inhibitor as FTY720 and 2-Acetyl-5-

tetrahydroxybutyl Imidazole (THI), which competitively inhibits pyridoxal-5-phosphate

(P5P), an essential cofactor for SPL activity, and observed a significant decrease in

pentadecanal relative to the untreated cells.

In conclusion, we developed of the high sensitivity of HPLC-FLD and use of

commercially available internal standard for pentadecanal quantification, heptadecanal, to

develop a simple, sensitive protocol for characterization of S1PL activity in vitro.

72





P 24

APPLICATION OF LIQUID CHROMATOGRAPHY UNDER LIMITING

CONDITIONS OF DESORPTION TO SEPARATION OF DIFFICULT

COMPLEX POLYMER SYSTEMS

A. Šišková, E. Macová, D. Berek

Polymer Institute, Slovak Academy of Sciences, Dúbravská cesta 9,

845 41 Bratislava, Slovakia

Email: [email protected], [email protected]

Liquid chromatography under limiting conditions of desorption, LC LCD is an

unconventional chromatographic technique, which employs the action of a barrier. Its

principle is based on the selective deceleration of adsorptive macromolecules eluted from

a liquid chromatographic column packed with the polar porous particles. The barrier is the

zone of a low-molecular substance, which is injected into the column in front of the sample

solution, and which promotes adsorption of particular macromolecules. Such pore permeating

and therefore tardily progressing zone is impermeable for the adsorbing macromolecules and

adequately decelerates their elution. The less-adsorptive macromolecules are not affected with

the barrier and they elute freely in the exclusion mode to be efficiently discriminated from the

decelerated, more-adsorptive macromolecules. In this way, macromolecules that exhibit

different adsorptivities within the LC LCD column can be mutually discriminated in a

selective, efficient and very rapid way. The important conditions for successful separation of

distinct kinds of macromolecules with help of LC LCD is the difference in adsorptivity of

particular sample constituents within column packing and the solubility of macromolecules in

solvents of different polarities so that the adsorption effects can be well controlled with

mixing solvents preventing adsorption with solvents promoting adsorption. Both low

solubility and too high adsorptivity of constituents of complex polymer systems represent

important challenges for a successful LC LCD application. It will be shown how these

limitations can be overcome. The low solubility polymers, poly(ethylene terephthalate), PET

and poly(butylene terephthalate), PBT were mutually separated at ambient temperature.

Above poly(terephthalate)s were also base-line separated from the aliphatic biodegradable

polyesters poly(L-lactic acid) and poly(butylene adipate) [1]. The separations of highly

adsorptive polymers such as poly(N-vinyl pyrrolidinone), PNVP and poly(ethylene oxide),

PEO from the less adsorptive polymers poly(ɛ-caprolactone), PCL, poly(methyl

methacrylate), PMMA or polystyrene were carried out employing very strong desorli

dimethylformamide and – in case of PNVP by substituting bare silica gel with the PEG coated

silica gel as the column packing. The results again demonstrate high selectivity of LC LCD,

which enables discrimination of macromolecules of well similar chemical structure,

irrespectively of their molar mass. Moreover, LC LCD allows identification of minor (<1 and

even <0.1%) constituents of multicomponent polymers and it can be used as an integral part

of the two-dimensional liquid chromatography for comprehensive molecular characterization

of complex polymer systems [2].

Acknowledgement

This work was supported by the Slovak Research and Development Agency, APVV

project 0109-10 and by VEGA 2-0001-12.

73






References:

1. A. Šišková, E. Macová, D. Berek, European Polymer Journal 48 (2012) 155-162.

2. D. Berek, A. Šišková, Macromolecules 43 (2010) 9627-9634.

74

http://www.scopus.com/authid/detail.url?origin=resultslist&authorId=36877056700&zone=

http://www.scopus.com/authid/detail.url?origin=resultslist&authorId=12038941800&zone=

http://www.scopus.com/source/sourceInfo.url?sourceId=25854&origin=resultslist




P 25

CHROMATOGRAPHY SEPARATION OF ESSENTIAL IONS IN

STEVIA

Ž. Zgorelec, A. Jurišić, M. Mesić, I. Šestak, B. Benko, S. Fabek, B. Novak, A.-M.

Špicnagel

Faculty of Agriculture, University of Zagreb, Svetošimunska cesta 25, 10 000 Zagreb


Stevia (Stevia rebaudianaBertoni) belongs to the family Asteracae and the intensive

sweet taste of its leaves provided the name „new age sugar“. Stevia is cultivated for the sweet

taste leaves, which are, after drying and grinding, used as a substitute for artificial sweeteners.

The stevia products are widespread as food additives and a natural calorie free sweetener. An

intensive sweetness originates from glycosides, i.e. termostable chemical compounds well

tolerated the low pH values. The subject of the whole interdisciplinary study was to determine

the effect of fertilizer dosesand the soil type (pH) on the fresh leaves yield.Furthermore,

content of glycosides (stevioside and steviol) and mineral composition of stevia leaves were

determined. Plants were grown in a 5 L pots on the two types of acid soil (pH 4.0 and 5.0),

which were different according to their supply with major nutrients. Five doses (from 0 up to

1.6 g per plant) of Multi-Comp Base mineral fertilizer 14:13:20+2MgO+ME were applied.

After harvest, leaves were dried at 70 °C for 72 h, ground, sieved and homogenized.The

concentrations of 7 anions (F-, Cl

-, NO2

-, Br

-, NO3

-, SO4

2-, H2PO4

-) and 6 cations (Li

+, Na

+,

NH4+, K

+, Ca

2+, Mg

2+) was simultaneously determined by suppressedion chromatography(two

Dionex 1000 systems) using an IonPac AS 17 and IonPac CS 16 separation columns fitted

with an IonPac AG 17 and IonPac CG 16 guard columns, the eluent solutionswere KOH and

MSA, respectively.Detection was done after 2h hot (at 90 °C) ultra-pure water extraction at

1:1000 (w/v) ratio.Simultaneous analysis was enabled by a splitter, installed on

autosamplerAS40, which split the sample in two pipelines, one for anion and other for cation

system. Slightly overshadowed by all other nutrient, sodium and chlorine, as mineral

components, play the very important role in Stevia. In this paper content of Na+ and Cl

- as

essential micronutrients in Stevia is shown and discussed.

75





P 26

CHARACTERISTICS OF VOLATILE COMPOUNDS IN THE

LIVERWORT ANEURA PINGUIS COLLECTED IN THE POLAND

R. Wawrzyniak1, W. Wasiak

1, K. Buczkowska

2, J. Ostrowska

1

1Faculty of Chemistry, Adam Mickiewicz University, Umultowska 89b, 61-614 Poznań,

Poland 2Institute of Experimental Biology, Adam Mickiewicz University, Umultowska 89,

61-614 Poznań, Poland


The liverwort Aneura pinguis is represented in Europe by 5 cryptic species: A. pinguis

– species A, A. pinguis – species B, A. pinguis – species C, A. pinguis – species D and A.

pinguis – species E. Four of them (A, B, C, E) were found in Poland and the fifth (D) in the

Great Britain. Identification of individual species based on the cryptic morphological

characteristics is difficult. For that we decided to carry out characteristics of populations

collected in Poland by identification of volatile compounds. We applied the headspace solid-

phase microextraction (HS-SPME) technique coupled with GC/MS analysis. We used the

SPME holder for manual sampling and the fused silica fiber (length 2cm; film thickness

50/30μm) coated with mixed DVB/CAR/PDMS. Frozen, wet plant material was detached

from its support, placed in hermetically closed vial with a Teflon/silicone septum. The SPME

fiber was then manually inserted into the sample vial headspace. The vial was heated to

improve sorption of the hardly volatile compounds. Component identification was confirmed

by comparison of the mass spectral fragmentation patterns with those reported in the

literature. In addition, retention indices on the apolar and polar columns, determined relative

to a homologous series of n-alkanes, were compared with published index data.

We have detected the presence of characteristic for liverwort pinguisane-type

sesquiterpenoids. Quantitative data of the components was obtained by integrating the

chromatogram and calculating the relative percentage of the peak areas.

76






P 27

CAPILLARY LIQUID CHROMATOGRAPHY OF SMALL

MOLECULES ON HYPERCROSSLINKED MONOLITHIC COLUMNS

J. Urban, V. Škeříková

Department of Analytical Chemistry, Faculty of Chemical Technology, University of

Pardubice, Studentska 573, Pardubice, Czech Republic, 532 10


Hypercrosslinking post-polymerization modification allows preparation of organic

polymer capillary monolithic columns suitable for fast and efficient separations of small

molecules in isocratic liquid chromatography.Three dihalogenic solvents differing in polarity

(1,2-dichloroethane, 1,4-dichlorobutane, and 1,6-dichlorohexane) with three Friedel-Crafts

alkylation catalysts varying in reactivity (AlCl3, FeCl3, and SnCl4) have been used to prepare

hypercrosslinked poly(styrene-co-vinylbenzyl chloride-co-divinylbenzene) columns. After

optimization of hypercrosslinking conditions including reaction time, temperature, and

catalyst concentration, we prepared a column with efficiency exceeding 80 000 theoretical

plates/m. Optimized hypercrosslinked columns enable fast separations at higher flow rates

without the loss of efficiency as demonstrated by fast isocratic separation of low molecular

compounds, such as barbiturates.

To extend the applicability of hypercrosslinked monolithic columns we have modified

residual reactive chloromethyl groups using 4,4′-azobis(4-cyanovaleric acid) initiator and

used zwitterionic N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl) ammonium betaine

monomer to prepare a column allowing a separation of polar compounds in hydrophilic

interaction chromatography. The prepared column did not lose its separation power even after

more than 10 000 injections which confirmed very high stability of the modification reaction.

Finally, the optimized capillary monolithic column has been applied in comprehensive two-

dimensional chromatography of polar phenolic acids and flavones.

Acknowledgement

The financial support of GACR project P206/12/P049 is gratefully acknowledged.

77





P 28

MAGNETO-TLC AS A TOOL FOR PLANT EXTRACT ANALYSIS

M. Studziński1, I. Malinowska

1, W. Jesionek

2, H. Malinowski

3

1Department of Planar Chromatography, Chair of Physical Chemistry, Faculty of Chemistry,

Maria Curie-Skłodowska University, Maria Curie-Skłodowska Sq. 3, 20-031 Lublin, Poland

E-mail: [email protected], [email protected] 2Department of Chromatographic Methods, Faculty of Chemistry Maria Curie-Skłodowska

University, Maria Curie-Skłodowska Sq. 3, 20-031 Lublin, Poland

E-mail: [email protected] 3Vexler and Baldin Laboratory of High Energy Physics, Joint Institute for Nuclear Research,

Dubna, Russia


Thin layer techniques proved their usefulness in analysis of plant extract material.

Thanks to their low operation costs, high sample throughput and ability to gather

chromatographic data for the whole sample in single act, they allowed to find, and in some

cases identify, active ingredients often hidden in sophisticated plant extract matrices.

Despite all their advantages, planar chromatography techniques have a few

deficiencies. The most serious accusation against them is poor efficiency of chromatographic

system, especially comparing to more advanced techniques as HPLC, and unbalanced

conditions of chromatographic separation. One can say that described disadvantages limits

seriously the applications of TLC as a modern chromatographic method of analysis, but in

fact, the solution of particular chromatographic separation problem is not gaining the highest

system efficiency (which may, but does not have to lead to final solution of the problem) but

optimalisation the whole chromatographic system in order to separate all ingredients of

investigated mixture (for example a plant extract.

As it was presented in our previous works [1,2], applying external magnetic field

during chromatogram development results in changes of retention of chromatographed solutes

what can be used for adjustment of their chromatographic behaviour, in order to optimize the

separation. In this work, we would like to present the influence of application of external

magnetic field during chromatogram development on TLC and HPTLC plates on separation

of ingredients of plant extracts, which are multicomponent mixtures and are very difficult to

separate in conventional chromatographic conditions.

References:

1. Malinowska I, Studzinski M, Malinowski H (2008) JPC-Journal of Planar

Chromatography-Modern TLC 21:379-385. DOI 10.1556/JPC.21.2008.5.11

2. Malinowska I, Studzinski M, Malinowski H (2011) Journal of Separation Science

34:1788-1795. DOI 10.1002/jssc.201100249

78








P 29

COMPARISON OF NON-AQUEOUS NORMAL PHASE LIQUID

CHROMATOGRAPHY WITH AQUEOUS NORMAL-PHASE LIQUID

CHROMATOGRAPHY ON HYDROSILATED SILICA-BASED

STATIONARY PHASES

J. Soukup, P. Jandera

Faculty of Chemical Technology, Department of Analytical Chemistry, University of

Pardubice, Studentská 95, Pardubice 2, 532 10, Czech Republic


The effects of mobile phase composition on the separation of phenolic acids in both

aqueous normal phase (ANP) and non-aqueous normal phase liquid chromatography (NANP)

modes were studied. In case of ANP, the analyte is distributed between a water rich stationary

layer and the bulk mobile phase with lower water concentartions, probably by a combination

of partition and adsorption mechanisms, while in NANP, the retention mechanism should be

preferentially based on adsorption.Phenolic acids elute in the order of increasing polarities i.e.

with increasing number of polar groups (OH, -COOH, etc.) opposite to the reversed-phase

mechanism.

We investigated four types of hydrosilated silica columns (hydrosilated silica,

diamond hydride, cholesterol and bidentate column) for separations ofthe phenolic acids in

both acetonitrile/water and acetonitrile/methanol mobile phases, both in the ANP and NA-

HILIC modes. We have found that methanol in buffered mobile phase can substitute water in

the HILIC mode. In both ANP and NANP modes, the elution order of the phenolic acids

selected is almost identical, except syringic and 4-hydroxyphenylacetic acid and the retention

of the phenolic acids decreases with increasing concentration of water or methanol in mobile

phase. All four hydrosilated silica-based columns showed higher retention of the phenolic

acids used in NA-HILIC mobile phases than in aqueous normal-phase (ANP) mobile phases,

but the resolution and selectivity are significantly better in buffered ANP water/acetonitrile

mobile phases.

Acknowledgement

This work was financially supported by the project Enhancement of R&D Pools of

Excellence at the University of Pardubice reg. Nr. CZ.1.07/2.3.00/30.0021.

79





P 30

HEADSPACE SOLID-PHASE MICROEXTRACTION

OF RARE CORIANDRUM SATIVUM L. HONEY

I. Jerković1, M. Obradović

1, P.M. Kuś

2, M. Šarolić

3

1Department of Organic Chemistry, Faculty of Chemistry and Technology, University of Split,

N. Tesle 10/V, HR 21000 Split, Croatia

E-mail: [email protected] 2Department of Pharmacognosy, Wrocław Medical University, ul. Borowska 211a, PL 50-556

Wrocław, Poland 3Department of Food Technology, Marko Marulić Polytechnic in Knin, Petra Krešimira IV

30, HR 22300 Knin, Croatia

The chemical analysis of rare Coriandrum sativum L. honey sample from Poland

reveals new data on low-molecular compounds that could be helpful for the honey botanical

origin specification. The compounds responsible for highly individual aroma profile of

Coriandrum sativum L. honey were isolated by headspace solid-phase microextraction (HS-

SPME) and analyzed by gas chromatography and mass spectrometry (GC-MS). In order to

obtain more complete headspace composition, HS-SPME was applied with two different

fibres recommended for volatiles/semi-volatiles: polydimethylsiloxane/divinylbenzene

(PDMS/DVB) and divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS).

Coriander honey exhibited unusual chromatographic profile of linalool derivatives in the

headspace and possesses highly individual aroma that is very well distinguished from other

honey types.

OCH2OH

lilac alcohols

HH OCHO

lilac aldehydes

H HO

anhydrolinalool oxides

HO OH

linalool oxides

H

The headspace of the honey was dominated by trans-linalool oxide (up to 15%)

followed by other linalool derivatives such as lilac aldehyde/alcohol isomers (total up to

15%), cis/trans-anhydrolinalool oxides (total up to 6%), cis-linalool oxide (up to 3.5%) as

well as linalool (up to 6.5%) and other compounds, structurally related to linalool (such as

diastereomers of p-menth-1-en-9-al (total up to 19.0%). Among the compounds identified,

cis/trans-dehydroxylinalool oxides can be useful as more specific chemical markers of

coriander honey headspace.

80





P 31

GC-MS PROFILING OF RARE UNIFLORAL

PRUNUS CERASUS L. HONEY HEADSPACE

P.M. Kuś1, I. Jerković

2, Z. Marijanović

3, M. Šarolić

3

1Department of Pharmacognosy, Wrocław Medical University, ul. Borowska 211a, PL 50-556

Wrocław, Poland 2Department of Organic Chemistry, Faculty of Chemistry and Technology, University of Split,

N. Tesle 10/V, HR 21000 Split, Croatia

E-mail: [email protected] 3Department of Food Technology, Marko Marulić Polytechnic in Knin, Petra Krešimira IV

30, HR 22300 Knin, Croatia

A sour cherry honey is rare honey type characterized by taste of bitter almond [1]. The

chemical analysis of its specific headspace reveals new data on low-molecular compounds

that may be helpful for the honey botanical origin specification. The headspace compounds of

Prunus cerasus L. honey were isolated by headspace solid-phase microextraction (HS-SPME)

using fibre coated with polydimethylsiloxane/divinylbenzene (PDMS/DVB) and analysed by

gas chromatography and mass spectrometry (GC-MS). Sour cherry honey exhibited

chromatographic headspace profile characterized mainly by lilac aldehydes (total 48.3%)

benzaldehyde (18.7%) and several minor compounds like thymol (2.1%), eugenol (2.0%),

phenylacetaldehyde (1.7%), linalool (1.7%), 3,4-dihydro-3-oxoedulan (1.7%), trans-β-

damascenone (1.6%), hotrienol (1.4%) and α-isophorone (1.2%).

OCHO

lilac aldehydes

H H

H O

benzaldehyde

Benzaldehyde is known to be responsible for characteristic aroma of bitter almonds

where it is generated as a product of amygdalin hydrolysis [2]. Its aroma can be expressed

with such descriptors as: “sweet, almond, marzipan”. The contribution of benzaldehyde may

explain specific bitter almond taste of Prunus cerasus L. honey. Lilac aldehydes possess

smells that were described as “pleasant, sweet, fresh, flowery” [3,4]. Since their odor treshold

is low, their impact on the aroma of sour cherry honey may be significant. Among the

compounds identified, high levels of lilac aldehydes and benzaldehyde may be the most

useful only as non-specific chemical markers of sour cherry honey headspace since they occur

also in other honey types.

References:

1. Farkas Á, Zajácz E. (2007) Nectar production for the Hungarian honey industry. The

European Journal of Plant Science and Biotechnology, 1, 125-151.

2. Sánchez-Pérez R, Howad W, Garcia-Mas J, Arús P, Martínez-Gómez P, Dicenta F.

(2010) Molecular markers for kernel bitterness in almond. Tree Genetics & Genomes

6, 237–245.

81





3. Wilkins AL, Lu Y, Tan ST. (1993) Extractives from New Zealand honeys. 4. Linalool

derivatives and other components from nodding thistle (Carduus nutans) honey.

Journal of Agricultural and Food Chemistry, 41, 873-878.

4. Alissandrakis E, Tarantilis PA, Harizanis PC, Polissiou M. (2007) Aroma

investigation of unifloral Greek citrus honey using solid-phase microextraction

coupled to gas chromatographic-mass spectrometric analysis. Food Chemistry, 100,

396-404.

82




P 32

GAS CHROMATOGRAPHIC HEADSPACE FINGERPRINT OF

VIRGIN OLIVE OIL FROM AUTOCHTHONOUS VARIETY

MAŠNJAČA

M. Šarolić1, M. Gugić

1, Z. Marijanović

1, M. Šuste

1, I. Jerković

2

1Department of Food Technology, Polytechnic Marko Marulić in Knin,

Petra Krešimira IV 30, 22300, Knin

E-mail: [email protected], [email protected],

[email protected], [email protected] 2Department of Organic Chemistry, Faculty of Chemistry and Technology,

University of Split, N. Tesle 10/V, 21000 Split


Virgin olive oils, extracted from fresh and healthy olive fruits (Olea europaea L.) and

properly processed, are characterized by a delicate and unique flavor highly appreciated by

consumers. Their peculiar delicious taste and aroma are closely related, both to some

nonvolatile compounds and to a number of volatile compounds [1].

Most of aromatic volatile compounds are formed through the action of enzymes that

are released when the fruit is crushed, and continue to form during malaxation through

number of enzymatic reactions known as lipoxygenase pathway [2]. Lipoxygenase pathway is

initiated by the release of enzymes when olive fruit tissues are disrupted. The reaction

pathway involves a series of enzymes that oxidize (lipoxygenase) and cleave (hydroperoxide

lyase) polyunsaturated fatty acids to yield aldehydes that may be reduced to alcohols (by

alcohol dehydrogenase) and esterified to esters (by alcohol acyltransferase) [2]. The aroma of

olive oil is attributed to aldehydes, alcohols, esters, hydrocarbons, ketones, furans and others.

The major volatiles reported in virgin olive oils were C6 and the C5 compounds. Influence of

the olive cultivar can be evidenced by different amounts of C6 compounds arising from the

enzymatic oxidation of linoleic and linolenic acids [1].

Mašnjača is Croatian autochthonous variety found only in north Dalmatia (subVelebit

area and Zadar hinterland). In the present study the volatile profile of virgin olive oil from

autochthonous variety Mašnjača was established using solid-phase microextraction (SPME)

with PDMS/DVB fibre coating followed by gas chromatography-mass spectrometry (GC-MS)

analysis. Predominant headspace compounds were: (E)-hex-2-enal (27.6%), (Z)-hex-3-enal

(25.0%), 3-ethylocta-1,5-diene (two isomers: 13.8%; 10.3%), pent-1-en-3-one (5.6%) and

pentan-3-one (5.2%). The main volatiles found in tested oil are quite similar to Tunisian

variety Che´toui where the percentage of (E)-hex-2-enal was 20.0% and (Z)-hex-3-enal was

25.4%, while French variety Cailletier contained 78.3% (E)-hex-2-enal and 3.6% (Z)-hex-3-

enal [3]. However, the study of a larger number of samples from various years of production

can be suggested for support the results obtained by first screening.

References:

1. Angerosa et al., Food Chemistry, 68 (2000) 283-287.

2. Kalua et al., Food Chemistry, 100 (2007), 273-286.

3. Haddada et al., Food Chemistry, 103 (2007), 467–476.

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P 33

p53 SUPERCOILED PLASMID PURIFICATION BY A L-

METHIONINE-AGAROSE MATRIX

J.F.P. Valente, I.J. Correia, F. Sousa

CICS-UBI – Health Sciences Research Centre, University of Beira Interior,

Av. Infante D. Henrique, 6200-506 Covilhã, Portugal


Cancer is still one of the most lethal diseases and one of the leading causes of death

worldwide. In order to treat this scourge, gene therapy and DNA vaccination have been

proposed as an alternative to the common treatments. Recently, highly enriched supercoiled

(sc) isoform of a p53 tumor suppressor encoding gene have been proved to be more efficient

for gene transfection than open circular plasmid DNA.

To successfully isolate this isoform different affinity chromatographic techniques

using amino acids had been successfully used however, it remains essential to develop and

study new matrices with higher specificity and robustness, enabling higher yield of the

purified sc plasmid to be obtained.

Accordingly, this work describes the successful use of a new chromatographic matrix

of L-methionine-agarose to purify the sc isoform of the p53 plasmid. To achieve this goal the

6.07 kbp plasmid pcDNA3-FLAG-p53 was amplified in a cell culture of E. coli DH5α. After,

these cells were lysed through a modified alkaline method and then the lysate was injected in

the L-methionine-agarose column and the purified sc isoform was obtained. To achieve this

goal, the salt concentration, pH of the mobile phase and also the temperature were changed

and combined.

Acknowledgment


Technology through the Project with reference “PTDC/EBB-BIO/114320/2009” and also by

PEst-C/SAU/UI0709/2011 COMPETE.

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P 34

DETERMINATION OF POLYPHENOLS AND INDOLE-3-ACETIC

ACID IN WINES BY HPLC-DAD-FLD

L. Maslov1, I. Tomaz

1, M. Medić-Šarić

2

1Faculty of Agriculture, University of Zagreb, Department of Viticulture and Enology,

Svetosimunska 25, Zagreb, Croatia

E-mail: [email protected] 2Faculty of Pharmacy and Biochemistry, University of Zagreb, Department of Medicinal

Chemistry, A. Kovačićeva 1, Zagreb, Croatia

Phenolic compounds are important components of grapes and essential to wine

quality. They are responsible for some sensorial characteristics of red wines and may play an

important role in the health benefits attributed to moderate wine consumption. The tryptophan

metabolite indole-3-acetic acid (IAA) is regarded as an important potential precursor of 2-

aminoactophenone (2-AAP), an aroma compound which is associated with so-called untypical

aging (UTA) off flavor in V. vinifera L. white wines. IAA plays an important role as a

metabolite in amino acids and polyphenol biosynthesis. HPLC method for analysis of 35

phenolic compounds and indole-3-acetic acid in red and white wines by UV-VIS photodiode

array (DAD) and fluorescence detector (FLD). The method uses acidified water and

acetonitrile as mobile phases. Separation is achieved on C-18 phenyl hexyl column.

Precisions, recoveries and LODs achieved for all the analytes were satisfactory. The proposed

method was applied to determination of these compounds in Gewürztraminner white wines

from Croatia and in red wines „Plavac mali“ from native Croatian grape variety. Solid phase

extraction (SPE) was used for isolation of phenolic compounds and indole-3-acetic acid in

wines. Different copolymer reversed-phase SPE cartridges were tested. HLB cartridges were

chosen and SPE method was optimized. Recoveries for all tested compounds were above 92

%. For the first time indole-3-acetic acid was determined in red wines from Croatia.

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P 35

THE STUDY OF DIFFERENCES IN METABOLOMIC PROFILES OF

GRASSHOPPER’S CHORTHIPPUS SPP. ABDOMINAL SECRETION

OBTAINED FROM THREE SPECIES OF INSECTS

M. Buszewska-Forajta, J. Raczak-Gutknecht, K. Walkowiak, M. Patejko, D. Siluk,

R. Kaliszan

Department of Biopharmaceutics and Pharmacodynamics, Medical University of Gdańsk,

ul. Gen. J. Hallera 107, 80-416 Gdańsk, Poland


Advantages of biologically active compounds of natural origin have been known since

ages as herbalists and folk healers used extracts or other herbal preparations in the treatment

and prevention of many diseases. Later on a substantial amount of drugs, isolated mainly from

plants were recognized by academic medicine or served as “lead compounds” in search for

new drugs. In recent years numerous research projects have been dedicated to discovery of

active substances isolated from insects. Particularly, many studies have been focused on

peptides with potential antifungal, antibacterial or myotropic activities. Therefore, it seemed

rational to undertake a study on grasshopper based on etnopharmacological premises. First of

all, it could be expected that identification of the secretion composition of the insect would be

original cognitive value. Identified agents, could then serve as a base for rational structure

modifications into potential drugs with activity as wound healing agents. According to

etnopharmacological observations an ointment-like material squeezed out from abdomen of

grasshoppers was used by villagers of the west central Poland to facilitate healing of wounds

and scars.

The aim of the project was to study grasshopper metabolome, including its qualitative

and quantitative analysis.

In the work we focused on identification of main components of fractions of

grasshopper abdomen using HPLC combined with MS detection. To cope with the problem of

interfering substances, both liquid-liquid extraction and solid-phase extraction (SPE)

pretreatment methods were used to concentrate and fractionate compounds from the insect

matrix. Compounds were separated by HPLC using gradient elution on Zorbax Extend-C18

column (2.1x50 mm, 1.8 micron). The analysis was performed with the use of LC-ESI-MS-

TOF system operated in fast polarity switching mode. The obtained data were on analyzed by

means the Molecular Feature Extraction Algorithm (Agilent Technologies, Inc, Santa Clara,

CA), which is included in MassHunter Qualitative Analysis Software.

The results obtained showed the differences in metabolomic profiles between the

studied grasshopper species. The analysis of a typical domestic insect species enabled us to

obtain a full screen of compounds present in the fractions of grasshopper abdomen’s material.

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P 36

COCAINE AND BENZOYLECGONINE SELECTIVE EXTRACTION

FROM WATER SAMPLES

USING MOLECULARLY IMPRINTED POLYMERS

J. Raczak-Gutknecht1, R. Bujak

2, R. Gadzała-Kopciuch

3, M. Buszewska-Forajta

1, A.

Nowaczyk4, E. Daghir

2, P. Kośliński

2, B. Buszewski

3, R. Kaliszan

1, M.J. Markuszewski

1

1Department of Biopharmaceutics and Pharmacodynamics, Medical University of Gdańsk,

Al. Hallera 107, 80-416 Gdańsk, Poland 2Department of Toxicology, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus

University in Toruń, dr A. Jurasza, 85-089 Bydgoszcz, Poland 3Department of Environmental Chemistry & Bioanalytics, Faculty of Chemistry, Nicolaus

Copernicus University, 7 Gagarin St., PL-87 100 Toruń, Poland 4Department of Organic Chemistry, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus

University in Toruń, dr A. Jurasza, 85-089 Bydgoszcz, Poland


Abuse of illicit drugs is a serious global problem. It is the cause of numerous diseases,

raised mortality and socioeconomic problems [1]. Assessing the real amount of used drugs is

rather difficult. Monitoring the cocaine and its metabolites exerted with urine in waste waters

is the method that provides to obtain realistic estimates of use of these compounds [2].

Samples of waste water are complex and impurities could interfere with quantification. To

clean up the sample before the analysis the new, selective sorbents for solid phase extraction

(SPE) are needed. Molecularly imprinted polymers (MIPs) are highly cross-linked synthetic

polymers having molecular recognition properties, towards the template molecules or even

group of similar compounds, with specificity and binding selectivity [3]. In this studies, MIPs

as a new cocaine and benzoilecgonine selective sorbents were presented.

The first step of the studies was the choose of polymers components by computational

approach: template, functional monomer, cross-linker and porogen. During MIPs synthesis as

the templates, atropine and scopolamine were used. The synthesis based on non-covalent

strategy. The synthesis of non-imprinted polymers (NIPs) has been performed simultaneously.

The selectivity of new synthesized polymers has been estimated in binding and adsorption

study. The concentrations of cocaine and its metabolite benzoylecgonine were measured in

liquid phase by HPLC method.

Estimation of cocaine and benzoylecgonine, a marker of cocaine use, is important in

biological and environmental studies. Selective sorbents used in this experiment appears to be

sufficient materials that can be used in extraction step before qualitative or quantitative

analysis of these compounds from wastewater. Selective recoveries obtained for cocaine and

benzoylecgonine on newly developed MIP materials were from 77.1% to 92.1%, and from

62.1% to 89.2% respectively. Further studies in real samples are needed to evaluate the

properties of sorbents tested as well as to optimize extraction conditions.

References:

1. C.P. O’Brien: Drug addiction. In: Brunton LL, Chabner BA, Knollmann BC, editors.

Goodman and Gilman’s the pharmacological basis of therapeutics. New York, NY:

McGraw-Hill, (2011) 649–68. S. Castiglioni, E.

87





2. S. Castiglioni, E. Zuccato, E. Crisci, Ch. Chiabrando, R. Fanelli, R. Bagnati,

Identification and measurement of illicit drugs and their metabolites in urban

wastewater by liquid chromatography-tandem mass spectrometry. Analytical

Chemistry 78 (2006) 8421-8429.

3. M. Lasakova, P. Jandera: Molecularly imprinted polymers and their application in

solid phase extraction. J. Sep. Sciences 32 (2009) 799 – 812

88




P 37

HYDROPHILIC INTERACTION CHROMATOGRAPHY OF URINARY

COMPOUNDS TOXIC TOWARD VIBRIO FISCHERI BACTERIA

E. Okrągła1, G. Gałęzowska

1, L. Wolska

1,2

1Medical University of Gdansk, Faculty of Health Sciences with Subfaculty of Nursing and

Institute of Maritime and Tropical Medicine, Department of Environmental Toxicology, 9b

Powstania Styczniowego Str., 81-519 Gdynia, Poland 2Gdansk University of Technology (GUT), Chemical Faculty, Department of Analytical

Chemistry, 11/12 G. Narutowicza Str., 80-233 Gdańsk, Poland


Kidneys generate 1-1.5 liters of urine per day. The urine is a biological material

including noxious, toxic or unwanted products of metabolism, both endogenous and

exogenous substances. In addition, it is known that urine in physiological conditions is sterile,

which means that urine does not contain bacteria. The 95% of urinary tract infection (UTI) are

caused by gram-negative bacteria, especially by Escherichia coli. There are also substances,

present in urine such as urea or organic acids, which are responsible for urinary tract

protection against uropathogens.

Our pilot studies have shown the toxicity of urine towards Vibrio fischeri bacteria,

which are gram-negative bacteria. The structure of gram-negative Vibrio fischeri bacteria is

similar to Escherichia coli, which are responsible for urinary tract infections. Therefore,

toxicity towards Vibrio fischeri bacteria has been chosen as one of parameters of urine.

The aim of the study is isolation and identification of compounds present in urine,

toxic to Vibrio fischeri bacteria. To isolate this compounds the high performance liquid

chromatography (HPLC) technique was applied. Urine (>75% toxicity) was fractionated in 28

fractions and toxicity of each fraction in the presence of Vibrio fischeri bacteria was

determined. The fractionation of urine allows for separation of the toxic fraction no.2. The

compounds contained in toxic fraction no.2 were identified with HPLC-MS technique.

Because of fractions no.2 matrix complexity identification of toxic compound become

impossible. Subsequently the toxic fraction no.2 was lyophilized and fractionated by ZIC-

HILIC. This method allowed for separation and identification toxic substances of urine.

The absence of toxic substances in urine can probably be responsible for more often

urinary tract infection (UTI).

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P 38

PLANAR CHROMATOGRAPHY –BIOAUTOGRAPHY AS A TOOL

FOR INVESTIGATION OF BIOLOGICALLY ACTIVE COMPOUNDS

IN PLANT EXTRACTS

W. Jesionek1, I. Choma

1, B. Majer-Dziedzic

2

1Department of Chromatographic Methods, M. Curie-Sklodowska University, Lublin, Poland

2Department of Veterinary Microbiology, University of Life Sciences, Lublin, Poland


Nowadays, return to an alternative medicine based on natural bioactive compounds is

increasingly observed. Searching for biologically active substances of plant origin which

would be less toxic and more effective than conventional drugs seems to be especially

important. Various methods can be applied, depending on properties of the plant extracts

under investigation. The most popular group of methods is so-called Effect Directed Analysis

(EDA) which gives specific information about biological activity of tested compounds, e.g.

antibacterial, antioxidant, antifungal, or anticancer. TLC-EDA is a combination of thin-layer

chromatography (TLC) with an effect directed analysis [1]. Not only does this hyphenation

allow to separate the tested compounds but it also helps to verify their biological activity.

Comparing to HPLC, TLC is an ideal method for analyzing samples needing sophisticated

pre-treatment procedure, like plant extracts. Thin-layer chromatography-direct bioautography

(TLC-DB) is a technique which hyphens separation of components with antimicrobial

detection directly on a TLC plate. Generally speaking, it gives information on biological

activity of a given zone on a chromatogram by measuring changes in microorganism

growth[2]. Plants such as: Melisa officinalis L., Viola tricolor L., Sambucus nigra L. and

Equisetum arvense are widely considered to be therapeutic ones. Biological properties of

tinctures of the above mentioned plants have been examined using TLC-DB technique. The

antimicrobial activities of the plant extracts against Bacillus subtilis were investigated.[3]

Additionally, the antioxidant activities of the essential oils and plant tinctures were assessed

by their ability to scavenge 2,2-diphenyl-1-picrylhydrazyl stable radicals (DPPH) [4]. The

experiments pointed to both antibacterial and antioxidant activities of many components of

targeted extracts.

References:

1. W. Jesionek, E.M. Grzelak, B. Majer-Dziedzic, I.M. Choma, J. Planar Chromatogr. 26

(2013) 109-113

2. Choma, E.M Grzelak., J. Chromator. A, 1218 (2011) 2684-2691

3. E.M. Grzelak, B. Majer-Dziedzic, K.M. Pilorz, I.M. Choma, JAOAC Int. 96 (2013)

386-391

4. A .Marston, J. Chromatogr. A 1218 (2011) 2676-83

90





P 39

NON-TARGETED METABOLOMIC ANALYSIS OF URINE IN

UROGENITAL TRACT CANCER DISEASES USING LC-MS

A. Yumba Mpanga1, W. Struck-Lewicka

1, M. Buszewska-Forajta

1, D. Szczesny

1, M.

Markuszewski2, M. Roslan

2, R. Kaliszan

1, M.J. Markuszewski

1


Al. Hallera 107, 80-416 Gdańsk, Poland 2Department of Urology, Medical University of Gdańsk, Mariana Smoluchowskiego 17,

80-214 Gdańsk, Poland

Email: [email protected]

Metabolomics focuses on complex analysis of metabolic profiles in biological samples

and is strictly linked to the pathophysiological states of the organism. One of the challenges in

metabolomics is a metabolic fingerprinting as a non-targeted tool to understand metabolic

perturbations that occur in urogenital tract cancer and as an approach for biomarker discovery

and screening. In this study, an LC-MS metabolomics method was used to compare patients

with urogenital tract cancer to healthy persons. The analyses were performed with a gradient

elution of mobile phase consisted of 0.1 % formic acid in water and 0.1% formic acid in

methanol on an 150 mm x 4.6 mm x 2.7 µm Ascentis Express C-18 column (Supelco

analytical, USA). The urine samples were collected from healthy persons and patients with

urogenital tract cancer and stored at –80 °C. Prior analysis urine samples were thawed,

centrifuged, diluted and filtrated. The data analysis have been performed by use of the

statistical methods (U-Mann Whitney, principal component analysis, partial least-squares

discriminant analysis). In result the statistically significant differences among metabolite

levels in groups of cancer patients and healthy controls have been found and identified using

different database such as HMDB, METLIN, KGG. The combination of high performance

liquid chromatography with TOF/MS detection is particularly attractive for analysis of

multicomponent mixtures of biological samples such as urine. LC-TOF/MS analysis and

advanced statistical methods can allow the identification of potential biomarkers based on the

differences in metabolites level.

91





P 40

EFFICIENT SEPARATION OF BIOMOLECULES AND SMALL

MOLECULES BY MONOLITHIC POLY (N-VINYLCARBAZOLE-CO-

1,4-DIVINYLBENZENE) CAPILLARY COLUMNS

R. Koeck1, M. Fischnaller

1, R. Bakry

1, R. Tessadri

2, G.K. Bonn

1

1Institute of Analytical Chemistry and Radiochemistry,

Leopold-Franzens University, Innsbruck, Austria 2Institute of Mineralogy and Petrography,

Leopold-Franzens University, Innsbruck, Austria


Bioanalysis, including drug discovery, drug screening and biomarker research, is an

ambitious analytical field that necessitates sophisticated enhancements in sample preparation,

chromatographic separation science and mass spectrometry. HPLC has to be regarded as a

universal tool for resolving complex biological mixtures, whereas novel stationary phase

designs with tailored separation properties and efficiencies are needed to match the high

demands of modern bioanalytical platforms. Organic monolithic stationary phases are known

to be a valuable class of HPLC supports with exciting resolving power that can serve a

number of bioanalytical applications. Here we present the newly developed poly (N-

vinlycarbazole-co-1,4-divinylbenzene) stationary phase, synthesized by free radical

copolymerization, applying AIBN as free radical initiator and a mixture of toluene/1-decanol

as inert diluents. By applying 24 hour polymerization time, the support could be optimized for

the separation of biomolecules and showed high chromatographic efficiency, high

permeability, high loading capacity and a specific surface area between 120-160 m2/g. Higher

retention characteristics were observed in IP-RP separation mode of oligonucleotides,

compared to the PS-DVB monolith. Theoretical plate height values below 6 µm were received

for oligonucleotides and a peak capacity of 96 and 127, respectively for protein and

oligonucleotide separations in a 60 min elution window. Moreover, the chromatographic

supports could also be optimized for the separation of small molecules by reducing the

polymerization time below 2 hours and by adjusting the monomer-crosslinker ratio and the

content of radical initiator. N-vinylcarbazole allowed specific surface areas of about 400 m2/g,

which is likely due to earlier or higher amount of phase separation. The supports showed

excellent efficiency for e.g. alkylbenzenes, phenols, parabens, acetophenones. The number of

theoretical plates was found to be up to 256000, applying isocratic paraben separations. A

long-term stability test procedures of 1000 consecutive runs attests this NVC/DVB stationary

phase to be highly stable and robust.

92





P 41

EXAMINATION OF DEGRADATION OF DIFFERENT MYCOTOXIN

AND DEGRADATION PRODUCTS WITH OZONE-ENRICHED

MEDIUM BY HPLC-MS TECHNIQUE

S.B. Tóth

Eszterházy Károly College, Department of Chemistry

H-3300 Eger, Leányka str 6.


The cereals (wheat, barley, maize), which are belong in food substrates, nowadays

during the growing of agricultural production come into view the organic farming. Therefore

significantly increased the colonizing fungis (molds) on plants due to their positive living

environment. The production of secondary metabolites (mycotoxins), which are produced by

molds, increased because of large rate spread of molds, which means significant risk in

qualify in foods.

The total number of mycotoxins is evaluated approx. 300-500.000, only 10-15 cause

major damage of these. During the development of food crops are more possibility to

terminate the presence of mycotoxins, but unfortunately many of the harvested corps has been

contaminated due to inadequate and deficient intervention.

The mycontoxins content mainly concentrated in external shell in the harvested crops,

so in cereals which use of food substrates. To reduce the quantity of toxins of contaminated

crops is a good resort to remove or demolish the toxins in the external shell. The basic of the

research is the destruction of the ozone.

The concentrated mycotoxins in the external shell of the corn react with the ozone

when the corn and with the simple technique produced ozone are contacted, as a result of

reaction the toxin loses toxicity, and other types of compounds produces. Two different

methods were used for the production of the ozone, first is arc discharge method, second

method is the UVC (254 nm) technique with Germicid lamp. From the comparison of the two

methods the arc discharge technique was the more productive which were used in the further

phase of the experiment. During the first experiments the rate of decomposition of ozone, than

the rate of decomposition of pure toxin was determined in model system. The quantity of

mycotoxins in contamined grain of wheat were examined between real circumstances on three

toxins. During degradation experiments the quantity and quality of the zearalenone,

deoxynivalenol and different kinds of aflatoxines were determined in ozone enriched agent

with HPLC-UV and HPLC-MS technique, were analised the possible degradation products

from degradation of toxins, which chemical structure and properties are significant because of

the toxicity of resulting products.

“This research was realized in the frames of TÁMOP 4.2.4. A/1-11-1-2012-0001

"National Excellence Program - Elaborating and operating an inland student and researcher

personal support system" The project was subsidized by the European Union and co-financed

by the European Social Fund.”

Keywords: ozone, degradation, aflatoxins, trichotechene, HPLC-MS

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P 42

UHPLC STUDY ON THE DEGRADATION PROFILES OF ACTIVE

SUBSTANCES IN THE EYE DROPS SUBJECTED TO HEAT AND

FILTRATION STERILIZATION METHODS

I. Valentić, M. Buratović, D. Štanfel

Research and Development, JGL d.d., Pulac bb, 51000 Rijeka, Croatia


Products intended to be sterile should be terminally sterilised by heating [1]. Where it

is not possible to carry out terminal sterilisation by heating due to formulation instability, an

alternative method of terminal sterilisation, like filtration, should be taken to utilise. The

purpose of this study was to determine the impact of heat sterilisation on the eye drops

stability and to assist in the selection of the optimal sterilisation method.

The ultra high performance liquid chromatography (UHPLC) method was developed

and validated for determination of degradation products of the active substance in the eye

drops.

The developed method was linear over the concentration range of 0.05 - 4 µg/ml with

acceptable correlation coefficient of ≥ 0.995. The accuracy was within the 5% bias (98.5 –

104.0%). The limit of qualification was 0.02 µg/ml. Repeatability yielded coefficient of

variation (CV) of less than 7% (0.47%) and intermediate precision provided CV value of less

than 20% (13.13%).

Two samples of the eye drops, one sterilized by heat and other sterilized by filtration

were conducted to the UHPLC study for degradation profiles.

All results of impurities were low and below ICH reporting threshold [2] which is

0.1% for tested eye drops. More impurities and higher content of impurities was determined

on the chromatogram of the sample sterilised by heat.

The method validation study proven that the method is accurate and precise. Thanks to

the high resolution and sensitivity of the UHPLC method, the method has been applied

successfully to determine the influence of the different sterilization procedures on the drug

product stability.

References

1. European Medicines Agency, Decision trees for the selection of sterilisation methods

(CPMP/QWP/054/98), 2000

2. European Medicines Agency, Note for guidance on impurities in new drug products

(CPMP/ICH/2738/99), 2006.

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P 43

SEPARATION AND DETERMINATION OF CARBOXYLIC ACIDS IN

WINE AND HUMAN URINE SAMPLES BY ION-EXCLUSION

CHROMATOGRAPHY

R. Halko, I. Hukelová

Department of Analytical Chemistry, Faculty of Natural Sciences,

Comenius University in Bratislava, 842 15 Bratislava, Slovakia


This work is focused on developing a new chromatographic method for the separation

and determination of thirty six carboxylic aliphatic and/or aromatic acids in single run. Ion-

exclusion chromatography with silica based analytical column Alltech PrevailTM

organic acid

5 µm (150 mm × 4,6mm I.D) was used for the solving of this problem. Developed method

was based on ion-exclusion and/or hydrophobic interaction chromatographic separation

mechanism. The effect of the concentrations of phosphate buffer and its pH as well as the

column temperatures on the retention of the test acids has been investigated. Gradient elution

of the mobile phase composed of aqueous phosphate buffer and methanol was used to achieve

a required separation of carboxylic acids within 45 minutes. All measurements were done at

220 nm. Column temperature was set at 30±0.1○C. The proposed method was successfully

applied for the determination of aliphatic and aromatic acids in three wine samples (white,

rose and red) and human urine sample.

Acknowledgments

This work was generously supported by the grant of project VEGA 1/0852/13 and the

grant of project APVV-0583-11. This work is partially outcome of the project VVCE-0070.

95





P 44

REDUCTION OF QUANTITY OF MYCOTOXINS IN FOOD

PRODUCTS BY DIFFERENT LEAVENING AGENTS, ANALYSIS OF

METABOLITE

S.B. Tóth

Eszterházy Károly College, Department of Chemistry

H-3300 Eger, Leányka str 6. Hungary


During the production of agricultural raw materials for food production efface the

application of the pesticides in the background because of in our days preferred organic

farming. As a result of this activity grown the colonizing fungi on the plants due to their

positive trends in living environment. Formation of secondary metabolites- mycotoxins-

produced by fungi, the total amount is directly proportional to wet weather, which is major

risk factor in the quality of the food.

The investigation and demonstration of the degradation of mycotoxins in alkaline

medium based on researches [1]. The aim of this examination is analysis of the effect of the

decrease of toxins content of different food additives.

Base of hypothesis is the article of Ryu D, Hanna MA, Eskridge KM, Bullerman LB:

Heat stability of zearalenone in an aqueous buffered model system. J Agric Food Chem. 2003

Mar 12;51(6):1746-8, examination of the toxin degradation in different pH media, the results

are that the zearalenone degradation in strongly alkaline medium, the zearalenone’s effect

ceases.

In focus of examinations were without the zearalenone, the aflatoxin B1 and patuline

toxins.

Significance of research is analyse qualitative effects of main materials, adjutants and

additives in baking the foods – breads- quantitative changes of various mycotoxins, and

determine the additives which reduce the amount of toxins in breads.

In the course of the research, the aim was analyses the degradation of different toxins

in breads, which were baked with an everyday baking bread recipe. Out of baking parameters

only the change of quality of leavenings agent determine the degradation of toxin in relations

baking powder and szalalkáli (ammonium-hydrogen-carbonate). During the earlier

examinations out of three leavening agent, two – baking powder and szalalkáli- promoted the

degradation of trichothecene toxins. The effect of szalalkáli for mycotoxins was more

productive as seen with baking powder.

Keywords: zearalenon, aflatoxin B1, leavening agents.

References

1. Ryu D, Hanna MA, Eskridge KM, Bullerman LB: Heat stability of zearalenone in an

aqueous buffered model system. J Agric Food Chem. 2003 Mar 12;51(6):1746-8

“This research was realized in the frames of TÁMOP 4.2.4. A/1-11-1-2012-0001 "National

Excellence Program - Elaborating and operating an inland student and researcher personal

96


http://www.ncbi.nlm.nih.gov/pubmed?term=%22Ryu%20D%22%5bAuthor%5d


http://www.ncbi.nlm.nih.gov/pubmed?term=%22Hanna%20MA%22%5bAuthor%5d

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Eskridge%20KM%22%5bAuthor%5d


http://www.ncbi.nlm.nih.gov/pubmed?term=%22Bullerman%20LB%22%5bAuthor%5d




http://www.ncbi.nlm.nih.gov/pubmed?term=%22Hanna%20MA%22%5bAuthor%5d








support system" The project was subsidized by the European Union and co-financed by the

European Social Fund.”

97




P 45

APPLICATION OF MANUAL AND AUTOMATED

MICROEXTRACTION BY PACKED SORBENT FOR ISOLATION OF

BENZODIAZEPINES FROM TWO ALTERNATIVE BIOLOGICAL

MATERIALS

A. Moos1, A. Bocheńska

1, R. Wietecha-Posłuszny

1, P. Kościelniak

1,2

1Jagiellonian University, Department of Analytical Chemistry, Laboratory for Forensic

Chemistry, Ingardena 3, 30-060 Krakow, Poland 2Institute of Forensic Research, Westerplatte 9, 31-033 Krakow, Poland


The proper sample preparation is still the most important entering analysis step,

especially when the matrices are very complex (e. g. biological materials). Some of the human

biological materials as oral or lachrymal fluid (OF or LF, respectively) are possible to be

collected only in small volume, so application microextraction techniques, like

Microextraction by Packed Sorbent (MEPS), is highly demanded.

The aim of this study was to apply the Microextraction by Paced Sorbent (MEPS) to

isolation of seven benzodiazepines (BZDs): alprazolam, clonazepam, diazepam, estazolam,

lorazepam, lormetazepam and tetrazepam from human OF and LF. Within this study the

comparison of manual and automatical modes of MEPS was done. The following internal

standards were used: alprazolam-D5, clonazepam-D4, diazepam-D5, estazolam-D5 and

lorazepam-D4. Samples were drawn through the MEPS sorbent in small volumes: 150 µl – OF

and 30 µl – LF. The extracts of studied compounds were analysed by sensitive and precise

method – ultrahigh performance liquid chromatography coupled with mass spectrometry and

positive electrospray ionization (UHPLC-ESI-MS). The separation was carried out using a

Hypersil Gold Phenyl column (50 x 2.1 mm I.D.), the mobile phase was prepared by mixing

of 0.1% HCOOH in acetonitrile with formic buffer of pH 3.4 in established gradient flow rate

of 0.4 mL/min and the column temperature equal to 25°C. The MS conditions were: nebulizer

pressure: 2.5 bar, dry gas: 5.5 L/min, temperature of drying gas: 200°C and capillary voltage:

-700 V. The MEPS/UHPLC-ESI-MS method was validated at three concentration levels of

analytes: 10, 50 and 70 ng/mL and 4, 8 and 12 ng/mL for OF and LF, respectively. The

following parameters were evaluated: limit of detection (OF: 1.10 – 5.77 ng/mL, LF: 0.41 –

1.34 ng/mL), limit of quantification (OF: 3.67 – 19.24 ng/mL, LF: 2.16 – 4.45 ng/mL),

precision (OF: 2.0 – 10.8%, LF: 3.5 – 10.7%) and accuracy (OF: -15% – 8%, LF: -15% –

10%).

Acknowledgement

A. Moos gratefully acknowledges the financial support from the project

Interdisciplinary PhD Studies “Molecular Sciences for Medicine” (co-financed by the

European Social Fund within the Human Capital Operational Programme).

The research was carried out with the equipment purchased thanks to the financial

support of the European Regional Development Fund in the framework of the Polish

Innovation Economy Operational Program (contract no. POIG.02.01.00-12-023/08).

98





P 46

APPLICATION OF DOEHLERT EXPERIMENTAL DESIGN FOR

OPTIMIZATION OF MICROWAVE-ASSISTED EXTRACTION

OF TROPANE ALKALOIDS FROM SOLANACEAE FAMILY PLANTS

J. Nowak1, M. Ciechomska

1, M. Woźniakiewicz

1, P. Kościelniak

1,2

1Jagiellonian University, Department of Analytical Chemistry, Laboratory for Forensic

Chemistry, Ingardena 3, 30-060 Krakow, Poland 2Institute of Forensic Research, Westerplatte 9, 31-033 Krakow, Poland


Hyoscyamine (atropine) and scopolamine are two tropane alkaloids naturally

occurring in many members of the Solanaceae family plants, which are widely spread

thorough the world. Due to their hallucinogenic properties and wide availability Solanaceae

are used by young people as recreational drugs, which frequently leads to poisonings, with

fatal cases being reported. For this reason, the assessment of tropane alkaloids concentration

in plants of the Solanaceae family is highly important for toxicological and forensic purposes.

A simple and rapid sample treatment method has been developed for quantitation of

atropine and scopolamine in such plants belonging to the Solanaceae family as Datura

stramonium and Brumansia aurea. Collected leaves and seeds were dried, ground and 0.3 g of

the sample was subjected to extraction in methanol by means of microwave-assisted

extraction (MAE). The extract were purified with graphitized carbon and analyzed by GC-MS

in SIM mode for quantitative analysis.

The MAE method was optimized with Doehlert uniform shell design and response

surface methodology. Separate optimization was performed for every kind of sample, that is:

datura leaves, datura seeds and brugmansia leaves. The optimized parameters were: volume of

extraction solvent – at 5 levels from 2 to 10 ml; time of extraction at maximum temperature –

at 7 levels from 0 to 18 minutes; maximum temperature – at 3 levels from 35 to 65 degrees.

Other parameters, such as extraction solvent to sample mass ratio and time of temperature

ramping have been chosen on the basis of initial experiments. The established optimal

conditions of MAE were found to be different for every kind of sample, that is: 5 ml, 12

minutes in 50 degrees for datura leaves, 10 ml, 3 minutes in 65 degrees for datura seeds and

10 ml, 9 minutes in 50 degrees for Brugmansia leaves.

The optimized MAE/GC-MS method was validated and successfully applied for

determination of atropine and scopolamine in leaves and seeds of plants from Datura and

Brugmansia genera.

99





P 47

THE USE OF HIGH LIQUID PERFORMANCE CHROMATOGRAPHY

FOR THE DETERMINATION OF PAHS IN THE FINE PARTICLE

FRACTION OF DIESEL EXHAUST

M. Szewczyńska, M. Pośniak, E. Dobrzyńska

Central Institute for Labour Protection – National Research Institut,

ul. Czerniakowska 16, 00-701 Warsaw, Poland

E-mail: [email protected]; [email protected]; [email protected]

Polycyclic aromatic hydrocarbons (PAHs) from mobile source exhaust due to their

mutagenic and carcinogenic potential have contributed to a substantial share of air toxics. In

order to characterize PAHs emissions of diesel engine fuelled with diesel (ON), and its blend

(B20, B40), an experimental study has been carried out on a direct-injection diesel engine.

The particle-phase and gas-phase PAHs in engine exhaust were collected by filters using

Electrical Low Pressure Impactor (ELPI) and Sioutas Personal Cascade Impactor (SPCI).

Subsequently PAHs were extracted from filters with cold dichloromethane (below 50C) to

prevent losses by volatilization. A sonic bath was also used with closed vials for 30 min.

Separation and identification of the PAHs were achieved using HPLC (Elite LaChrom,

Merck Hitachi) with fluorescence detection (FL). A reversed phase HPLC column Pinnacle II

PAH, was used with a pre-column. The flow rate was 0.97 mL min-1

and the injection volume

was 10 µL. LOD results for selected PAHs, determined based on blank analysis, did not

exceed 0.1 ng m-3

In result of the conducted studies it was determined that the main contents in exhaust

gases from Diesel engine, independently of the type of applied fuel, is constituted by the

particles fraction of diameter <0.25 µm. The analysis of chemical composition of <0.25 µm

exhaust gas fraction showed that there are mainly 3- and 4-ring aromatic hydrocarbons in

exhaust gas of Diesel fuel, while in B40, single PAHs with the number of rings of 4 and 5

were detected.

Quantitative analysis showed that the mean total PAH content in the exhaust of the

engine in the fraction <0.25 µm is properly 910 (ON), 746 (B20) i 340 (B40) ng/m3.

The publication was prepared based on the results obtained within the National Programme:

“Improvement of Safety and Working Conditions” (2011–2013). The Central Institute for

Labour Protection – National Research Institute is the co-ordinator of the programme.

100







P 48

HIGH LIQUID PERFORMANCE CHROMATOGRAPHY AS A

METHOD FOR OCCUPATIONAL EXPOSURE ASSESSMENT OF

ACTIVE SUBSTANCES IN THE PHARMACEUTICAL INDUSTRY

M. Pośniak, E. Dobrzyńska, M. Szewczyńska

Central Institute for Labour Protection – National Research Institut,

ul. Czerniakowska 16, 00-701 Warsaw, Poland

E-mail: [email protected]; [email protected]; [email protected]

The occupational exposure to several hundred pharmacological active substances

applied in medicines production can caused harmful effects in health of workers in

pharmaceutical industry. The assessment of risk posed by these agents is a very large problem

for OSH expert because of lack of occupational exposure limits and the methods for

measurement of their concentrations in the workplaces air.

The aim of this study was to provide tools and criteria for assessment of occupational

exposure to selected dangerous pharmacological active substances – n-hydroxyurea (NHU)

classified as harmful agent, sulpiryde (SP) – irritant agent and warfarin sodium (WAS) –

harmful, irritant and reptrotoxic category 1. Base on toxicological data and procedures of

establishing reference values of occupational exposure limits for pharmacological substances,

value OEL - 0,01 mg m-3

were proposed for these compounds and methods based on

adsorption of NHU, SP and WAS on glass fiber filters, elution by water and analysis using

high-performance liquid chromatography with UV detector. Selectivity determination these

compounds enable the following conditions: WAS – column LiChrospher 100, modified by

cyanopropyl group, mobile phase: acetic acid/acetonitryle/water (1:25:74), flow rate 1,5 mL

min-1

, λ= 260 nm; NHM - column Partisil 10 ODS, mobile phase: aqua/methanol (95:5), flow

rate: 0,5 ml/min; λ= 214 nm and SP - Nucleosil 100-C18; mobile phase: buffer phosphate

/acetonitryle (85:15), flow rate - 1,0 mL min-1

; λ= 240 nm. The limit detection of analytical

method for determination NHU, SP and WAS was 0,001 mg m-3

for 480 L air sample.

The publication was prepared based on the results obtained within the National

Programme: “Improvement of Safety and Working Conditions” (2011–2013). The Central

Institute for Labour Protection – National Research Institute is the co-ordinator of the

programme

101







P 49

SIMULTANEOUS DETERMINATION OF 29 ENDOCRINE

DISRUPTING PESTICIDES IN SEDIMENTS FROM RIA DE AVEIRO

USING QuEChERS BY GC-MS

J.L. Vera1,2

, V.F. Domingues1, A. Almeida

3, J.M. Costa

3,4, C. Mansilha

4,5,

C. Delerue-Matos1

1REQUIMTE, Instituto Superior de Engenharia do Porto, R. Dr. António Bernardino de

Almeida, 431, 4200-072 Porto, Portugal 2REQUIMTE, Faculdade de Ciências da Universidade do Porto, 4169-007 Porto, Portugal

3Centro de Estudos de Ciência Animal (CECA). Vairão, Portugal

4Instituto Nacional de Saúde Dr. Ricardo Jorge, Porto, Rua Alexandre Herculano, 321

4000-055, Portugal 5Requimte, Universidade do Porto, Portugal


A multi-residue method was developed to analyzed 29 endocrine disrupting pesticides

(EDPs) applying QuEChERS and Gas Chromatography-Mass Spectrometry. Sediments

samples were taken from seven points of Ria de Aveiro, that is located in the northern west

region of Portugal and receives inputs from agriculture, urban and industrial activities. This

environment is particularly susceptible to pollution due to intentional and accidental release of

pesticides. An optimization was carried out according to soils organic carbon level, the

samples were divided in two groups: Ma (organic carbon<0.4) and Mb (organic

carbon>0.7%). The analytical method was developed and validated and showed good

linearity, with correlation coefficients (R) higher than 0.9949 for all compounds.The

quantification was carried out using a matrix matched calibration to minimize the existence of

the matrix effect. The ranges of the limits of detection (LOD) and of the limits of

quantification (LOQ) in Ma sediments were from 0.22 to 29.52 µg kg−1

and from 0.74 to

98.42 µg kg−1

, respectively. For Mb sediments, the LODs ranged from 0.23 to 32.19 μg kg−1

and the LOQs from 0.77 to 107.30 μg kg−1

. Results showed the presence of atrazine desethyl,

HCB, aldrin, α-endosulfan, bifentrhin, iprodione, deltamethrin in several samples, with

concentrations ranging from 3 to 388 µg kg−1

.

102





P 50

TARGETED AND UNTARGETED STUDY OF URINARY

METABOLITES AS POTENTIAL CANCER MARKERS

W. Struck-Lewicka1, D. Siluk

1, A. Yumba Mpanga

1, M. Markuszewski

2, R. Kaliszan

1,

M.J. Markuszewski1


Poland, ul. Gen. Hallera 107, 80-416 Gdańsk 2Department of Urology, Medical University of Gdańsk, Poland,

ul. M. Smoluchowskiego 17, 80-214, Gdańsk


The levels of metabolites, similarly to transcriptomes and proteins, can reflect

information about human’s health status. According to the literature, it has been noticed that

RNA’s metabolites, namely nucleosides can play a significant role in cancer diagnosis [1].

During RNA turnover, nucleosides are excreted intact into the urine so their levels are higher

in cancer patients in comparison to the healthy ones. In the present work we focused on

determination of nucleosides and other cis-diol compounds using two approaches, targeted

and untargeted analysis. The targeted determination was performed with the use of HPLC

technique coupled with triple quadrupole mass spectrometer (6430 LC-MS/MS, Agilent

Technologies), whereas the untargeted method was evaluated using HPLC technique coupled

with mass spectrometer with time-of-flight analyzer (6240 LC-TOF/MS, Agilent

Technologies). In order to selectively analyze cis-diol compounds from urine samples

(n=129), sample pretreatment procedure with solid phase extraction was applied (Varian

PBA, Agilent Technologies). The metabolites were separated using gradient elution

composed of 0.05 % formic acid in water (A) and 0.05 % formic acid in methanol (B), pH

2.7, on Zorbax Extend C-18 column (2.1 x 50 mm, 1.8 µm) at 8ºC. The obtained data sets

were analyzed using univariate and multivariate chemometric techniques (Mass Profiler

Professional Software, Agilent Technologies, USA) as well as Matlab 9.1 (The MathWorks,

Inc., USA). As a result of statistical analyses based on LC-MS/MS data, five statistically

significant nucleosides have been extracted (3-methyluridine, 6-methyladenosine, inosine,

N2-methylguanosine and NN-dimethylguanosine), (p<0.05). Concerning data obtained from

untargeted analysis, 19 metabolites were statistically significant (p<0.05). Furthermore the

obtained two data sets were classified using PCA. The results showed that healthy samples

were better clustered together than cancer samples. That confirmed higher diversity among

samples belonging to different types of cancer. The sensitivity as well as specificity of the

statistically significant metabolites calculated with the use of PLS-DA, K-NN, SVM or

logistic regression for data obtained from LC-TOF-MS were in the range from 53.9 to 92.3%,

and from 37.5 to 85.7%, respectively. The sensitivity and specificity for data obtained from

LC-MS/MS were lower and ranged from 61.9 to 88.9% as well as from 27.8 to 66.7%,

respectively.

References

1. W. Struck, D. Siluk, A. Yumba Mpanga, M. Markuszewski, R. Kaliszan, M.J.

Markuszewski, J Chromatogr A, 2013, 1283, 122-131.

103





P 51

DETERMINATION OF DEXPANTHENOL DEGRADATION

PRODUCTS IN SEMISOLIDS

M. Mavrinac, S. Kamber, D. Štanfel, I. Valentić, K. Mihaljević

Research and Development, JGL d.d., Pulac bb, 51000 Rijeka, Croatia


Introduction

Degradation pathway of dexpanthenol depends significantly on environmental

conditions. If environment is mostly acid, a 3-aminopropanol and pantoic acid are formed. In

mostly basic environment, 3-aminopropanol and pantolactone are formed.

The aim of this study was to create an HPLC method that would detect pantoic acid,

pantolactone and all other possible dexpanthenol degradation products in semisolid drug

forms and determine their exact quantity.

Results & Discussion

First an appropriate pH of the solvent had to be chosen in order to keep both

degradation products in their stable form, because pantoic acid is transformed into

pantolactone, and vice versa, at different pH values. Since pantolactone is stable at pH about

4.0 [1], the buffer with pH 4.2 was chosen for the analysis.

Secondly, semisolid drug forms, e.g. cream and ointment, have very complex matrixes

and it is a challenge to extract and separate the substances of interest in order to analyze them.

Also dexpanthenol and most of its degradation products have absorption maxima at very low

wavelengths (<200 nm); except pantolactone that has λmax at 215 nm. In this case, detection is

rather difficult because there is much more baseline noise and many components that are not

of interest can also be detected. Based on empirical proofs, λ = 210 nm was chosen for the

analysis.

Despite the lack of literature data regarding detection of pantoic acid and pantolactone

[2-5], the appropriate HPLC method for detection and quantitation of pantoic acid and

pantolactone in semisolids was created: appropriate mobile phase mixture (0.05 M phosphate

buffer, pH 4.2 : acetonitrile = 99:1) and gradient (up to 0.05 M phosphate buffer, pH 4.2 :

acetonitrile = 50:50) at λ=210 nm were chosen for the analysis of dexpanthenol degradation

products in semisolids (cream and ointment).

References

1. DL-lactone, CAS N°: 79-50-5 (2006); UNEP publications.

2. Dexpanthenol and Dexpanthenol Preparation official monographs. U.S. Pharmacopeia

34 2: 2515-2516.

3. Dexpanthenol monograph. British Pharmacopoeia 2012 1: 667.

4. Dexpanthenol monograph. European Pharmacopoeia 7.0 2: 1815.

5. Woollard, D. C.; Indyk, H. E.; Christiansen, S. K. (1999) The analysis of pantothenic

acid in milk and infant formulas by HPLC. Food Chem 2000: 69, 201-208.

104





P 52

SIMULTANEOUS DETERMINATION OF PHENYLUREA AND

TRIAZINE HERBICIDES IN RIVER WATER BY SOLID-PHASE

EXTRACTION AND LIQUID CHROMATOGRAPHY

G. Mendaš, B. Tariba, S. Stipičević, M. Dvoršćak, V. Drevenkar

Institute for Medical Research and Occupational Health, Zagreb, Ksaverska cesta 2

E-mail: [email protected]; [email protected]; [email protected]; [email protected];

[email protected]

Three different solid-phase extraction (SPE) procedures were investigated and

compared for the simultaneous extraction of frequently used phenylurea and triazine

herbicides from water. Diuron, isoproturon, chlorotoluron, linuron, terbuthylazine, and its

degradation product deethylterbuthylazine were extracted from water samples by a single SPE

procedure using either octadecylsilica (C18) or poly(divinylbenzene-co-N-vinylpirrolidone)

(Oasis HLB) or styrene-divinylbenzene (SDB-1) sorbent cartridges and acetone (C18 and

SDB-1) or methanol (Oasis HLB) as elution solvents. The sample preparation was optimized

for final analysis by high performance liquid chromatography with UV diode array detection.

The accumulation efficiency of compounds from deionised water was determined by

extracting 500 ml water samples spiked with 34 ng l-1

to 166 ng l-1

of each analyte. Good

extraction recoveries were achieved with all of the three sorbents and ranged from 81 % for

terbuthylazine to 110 % for isoproturon, with RSD values between 4 % and 12 %. The

detection limit for triazine and phenylurea compounds in the water was 10 ng l-1

. However,

the most uniform recoveries (>87 % for all compounds) with an RSD of 5 % to 10 % were

achieved with the SDB-1 extraction procedure, which was therefore chosen for further

experiments. Extraction of triazine and phenylurea compounds on the SDB-1 sorbent from

500 ml and 1000 ml water samples was comparably efficient, allowing for a decrease in the

analyte detection limits by treating the larger sample volume. The influence of the sample

matrix on the accuracy, precision, and sensitivity of the SDB-1 procedure was tested by

analysing different river water and deionised water samples spiked with phenylurea and

triazine compounds at the same mass concentration levels. The analytical quality of the SDB-

1 procedure was additionally confirmed by a successful participation in international

proficiency testing exercises. The procedure was applied for monitoring phenylurea

herbicides, terbuthylazine, and deethylterbuthylazine in river waters of Croatia. The mass

concentrations of target pollutants ranged from 0.01 μg l-1

to 2.9 μg l-1

.

105




mailto:%[email protected]





P 53

PHARMACOKINETICS BEHAVIOUR OF VERAPAMIL AFTER

INTRAVENOUS AND ORAL ADMINISTRATION OF

DIAZEPAM IN RATS BY HPLC

H. Rechak1, M. Lahouel

1, A. Hamdi

2

1Laboratoire de toxicology, université de Jijel

2Université USTHB, faculté de chimie, BP32 El-Alia, Bab Ezzouar 1611 Alger, Algeria

Email: [email protected]

In present studies, pharmacokinetics behaviour of verapamil, a Calcium channel

antagonist, after intravenous and oral administration of diazepam were investigated in rats.

Rats were given verapamil alone or verapamil in the presence of diazepam and the

plasma samples were collected at different time intervals and then purified using liquid-liquid

extraction procedure.

The plasma concentration of both verapamil and diazepam were measured by using R-

HPLC, after oral (10 mg/kg) or intravenous (1mg/kg) administration of verapamil alone or in

the presence of diazepam, after oral (1 mg/kg) or intravenous (0.1 mg/kg) administration.

Verapamil and diazepam were separated using reversed phase column (125x4.6 mm.

i.d.) nucleosil 100-5 C18, with mobile phase consisting of ACN/butter phosphate, 75 mmol,

pH 3 (40/60 v/v%) and λ=230nm.

Chromatographic analysis has been carried out with reasonable retention times.

Keywords: verapamil, diazepam, hplc, pharmacokinetics

106





P 54

COMBINATION QuEChERS, DISPERSIVE LIQUID-LIQUID

MICROEXTRACTION AND HPLC WITH FLUORESCENCE

DETECTION FOR SIMULTANEOUS EXTRACTION AND

DETERMINATION OF EIGHT MYCOTOXINS IN CEREAL AND FEED

N.M Karaseva, V.G. Amelin, A.V. Tretaykov

Federal Center for Animal Health, Russia, Vladimir


Mycotoxins producing by molds of the genus Aspergillus, Fusarium and Penicillium

are the most dangerous and commonly observed in the world. Contamination of mycotoxins is

serious problem concerning food and feed safety. The control of these compounds in the

product is very important.

Analytical method have been developed for the determination of 8 mycotoxins

producing by Aspergillus, Fusarium, and Penicillium in cereal and feed. It are aflatoxins B1,

B2, G1 and G2, ochratoxins A and B, zearalenone and patulin.

QuEChERS and dispersive liquid-liquid microextraction (DLLME) was used for

extraction and purification of samples. Mycotoxins were extracted from cereal and feed

samples by using water and acetonitrile with added salts. Clean up carried out with using C18

and PSA, this step is not necessary for ochratoxins A and B. The extract was divided into

three parts and then it was carried out DLLME step. Different parameters of DLLME have

been investigated and optimized for aflatoxins, ochratoxins, zearalenone and patulin.

HPLC with fluorescence detection was used for determination mycotoxins. Parameters

of HPLC––FLD (fiber polarity, temperature, pH, mobile phase) have been optimized for each

group of mycotoxins. Chromatographic separations were performed on a column

SUPELCOSIL LC18 4.6 x 250 mm 5 µm. Pre-column derivatization with solution iodine was

used for aflatoxins for more a sensitive determination. Satisfactory recoveries were obtained

80–100% and precision (expressed as relative standard deviation) was 10%. Duration of the

analysis was 1.5 – 2 h.

107





P 55

THE ANALYTICAL USE OF NEOCUPROINE FOR DETERMINATION

OF COPPER IN HUMAN URINE

Simona Čurmová, Lenka Okenicová, Radoslav Halko

Department of Analytical Chemistry, Faculty of Natural Sciences, Comenius University in

Bratislava, Mlynská dolina, CH2-312, 842 15 Bratislava, Slovakia,


This work characterizes newly developed methods for the determination of trace

concentration of copper in human urine. 2,9-dimethyl-1,10-phenanthroline (neocuproine) was

used as a selective chelating reagent for Cu(I). In Reversed-Phase High Performance Liquid

Chromatography (RP-HPLC) and Cloud Point Extraction combined with Flame Atomic

Absorption Spectrometry (CPE-FAAS) the reduction Cu(II) on Cu (I) was carried out by

adding appropriate amounts of ascorbic acid. These methods were based on the reaction of

neocuproine with Cu(I) which forms orange-yellow, hydrophobic chelate. RP-HPLC was

established for the determination of copper. Separation was accomplished on C8 column, and

the elution condition was optimized by changing the composition of the mobile phase. A good

resolution of all of the relative components in the reaction solution was achieved when the

mobile phase was composed of aqueous buffer solution (ascorbic acid/ammonium acetate)

and methanol by gradient elution. A required separation of chelate was within 9 minutes.

Second a simple and sensitive method was desribed for the pre-contretation by CPE-FAAS

determination of copper. Neocuproine and Triton X-114 were used as hydrophobic ligand and

non-ionic surfacant. The effects of experimental conditions such as pH, concentration of

chelating agent and surfactant, equilibration temperature and time on recovery were studied.

The proposed methods were applied to the determination of trace copper in human urine

samples.

Keywords: copper, neocuproine, RP-HPLC, CPE-FAAS

Acknowledgements

This work was generously supported by the grant of project VEGA 1/0852/13, Grant

Comenius University UK/308/2013 and the grant of project APVV-0583-11. This work is

partially outcome of the project VVCE-0070.

108





P 56

DEVELOPMENT OF A RAPID QUANTIFICATION METHOD OF 10

PESTICIDES IN WHOLE BLOOD BY LIQUID CHROMATOGRAPHY

/TANDEM MASS SPECTROMETRY LC-MS2

O. Ferroukhi, S. Bouanani, M. Bensebaa, H. Mebarki

Laboratoire de chromatographie, faculté de Chimie, BP 32 El-Alia,

Bab-Ezzouar, Alger, Algérie.


Intoxication with pesticides is one of modes of poisoning after intoxication by carbon

monoxide and drugs. This work aims to develop a protocol dedicated to the research and

determination of 10 most used pesticides, belonging to different chemical families in a single

analysis: Acétamipride, Carbendazime, Chlorpyrifos, Dichlorvos, Diméthoate, Phosalone,

Méthomyl, Fenitrothion, Imidaclopride, Malathion, in biological matrices since pretreatment

with Solid Phase Extraction (SPE) to the qualitative and quantitative analysis of these

molecules in whole blood by Liquid chromatography coupled to tandem mass spectrometry

(LC-MS/MS).

This study reveals the ease of application of this technique as a routine method in

toxicology laboratories for research of a large number of pesticides in a single analysis in

forensic cases.

Keywords: pesticides, SPE, LC-MS/MS

109






P 57

EVALUATION OF THE UPLC METHOD FOR IGG GLYCAN

ANALYSIS FOR IT'S ROBUSTNESS AND REPEATABILITY

I. Trbojević Akmačić1, J. Štambuk

1, F. Vučković

1, M. Novokmet

1,

M. Pučić Baković1, G. Lauc

1,2

1Genos Ltd. Hondlova 2/11, 10 000 Zagreb, Croatia

E-mail: [email protected] 2Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovačića 1,

10 000 Zagreb, Croatia

Changes in glycosylation of human immunoglobulin G (IgG) alter its function, and

therefore have been related with various pathological states. In recent years several large-scale

studies on IgG glycosylation have been performed and new possibilities for applications in the

field of glycan biomarker discovery and disease prognosis have emerged. Since there are long

term demands for rapid UPLC analysis of high number of samples, it is neccessary that

developed method is evaluated for it's robustness and repeatability. This way, data collected

during longer time periods will be more comparable. IgG glycans were analysed by

hydrophilic interaction liquid chromatography (HILIC) on ultra performance chromatography

system (UPLC) with acetonitrile and 100 mM ammonium formate as mobile phases. Prior

analysis, glycans were derivatized with fluorescent dye, 2-aminobenzamide, and purified with

solid phase extraction (SPE). The experimental conditions like column temperature, flow rate

and wavelength of detection were deliberately changed to determine the robustness of the

method. Same sample was analyzed several times in a row from the same vial, and also from

separate vials to check for repeatability. Maximum number of samples analyzed in one

sample set and stability of mobile phases were also investigated. Analysis of five samples by

two different analysts on different days was performed to determine ruggedness. Also,

different UPLC systems and columns from different production lots have been compared to

exclude possible differences in results. Repeatability and high robustness are of highest

importance in large-scale population studies, when several UPLC systems are being used.

110





P 58

QuEChERS SAMPLE PREPARATION IN SIMULTANEOUS

DETERMINATION OF RESIDUAL AMOUNTS OF ANTIBIOTICS

QUINOLONE SERIES AND CHLORAMPHENICOL

IN FOOD BY HPLC-DAD

N.M. Volkova, A.A. Timofeev, V.G. Amelin, A.V. Tretaykov

Federal Center for Animal Health, Russia, Vladimir


Quinolone antibiotics and chloramphenicol are widely used in veterinary medicine and

animal husbandry. Veterinary drugs, when used in food animals, have the potential to

generate drug residues in animal products. The regulatory agencies around the world set

tolerances or maximum residue levels to ensure residues are not present in excess of the set

tolerance levels and that no unapproved drugs are used. Efficient methods are needed for

determining these residue levels in food. A large number of available methods have been

developed for drug residues. However, these methods are long and require the use of

sufficiently large amounts of toxic organic solvents for extraction and concentration of

quinolones and chloramphenicol.

A new method has been developed which allows for the simultaneous determination of

six quinolone and chloramphenicol antibiotics residues in food by high-performance liquid

chromatography with diode array detection of enoxacin, danofloxacin, lomefloxacin,

enrofloxacin, oxolinic acid, difloxacin and chloramphenicol. The samples were prepared

using simplified, quick and safe sample preparation QuEChERS. The detection limits of

quinolones and chloramphenicol with sample weight of 5 g were 0.002-0.04 mg/kg. The

relative standard deviations analysis results is less than 0.09. The analysis time is about 1

hour.

111





Figure 1. The chromatogram of a mixture of solutions of antibiotics (10 μg / ml).

1 - Enoxacin, 2 - Danofloxacin, 3 - Lomefloxacin, 4 - Enrofloxacin,

5 - Oxolinic acid, 6 - Chloramphenicol, 7 - Difloxacin

112




P 59

IDENTIFICATION OF VOCS EMISSION FROM INDOOR

MATERIALS AND THE ATTEMPT OF BIOTESTS

IMPLEMENTATION IN TOXICITY ASSESSMENT OF

EMISSION STREAM

K. Sosnowiec1, G. Gałęzowska

1, M. Cieszyńska

1, L. Wolska

1,2

1Medical University of Gdansk, Faculty of Health Sciences with Subfaculty of Nursing and

Institute of Maritime and Tropical Medicine, Department of Environmental Toxicology,

9b Powstania Styczniowego Str., 81-519 Gdynia, Poland 2Gdansk University of Technology (GUT), Chemical Faculty, Department of Analytical

Chemistry, 11/12 G. Narutowicza Str., 80-233 Gdańsk, Poland


The Indoor Air Quality (IAQ) is determined by the emissions of Volatile Organic

Compounds (VOCs) from indoor materials. The analytical methods applied so far, did not

provide the direct information about the biological effects. The implementation of biotests to

assess the quality of indoor materials, can provide information about possible result of the

interaction of many substances existing on various levels of concentration.

Indoor materials (polystyrene plates, roller blind) emitting VOCs were the subject of

research. The stream of emissions was generated in the toxicological chamber (volume 1m3).

Samples for the identification of VOCs and ecotoxicological studies were collected

simultaneously. The adsorbed of VOCs on the solid sorbent Tenax GC, were thermally

desorbed into the gas chromatography - mass spectrometry device. The polar compounds,

which were absorbed in the aqueous extract were ecotoxicologically tested. The examinations

were conducted with application of biotests: Microtox® and ThamnotoxkitTM. In the studies

the following compounds were identified from polystyrene plates and roller blind: styrene,

hexanol, decane, undecane, benzene, p-xylene. Most of the compounds identified from indoor

materials have a negative impact on human health.

After 24 hours, of the material (roller blind) incubation in the chamber, toxicity of

emission stream, assessed in the Microtox® test was 93%. The samples after 48 and 72 hours

indicated lack of the toxicity with toxicity values of 2% and 7% respectively. The results in

the Microtox® test, were however of low repeatability. The ThamnotoxkitTM test was in turn

too insensitive towards the compounds tested.

It should be emphasized that there is a scarcity of research on VOCs toxicity

assessment based on biotests application and therefore there is still a huge requirement for

further research.

Keywords: biotests, VOCs, emission, identification

Partial support of this study was provided by project No. MN37, 01-0037/08.

113





P 60

COMPARISON OF DIFFERENT CHROMATOGRAPHIC METHODS

FOR THE DETERMINATION OF HYDROCARBON TYPES, BENZENE

AND OXYGENATES IN MOTOR GASOLINE

M. Miliša Gregurić1, Z. Kauzlarić

2

1INA d.d. Sektor Rafinerija nafte Rijeka, Urinj bb, 51221 Kostrena

E-mail: [email protected] 2INA d.d. Sektor Rafinerija nafte Sisak, Ante Kovačića 1, 44000 Sisak


Oxygenated gasoline is a mixture of conventional hydrocarbon-based gasoline and one

or more oxygenates. The hydrocarbons vary by type – paraffins, olefins, naphthenes, and

aromatics, and, within each type, by size. Current oxygenates belong to one of two classes of

organic molecules: alcohols and ethers .The mixture of hydrocarbons and oxygenates in a

gasoline determines its physical property and engine performance characteristics.

Multidimensional gas chromatography is extensively used in the analysis of gasoline

range petroleum fractions. Commercially available „micropacked/packed/capillary column

PIONA column system“ is used for determination of all hydrocarbon types: paraffins,

isoparaffins, olefins, naphthenes, and aromatics in finished gasoline and gasoline-related

streams. In addition this system is extended to quantitative oxygenates in reformulated

gasoline.

Capillary column multidimensional GC systems are used to determine hydrocarbon

types as well as distribution of individual hydrocarbons. Configuration presented has both

columns–polar capillary column and nonpolar capillary column placed in the same oven. Two

capillary columns are connected via a pressure switching device „heart cut“ switch.

Application is used to determine benzene and oxygenates. Benzene or oxygen-containing

fraction is isolated from the injected sample using a first capillary column. The isolated

fraction is further separated on a second capillary column with a different polarity.

In the Fluorescent Indicator Adsorption (FIA) method oxygenated gasoline samples

are separated using a special glass adsorption column packed with activated silica gel and a

small layer of fluorescent indicator dyed gel. Pressurized isopropyl alcohol (IPA) promotes

the vertical migration of the sample down the column (open column liquid chromatography).

The dyes are also separated selectively with the hydrocarbon types, which differentiate the

boundaries of the saturate, aromatic and olefinic fraction under UV light. The separated bands

are measured and corrected for oxygenate content determined with capillary column

multidimensional GC.

In INA refineries test methods used for composition determination are in accordance

with the European EN 228 gasoline specification.

Different chromatographic methods are described to discuss results precision, analysis

run time and analysts needed for this type of test.

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P 61

A CHROMATOGRAPHIC STUDY OF THE NEW HETERONUCLEAR

COMPLEXES WITH THE SCHIFF BASE AS A MAIN LIGAND

A. Wronka, I. Malinowska

Department of Planar Chromatography, Chair of Physical Chemistry, Faculty of Chemistry,

Maria Curie-Skłodowska University, Maria Curie-Skłodowska Sq. 3, 20-031 Lublin, Poland


The chemistry of coordination compounds is an important and challenging area of

modern chemistry. Schiff base ligands obtained from salicylaldehyde and its derivatives are

largely used for the synthesis of metal complexes having application in bioinorganic

chemistry, catalysis and magnetochemistry.

Significant research progress in this area has been observed in last few years.

Exploring physical properties of coordination compounds such as solubility, structural and

magnetic features. Complex compounds have been investigated using various methods, such

as: IR, Raman spectroscopy and X-ray analysis. In our work, thin layer chromatography

combined with magnetic and electric field has been proposed as complementary method for

determination of physicochemical properties of investigated compounds. The retention

analysis of those complexes may give us some information about their affinity to different

stationary phases and the influence of the central ion on it.

In our research, retention of 12 new heteronuclear coordination compounds (Fig. 1)

and their ligand in RP chromatographic systems were investigated. Taking into account the

fact, that in present times devices generating static or dynamic electromagnetic field are used

in many places and this compounds are very interesting regarding their magnetic properties, it

justified to examine, how the presence of magnetic or electric field influences the properties

of investigated compounds. Therefore, the chromatograms were developed simultaneously in

three identical chromatographic chambers. One of them was placed in external magnetic field

of 0.4 T inductivity and the second in external electrical field.

In magnetic and electric field, retention of some complexes have been changed, what

means, that this conditions influences the physicochemical properties of the analyzed

compounds and their interactions with the stationary phase.

Figure 1. Heteronuclear coordination compounds.

115






P 62

PURIFICATION OF HPV16 E6/E7 PLASMID DNA-BASED VACCINE

USING A MODIFIED MONOLITHIC SUPPORT

A. Soares, J.A. Queiroz, F. Sousa, Â. Sousa

CICS-UBI – Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior,

Av. Infante D. Henrique, 6200-0506 Covilhã, Portugal


The plasmid DNA (pDNA) application as a non-viral vaccine has become an attractive

pharmaceutical strategy in the non-invasive treatment of diseases, like cervical cancer caused

by Human Papillomavirus (HPV) type 16. This approach is safer than using viral vectors.

These therapeutic vaccines generate immune responses through the plasmid inoculation

encoding E6 and E7 antigenic proteins, which are subsequently expressed by the cellular

machinery of transfected cells in the immunized patient. Afterwards, the immune system

develops a primary response to destroy the pathogen and to acquire a memorized response

against HPV. In this way, it is important to develop purification processes to achieve

maximum recovery of the most biologically active conformation of pDNA, the supercoiled

(sc) isoform. Recently, the application of chromatographic operations based on affinity

interactions between plasmid DNA or impurities with specific amino acids immobilized in

stationary phases has demonstrated good results in the sc pDNA purification. Despite of

selectivity achieved with these ligands, conventional matrices present limitations such as the

low binding capacity and diffusivity for plasmid DNA samples. Owing to bottlenecks

associated to conventional matrices, monolithic supports have emerged as interesting

alternatives due to the versatility of their structural characteristics. This work reports a new

strategy that combines the selectivity of arginine as affinity ligand with the versatility of the

epoxy-based monoliths to specifically purify the supercoiled HPV-16 E6/E7 plasmid from

other plasmid isoforms and Escherichia coli impurities present in a clarified lysate. The

quality control tests of the final plasmid product indicated that the RNA and proteins were

undetectable while the gDNA and endotoxins were below the generally accepted

specifications. The combination of the selectivity of arginine ligands with the versatility of

epoxy-based monoliths is thus an interesting strategy to be used as plasmid purification step,

obtaining an adequate non-viral vaccine against a HPV infection.

Keywords: affinity chromatography, arginine ligand, Human papillomavirus, modified

monolithic support, plasmid DNA vaccines

Acknowledgments


Technology (PTDC/EBB-BIO/114320/2009) and PEst-C/SAU/UI0709/2011 COMPETE. A.

Sousa also acknowledges a post-doctoral fellowship (SFRH/BPD/79106/2011) from FCT.

The authors acknowledge to BIA Separations for having kindly provided the monolithic

support and especially to Dr. Urh Černigoj for the valuable help in the arginine amino acid

immobilization to the epoxy monolithic support. The authors also acknowledge to Karl

Münger for the HPV16 E6/E7 Addgene plasmid.

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P 63

EXPLOITING MULTIPLE INTERACTIONS IN PRE-miR-29

PURIFICATION BY ARGININE-AFFINITY CHROMATOGRAPHY

P. Pereira1, A. Sousa

1, I.J. Correia

1, A. Figueiras

1,2, F. Sousa

1

1CICS-UBI – Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior,


E-mail: [email protected] 2CEF-FFUC – Centro de Estudos Farmacêuticos, Faculdade de Farmácia, Universidade de

Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba,

3000-548, Coimbra, Portugal

Alzheimer´s disease is an incurable neuropathology that affects millions of people,

posing a heavy economic and social burden. Recent studies demonstrated that the miR-29 is

significantly decreased in Alzheimer’s disease patients displaying abnormally high levels of

beta-amyloid precursor protein-converting enzyme 1 (BACE1). Therefore, microRNA is

arising as a new tool for gene silencing since it can act as powerful mRNA degrading

molecules. RNA biochemical or structural studies often require a RNA sample that is

chemically pure and biologically active. Thereby, this study aims the development of a new

affinity chromatographic method using an arginine support to specifically purify pre-miR-29

from a total RNA mixture with high purity degree and yield, envisioning their application in

gene therapy. The arginine-agarose support demonstrated the ability to bind sRNAs, thus this

interaction can be exploited to specifically purify the pre-miR-29. Furthermore, the selectivity

found for the RNA molecules with this support suggests that the interaction is accomplished

by a biologically-based recognition of the individual chemical structure. The RNA samples

obtained from recombinant Rhodovulum sulfidophilum growth were applied onto the arginine-

agarose support with different sodium chloride and ammonium sulfate concentrations, using

stepwise gradients. To better understand the mechanism for the specific recognition of pre-

miR-29 by arginine–agarose, some experiments of competitive elution with arginine were

also performed. The successful isolation of pre-miR-29 by arginine affinity chromatography

has a potential applicability on RNA structural and functional studies which can provide

nearly untapped opportunities on pharmaceutical applications.

Acknowledgements

This work was supported by FCT (EXPL/BBB-BIO/1056/2012 and PTDC/EBB-

BIO/114320/2009) and PEst-C/SAU/UI0709/2011 COMPETE. P. Pereira also acknowledges

a fellowship (SFRH/BD/81914/2011) from FCT.

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P 64

IMPROVED NATIVE PRE-miR-29 PURIFICATION WITH LYSINE-

AFFINITY CHROMATOGRAPHY

P. Pereira1, A. Sousa

1, I.J. Correia

1, A. Figueiras

1,2, F. Sousa

1



E-mail: [email protected] 2CEF-FFUC – Centro de Estudos Farmacêuticos, Faculdade de Farmácia, Universidade de

Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba,

3000-548, Coimbra, Portugal

MicroRNAs usually induce gene silencing by binding to target sites found within the

3’UTR of the targeted messenger RNA (mRNA). This interaction prevents protein expression

by suppressing protein synthesis and/or by initiating mRNA degradation. MicroRNA-based

biochemical or structural studies often require a RNA sample that is chemically pure, and

most protocols for its in vitro production use denaturing polyacrylamide gel electrophoresis or

chemical synthesis. Unfortunately, many RNAs do not quantitatively refold into an active

conformation after denaturation or in the presence of contaminants in these synthesized

formulations, creating significant issues for downstream characterization or use. Thus, the

interest in producing large quantities of RNA has increased with the rapid development of

gene therapy. Owing to the commercial interest in these approaches, the development of

innovative procedures to easily and efficiently purify the RNA is enforced. Several

chromatographic and non-chromatographic methods have been reported to accomplish this

purpose, but not all strategies allow the efficient separation of pre-miR-29 from a complex

small RNAs mixture. In addition, pre-miR-29 deficiencies or excesses have been related to a

number of clinically important diseases including Alzheimer’s disease. The recent application

of amino acids (histidine and arginine) as immobilized ligands in affinity chromatography has

lead to interesting results in nucleic acids purification field. The present study describes a new

strategy that uses a lysine ligand in affinity chromatography to efficiently separate pre-miR-

29. The retention behaviour of pre-miR-29 was characterized and adjusted to achieve higher

specificity in this chromatographic operation, using a stepwise ammonium sulfate gradient at

room temperature. Overall, it was verified that lysine-agarose support can promote a specific

interaction with RNA favoring the total pre-miR-29 separation. The results suggest that the

underlying mechanism involves biorecognition between the lysine matrix and pre-miR-29,

including hydrogen, hydrophobic interactions, among others.

Acknowledgements This work was supported by FCT (EXPL/BBB-BIO/1056/2012 and PTDC/EBB-

BIO/114320/2009) and PEst-C/SAU/UI0709/2011 COMPETE. P. Pereira also acknowledges

a fellowship (SFRH/BD/81914/2011) from FCT.

118





P 65

DETERMINATION OF AFLATOXIN M1

IN MILK AND MILK PRODUCTS

BY UHPLC-MS/MS

V. Stankov, H. Farkaš, A. Bognar, B. Marošanović

SP Laboratorija A.D., Industrijska 3, 21220 Bečej, Serbia


Mycotoxins are fungal secondary metabolites that if ingested can cause a variety of

adverse effects on both humans and animals [1]. Aflatoxins are a group of structurally-related

toxic compounds produced by certain strains of the fungi Aspergillus flavus and A. parasiticus

[2,3]. Lactating cows that eat feed containing 20ppb or greater aflatoxins may produce milk

that exceeds the tolerance level for aflatoxins in milk. Aflatoxin M1 contamination of milk

results primarily from the conversion of aflatoxin B1 that is metabolized by enzymes found

primarily in the liver. After aflatoxin M1 is formed, it is excreted in the urine and milk of the

cow [4].

A simple method for determination of aflatoxin M1 in milk and milk products was

developed by UHPLC-MS/MS. All samples were prepared using clean-up immunoaffinity

column. Chromatographic analysis was performed using the Ultimate 3000 U-HPLC system.

Analysis of aflatoxin M1 was performed on a column Thermo Scientific Hypersil GOLD aQ

(50 x 2.1mm, 1.9μm particle size). Mobile phase was 5mM ammonium formate in water with

0.1% formic acid (A) and 5mM ammonium formate in methanol with 0.1% formic acid (B)

flowing under gradient elution.

Validation results of this method were compared with the results obtained by standard

method of determing aflatoxin M1 by HPLC with fluorescence detector [5]. LOQ is

0.01µg/kg and for standard method it is 0,008µg/kg for milk and 0,08µg/kg for whole milk

powder. Repeatability was 15% and 14% for standard method and reproducibility was 25%

and 23% for standard method.

During the 2013, it was analyzed 1126 samples of milk and 234 samples of milk

products. 68% of milk samples were below 0.05µg/kg and 32% of milk samples contained

aflatoxin M1 whose concentration exceeded the MRL of 0.05µg/kg. 82% of milk products

samples were below 0.05µg/kg and 18% of milk products samples exceeded 0.05µg/kg.

References

1. H. Hampikyan, E. Baris Bingol, O. Cetin, and H. Colak. Determination of aflatoxin

M1 levels in Turkish white, kashar and Tulum cheeses, Journal of Food Agriculture

and Environment, 8 (2010), 13-15.

2. R. Baskaya, A. Aydin, A. Yildiz and K. Bostan. Aflatoxin M1 levels of some cheese

varieties in Turkey. Medycyna Wet, 62 (2006), 778-780.

3. C.Y. Chen, W.J. Li and K.Y. Peng. Determination of Aflatoxin M1 in Milk and Milk

Powder Using High Flow Solid Phase Extraction and Liquid Chromatography Tandem

Mass Spectrometry. J. Agric. Food Chem, 53 (2005), 8474-8480.

4. J.A. Pennington. Aflatoxin M1 in Milk. Agriculture and Natural Resources, FSA4018.

119





5. ISO 14501:2007 Milk and milk powder ― Determination of aflatoxin M1 content ―

Clean-up by immunoaffinity chromatography and determination by highperformance

liquid chromatography

120




P 66

REVIEW OF THE RATIO OF OMEGA-6/OMEGA-3 FATTY ACIDS

IN OILS AND FATS

K. Sabo, M. Pandurević Todorović, B. Marošanović

SP Laboratorija A.D., Industrijska 3, 21220 Bečej, Serbia


Omega−6 and omega−3 fatty acids cannot be synthesized by the human body, humans

must consume them through food.

In SP Laboratory, we have analysed the fatty acids in oils and animal origin foodstuffs

[1,2]. The obtained results allow to calculate the ratio of omega-6/omega-3 fatty acids in this

foodstuffs.

The vegetable and cereal oils contain generally more omega-6 fatty acid like as

Linoleic acid (C18:2n6). In the sunflower oil the content of Linoleic acid ranged is 55-67%,

the Linolenic acid (C18:3n3) is 0.05-0.16%. Taking average value of these acids the ratio of

omega-6/omega-3 is approximately 600:1, which is quite high. In potato and pumpkin seed

the result was also relatively high about 240:1 and 150:1, respectively. This ratio is slightly

reduced in wheat and soybean oil and even more in rapeseed oil. The ratio of omega-

6/omega-3 fatty acids in wheat was about 16:1, in soybean oil was about 8:1. In contrast to

these cereals in the rapeseed oil the result was quite low, about 2.5:1. Surprisingly, the result

of linseed was about 0.3:1.

In samples of animal origin the situation was different. In these samples were present

other omega fatty acids besides C18:2n6 and C18:3n3: Eicosatrienoic acid (C20:3n6),

Arachidonic acid (C20:4n6), γ-Linolenic acid (C18:3n6), Eicosapentaenoic acid (C20:5n3)

and Docosahexaenoic acid (C22:6n3). In milk, butter and cheese the ratio of omega-6/omega-

3 fatty acids was approximately 10:1. The values of ratio in eggs were about 5:1, in meat

products such as chicken breasts and pork the ratios were 4-8:1 and 3-8:1, respectively. In the

fish samples there was a great content of EPA and DHA omega-3 fatty acids. The sume of

omega-3 was varied in extracted fish oil: mackerel 30%, sprat 21%, tuna 9.5%, salmon 15%,

shark 2.5%, and the ratios of omega-6/omega-3 were 0.14:1; 0.31:1; 0.26:1; 1:1 and 3:1,

respectively.

References

1. ISO 5508:1990 Animal and vegetable fats and oils - Analysis by gas chromatography

of methyl esters of fatty acids

2. ISO 12966-2:2011 Animal and vegetabes fats and oils-Gas Chromatography of fatty

acids mehyl esters- Preparation of methyl esters of fatty acids. Rapid method 4.2

121





P 67

DEVELOPMENT OF LC-MS-Q-TOF-MS METHOD FOR

COMPREHENSIVE NONTARGETED LIPID PROFILING OF

STAPHYLOCOCCUS AUREUS

W. Hewelt-Belka1, J. Nakonieczna

2, A. Kot-Wasik

1, J. Namieśnik

1

1Department of Analytical Chemistry, Faculty of Chemistry, Gdańsk University of

Technology, Narutowicza 11/12, 80-233 Gdańsk, Poland 2Laboratory of Molecular Diagnostics, Department of Biotechnology, Intercollegiate Faculty

of Biotechnology University of Gdansk and Medical University of Gdansk,

Kładki 24, 80-822 Gdańsk, Poland


Staphylococcus aureus is one of the most common human infectious agents,

responsible for nosocomial and community-acquired infections. S. aureus is characterized by

a great genetic diversity, which results in a phenotype variability, including various sensitivity

for antibiotics. One of the features that can possibly differentiate bacterial strains of this

species is the cellular lipid content.

Advanced technologies in the field of HPLC and mass spectrometry allowed for

comparative quantitative and qualitative analysis of the proteome between S. aureus strains.

In many investigations, differences in protein expression between S. aureus strains with

varying degrees of sensitivity to antibiotics were demonstrated. Moreover, changes in

membrane phospholipids profiles in the strain resistant to daptomycin against strains sensitive

to this antibiotic were demonstrated. In this respect, it is appropriate to supplement the

knowledge of the differences between the strains at the level of lipid metabolism.

The goal was to develop LC-MS-Q-TOF method for comprehensive nontargeted lipid

profiling of S. aureus cellular lipids. Combination of high performance liquid chromatography

in reversed phase mode with high resolution Q-TOF-MS allowed for separation and sensitive,

accurate detection of total S. aureus lipidome.

The chromatographic separation of lipid extract was achieved on Kinetex® C18

column (50 mm×2.1 mm, 1.7 μm particle size) with methanol, isopropanol and 20 mM

ammonium formate mixture as the mobile phase at a flow rate of 0.5 mL/min and with

column temperature 60°C. Ions were monitored in positive ion mode. During the optimization

process, parameters such as eluent composition, column temperature, gradient steps, injection

volume and ionization parameters were investigated.

Preliminary results proved that developed methodology is suitable for comparative

lipidomic analyses of S. aureus.

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P 68

COMPARISON OF MOBILE PHASE ADDITIVES FOR

DETERMINATION OF AMINO ACIDS IN REVERSED PHASE MODE

W. Hewelt-Belka, P. Kubica, K. Wilczewska, A. Kot-Wasik, J. Namieśnik

Department of Analytical Chemistry, Faculty of Chemistry, Gdańsk University

of Technology, Narutowicza 11/12, 80-233 Gdańsk, Poland


Changes in physiological concentrations of protein and non-proteinogenic amino acids

(AAs) are in most cases connected with amino acids metabolism disorders and can lead to

appearance of clinical symptoms. Furthermore, the disrupt content of some AAs in biological

samples may be treated as potential indicator. There are many well-known diseases connected

with metabolism disorders including phenyloketonuria or homocystinuria. Thus, rapid and

reliable quantitative analysis of AAs in body fluids are needed for the diagnosis process.

Use of tandem mass spectrometry detectors and ion pair-reagents allows the

comprehensive analysis of most common AAs by HPLC technique. The advantage of the

proposed approach is the needless of derivatization.

The goal was to compare different mobile phase additives for determination of AAs

content in reversed phase mode. Influence of type and concentration additives including

formic acid or N,N-Diisopropylethylamine and uncommon additives therein

heptafluorobutyric acid, pentafluoropropinic acid, perfluoropropionic acid on amino acids

retention time, peak shape and MS signal abundance was investigated.

The idea was to set up a series of methods with the use of one chromatographic

column and comparison of them. The chromatographic separation of amino acids was

achieved on LiChroCART® LiChrospher

® RP18 analytical column (250 mm × 2.1 mm, 3 μm

particle size), with acetonitryl and water with different additives as the mobile phases.

Ion transitions for each of the AA were chosen, optimized and monitored in positive

multiple reaction monitoring (MRM) ion mode. Parameter which has the most influence to

the intensity of the signal of the individual transitions of AAs were collision energy and

declustering potential.

Chromatographic conditions were optimized regarding to the specific mobile phase

additive to set up specific methods. In summary there is no “the best” additive for the AAs

analysis, however different additives allows to increase sensitivity, resolution, enhance peak

shape due to the interactions between AAs-additive.

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P 69

ELUCIDATION OF DEGRADATION PATHWAY OF FUROSEMIDE

BY UPLC-QTOF-MS

A. Jakimska, W. Hewelt-Belka, A. Kot-Wasik, J. Namieśnik




Research focuses on the presence of drugs in the water and on the efficiency of their

elimination in wastewater treatment plants (WWTPs). The absence of the original substance

in the effluent stream is not equal to the total elimination, because they can be transformed in

stable compounds. Therefore, one must keep in mind is that the drugs are present in the

environment in conjunction with their products of bio- or photodegradation, which also

includes the so-called emerging pollutants.

Due to the high consumption and wide application for many diseases such compound

which requires special attention is furosemide. This compound belongs to a class of drugs that

cause increased production of urine (diuretic). The currently used diuretics include

benzothiazids and their derivatives, potassium-sparing diuretics and loop diuretics. Loop

diuretics, which include furosemide, discloses action in a thick ascending arm of Henle’s loop

(in nefron) by blocking (quickly and reversibly) media Na+ / K

+ / Cl

- and thus inhibiting the

resorption of sodium ions, potassium and chloride.

Furosemide in clinical practice is used in the treatment of edema associated with

congestive heart failure, cirrhosis of the liver and kidney disease, when it is advisable to

provide a strong diuretic and fast action. The drug is also used in the treatment of

hypertension.

The photodegradation experiment, performed in natural waters with the application

of UPLC-QTOF-MS, led to the determination of a few degradation products. Furosemide was

degraded to the following major products: 4-chlor-5-sulfonamide anthranilic acid, 2-[(2 -

furylmethyl)-amine-3-hydroxy-5-sulfamoyl] benzoic acid, 2-[(2-phenyl-methyl)-amino-5-

sulfamoyl-benzoic acid, 5-sulfonamide anthranilic acid, [5-sulfonamide-3-hydroxy]

anthranilic acid. The studies allowed to specify the fate and the transformation pathway of the

selected diuretic in the environment by investigation of their degradation products in different

waters (e.g. wastewater, natural water, treated water, drinking water).

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P 70

OCCURRENCE OF CAFFEINE IN THE AQUEOUS ENVIRONMENT

BY SPE-HPLC-APCI-MS/MS

A. Jakimska, W. Hewelt-Belka, A. Kot-Wasik, J. Namieśnik




Caffeine is the most widely used psychoactive substance in the world. Caffeine is

increasingly consumed by society and enormous amounts of the compound are introduced

into the environment. Accordingly, developing of sensitive analytical methods is desired. The

fact that caffeine is present in the environment proves badly of the technological capacity of

wastewater treatment plants. Articles describing the analysis of the caffeine content relate

primarily to commonly consumed beverages, such as coffee, tea, energy drinks. While

performing the monitoring of aqueous environment caffeine is not considered but due to the

fact that caffeine interferes with the behavior and human health the concentration of the

alkaloid in the environment should be determined. Little research was carried out so far where

the main interest was focused on caffeine in environmental water samples. Its concentration

was determined on the occasion of the determination of other pharmaceuticals.

In light of these concerns, our goal was to develop a fast, sensitive and robust

analytical procedure for the determination of caffeine in different water samples (effluent,

influent, treated water, untreated water, river water). The methodology include the application

of solid phase extraction as sample preparation technique followed by LC-APCI-MS/MS. The

recoveries were higher than 98% for all the matrices. MDLs were in range 0.058 (untreated

water) to 0.51 (river water). The trueness was below 10.7%, while the inter-day and intra-day

precision were below 8 and 7.4%, respectively, considering this method as sensitive and

reliable. Different water samples were monitored. The highest concentrations were

determined in influent wastewater (average 2142 ng/mL); however, caffeine was proved to be

present in treated water (drinking water) as well (average 49ng/mL), what shows that the

environment as well as humans and animals are continuously exposed to this alkaloid.

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P 71

QUANTITATIVE STRUCTURE - RETENTION RELATIONSHIP AS A

SUPPORTIVE TOOL FOR IDENTIFICATION OF CIS- AND TRANS-

ISOMERS IN STABILITY STUDIES OF A SERIES OF NOVEL

BENZENSULFONAMIDE DERIVATIVES – POTENTIAL

ANTICANCER COMPOUNDS

M. Belka1, J. Sławiński

2, T. Bączek

1

1Department of Pharmaceutical Chemistry, Medical University of Gdańsk,

Hallera 107, 80-416, Gdańsk, Poland

E-mail: [email protected] 2Department of Organic Chemistry, Faculty of Pharmacy, Medical University of Gdańsk,

Hallera 107, 80-416, Gdańsk, Poland

Liquid chromatography is a separation technique widely used for quantitative and

qualitative analysis of diverse compounds, based on retention behavior and partitioning

between mobile and stationary phase. Retention data is also used for determination of

common drug-like properties as lipophilicity and dissociation constant [1]. Recently, mass

spectrometry has gained a great popularity as an identification tool, however MS is powerless

when it comes to differentiate geometric isomers characterized by the same mass and

fragmentation pathway.

QSPR and QSRR are chemometric tools designed to quantitatively describe the

relationship between some property (e.g. retention factor) and chemical structure of a

compound. Modern approaches involves molecular modeling step to obtain most favorable

low energy conformation and specialized software used to calculate thousands of so called

molecular descriptors – variables that can potentially describe retention behavior of a

particular chemical structure.

Hydrazones, including studied thiohydrazones isomerize in water solutions, thus it is

important to develop a tool which supports identification of chromatographic peaks during LC

analysis. For studied benzensulfonamides we have already investigated structure-activity

relationship [2,3]. In this study, we have developed a regression model, which is able to

describe and predict retention factor of benzensulfonamide derivatives, both cis- and trans-

isomers. 29 compounds were divided into training and validation sets. Statistical performance

of obtained equation includes: R = 0.95, F = 52.4, p < 0.0001, s = 0.036.

Developed model can be a valuable tool during metabolic stability studies of drug

candidates. The proposed methodology can be easily transferred to other groups of

compounds.

References

1 Koba M, Belka M, Ciesielski T, Bączek T: Determination of lipophilicity for

antitumor acridinone derivatives supported by gradient high-performance liquid

chromatography method. Central European Journal of Chemistry 2012;10:216-223.

2 Belka M, Konieczna L, Kawczak P, Ciesielski T, Slawinski J, Bączek T: The

chemometric evaluation of antitumor activity of novel benzensulfonamide derivatives

based on their physiochemical properties. Letters in Drug Design & Discovery 2012;9.

126





3 Belka M, Slawinski J, Konieczna L, Kawczak P, Ciesielski T, Bączek T: Antitumor

activity of novel benzensulfonamide derivatives in view of their physiochemical

properties searched by principal component analysis. Medicinal Chemistry

2013;9:517-525.

127




P 72

CHARACTERIZATION OF SELECTED

ENVIROMACROMOLECULES BY COMBINATION OF REVERSED-

PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY AND

NARROW-BORE SIZE-EXCLUSION CHROMATOGRAPHY

R. Góra, M. Hutta, P. Rohárik, N. Bielčíková

Comenius Univerzity in Bratislava, Faculty of Natural Sciences, Department of Analytical

Chemistry, Mlynská dolina CH-2, 84215 Bratislava, Slovak Republic;


Multidimensional chromatography has a proven to be useful for the analysis of

complex samples such as HS or L samples. From the point-of-view of chemical analysis,

characteristic feature of these analytes is diffuse non-distinct analytical signal produced by

many detection principles. This signal does not usually result in an exact numerical physical-

chemical data, but is described also by their distribution function or range of validity. This

dictates the necessity of development of automated complex separation procedures with

minimal sample pre-treatment, and the use of on-line (off-line) multidimensional

chromatographic techniques is a logical solution to these requirements.

The aims of the presented work is design and development of novel methods of liquid

chromatography for analysis and characterization of some from industrial point-of-view

distinct biomacromolecules, e.g. humic substances (HS), lignin (L) by utilization of

combinations of two or more liquid chromatography methods. HS belong to the most spread

envirobiomacromolecules and they have direct influence to various processes playing

significant role in an environment. They are created by a complex mixture of amorphous,

yellow to black coloured, hydrophilic, polyelectrolyte poly-disperse macromolecules.

With respect to the non-common approach we focused to evaluation of its potential to

create orthogonal, i.e. on different separation principles working two dimensional

comprehensive separation methods. The coupling of two chromatographic methods, RP-

HPLC and SEC was evaluated using the statistical program calculated the Pearson Product

Moment Correlation. Comparison of the calculated values of Pearson correlation coefficients

for characterization of the examined samples of HA and their fractions by coupling of the RP-

HPLC and SEC methods led to the conclusion that the values show a very low level of

correlation and the separation system employed behaves as orthogonal.

This work was supported by the financial support of projects VEGA 1/0852/13, APVV-0583-

11 within the frame of VVCE-0070-07

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P 73

AMINO ACIDS EVALUATION IN CEREBROSPINAL FLUID IN

LEUKEMIA CHILDREN USING HILIC-ESI-MS METHOD

L. Konieczna1, M. Belka

1, M. Niedźwiecki

2, T. Bączek

1

1Department of Pharmaceutical Chemistry, Medical University of Gdańsk,

80-416 Gdańsk, Hallera 107, Poland

E-mail: [email protected] 2Department of Pediatrics, Hematology, Oncology and Endocrinology,

80-416 Gdańsk, Hallera 107, Poland

Background. Qualitative changes in cerebrospinal amino acid have been observed in patients

with leukemia [1]. It was found that the amino acid increased or decreased in a fairly

consistent manner according the number of white blood cells present in patients with acute,

chronic lymphatic and chronic granulocytic leukemia [2]. Until now, the lack of more specific

methods for the quantitative determination of the individual amino acids present in CSF has

been a limiting factor in the further understanding of amino acid metabolism in leukemia

diseases as well as its consideration as biomarker of cancer allowing their earlier diagnosis

and monitoring of diseases [3].

The aim of this study was to propose new approach to analyze underivatized amino acids by

hydrophilic interaction chromatography (HILIC) coupled to electrospray ionization mass

spectrometry (ESI-MS) and to assess the differences between amino acid profiles in children

with leukemia patients under chemotherapy and control group.

Method: Amino acid concentrations in cerebrospinal fluid (CSF) were measured in 44

children with acute lymphoblastic leukemia (ALL) under chemotherapy (at the moment of

leukemia diagnosis, day 14 and day 33 of treatment) versus control group by the LC-ESI-MS

method. The amino acid values for the leukemic subjects under chemotherapy were

statistically compared with the control group.

Results: Results from this study show that glutamine levels at day 0 were significantly higher

in patients than in controls. The significance of the differences were determined by using U-

Mann-Whitney test with p values less than 0.05 were considered significant. The acute

leukemia patients have increased levels of glutamine and phenylalanine whereas the

concentrations of asparagine and threonine were lower than normal. Serine values had a

tendency to be low but were at the borderline of significance. In comparison, at Day 14,

concentrations of glutamine significantly decreased comparing to control group while

glutamic acid amounts increased. Glutamine levels significantly fell at Day 33 comparing to

diagnosis (day 0), but no significantly comparing to control group which may have resulted

from more intensive treatment. From this study we hypothesise that higher baseline glutamine

levels are indicative of a greater risk for CNS leukemia.

Conclusion: Large-scale prospective trials are required to confirm increased baseline CSF

glutamine levels in ALL patients, to identify glutamine as a marker for leukemia disease and

to clarify underlying mechanisms regulating glutamine in ALL.

129





References

1 C.T. Peng, K.H. Wu, S.J. Lan et al.: Eur J Cancer, 41 (2005) 115–l 1163.

2 S. Scholl-Burgi, E. Haberlandt et al.: Pediatrics, 121 (2008) e1–e7.

3 T. Bączek, L. Konieczna, M. Belka, M. Niedźwiecki et al.: J Pharm Biomed Anal, 70

(2012) 330–336.

130




P 74

DEVELOPMENT OF BIOANALYTICAL LC/MS/MS ASSAY FOR

QUANTIFYING 5-FLUOROURACIL AND ITS TWO METABOLITES

IN HUMAN PLASMA

M. Szafarz2,1

, A. Zakrzewska1, G. Koralewicz

2, A. Gonciarz

1,2, A. Kij

1, K. Kuś

1,2,

J. Suraj1,2

, M. Walczak2,1

1Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University,

Bobrzyńskiego 14, 30-348 Kraków, Poland 2Department of Pharmacokinetics and Physical Pharmacy, Jagiellonian University Collegium

Medicum, Medyczna 9, 30-688 Kraków, Poland


Fluorouracil (FU) is the most widely used antineoplastic agent for the treatment of

several malignancies, including carcinomas of the colon, breast and skin. Activation of FU is

a prerequisite for achieving its pharmacological effects. The first steps of its conversion

include the addition of a ribose by uridine phosphorylase to yield 5-fluorouridine (FUrd) and,

quantitatively less important, path which consist of an addition of deoxyribose-1-phosphate to

FU by thymidine phosphorylase leading to 5-fluorodeoxyuridine (FdUrd).

The aim of this work was to develop and validate the analytical method for

simultaneous determination of FU, FUrd and FdUrd in human plasma using LC/MS/MS

technique for the purpose of therapeutic monitoring.

Chromatographic separation of investigated compounds was achieved on Kinetex™

2.6 µm PFP 100 Å, 100x4.6mm (Phenomenex) analytical column. Mobile phase consisting of

methanol and water (60:40, v/v) with an addition of formic acid (0.1%, v/v) was delivered

isocratically by the Nexera UHPLC system with the constant flow of 0.25 mL/min. Hybrid

triple quadrupole ion trap mass spectrometer QTRAP 5500 with electrospray ionization

interface was employed as a detector and thymine was used as an internal standard (IS). All

compounds were scanned in the SRM mode, and the following parent to fragment transitions

were monitored: 12942 m/z for FU, 245.1155 for FdUrd, 260.9171 and 260.9129

for FUrd and 125.3 42.2 for IS. Data acquisition, data processing and instrument control

were performed through Analyst version 1.6 software.

The linear answer of the detector was observed for the concentrations from 50 to 2000

ng/mL for all analyzed compounds. The validation parameters such as accuracy and inter and

intra-batch precisions were within the limits set by the FDA guidelines.

Presented method was used for the quantification of steady state concentrations of FU

and its metabolites in the cancer patient.

Acknowledgments

This work was partially financed by the European Union from the resources of the European

Regional Development Fund under the Innovative Economy Programme (grant coordinated

by JCET-UJ, No POIG.01.01.02-00-069/09).

131





P 75

DIRECT RESOLUTION AND QUANTITATIVE ANALYSIS OF

FLURBIPROFEN ENANTIOMERS USING MICROCRYSTALLINE

CELLULOSE TRIACETATE PLATES: APPLICATIONS TO THE

ENANTIOMERIC PURITY CONTROL AND OPTICAL ISOMER

DETERMINATION IN WIDELY CONSUMED DRUGS

L. Checchini, C. Ancillotti, L. Ciofi, S. Furlanetto, M. Del Bubba

University of Florence, Department of Chemistry,

v. della Lastruccia 3 Sesto F.no (Florence) Italy


Flurbiprofen is a chiral non-steroidal anti-inflammatory and analgesic drug of the 2-

arylpropionic acid class, widely marketed in different pharmaceutical dosage forms, such as

mouthwashes, tablets and suppositories. The two flurbiprofen enantiomers have very different

pharmacological properties, since the (S)-(+) form has a much higher anti-inflammatory

activity than the (R)-(-) isomer [Davies, 1995], the latter being responsible for very

undesirable side effects, such as gastrointestinal irritation [Jamali et al., 1989; Lotsch et al.,

1995]. Based on the different biological properties of flurbiprofen enantiomers, the

development of chiral chromatographic methods for the control of the enantiomeric purity is a

very important topic. Even though gas chromatography and column liquid chromatography

are certainly the most widely employed chromatographic techniques for chiral analysis, thin-

layer chromatography (TLC) has been also quite extensively applied to the resolution of chiral

compounds, including a number of molecules with pharmacological activity [Del Bubba et al.,

2013]. In this study the separation of flurbiprofen enantiomers was achieved using for the first

time non-commercial MCTA layers with polyvinyl alcohol as binder, that gives to these

plates a mechanical stability equivalent to that of marketed ones. Baseline resolution (α=1.31;

RS=2.0) was obtained with ethanol-acetic acid solution (pH=3.0±0.1) (60:40, v/v) as eluent

and a migration distance of about 14.5 cm. Under these experimental conditions, the TLC

determination of the enantiomeric purity of the pharmacologically active (S)-(+)-flurbiprofen

in the presence of 1% of the undesired (R)-(-) form has been demonstrated. Moreover, the

quantitative analysis of flurbiprofen enantiomers was achieved, obtaining quantification limits

and detection limits of 50 ng and 25 ng of each enantiomer applied to the plate, respectively.

The method was successfully applied to the enantiomer determination in widely consumed

drugs, obtaining results consistent with the flurbiprofen content declared in the drug facts.

References

1 Davies NM. Clinical Pharmacokinetics of Flurbiprofen and its Enantiomers. Clinical

Pharmacokinetics 1995; 28: 100-114.

2 Jamali F, Mehvar R and Pasutto FM. Enantioselective aspect of drug action and

disposition: therapeutic pitfalls. Journal of Pharmaceutical Science 1989; 78: 695-715.

3 Del Bubba M, Checchini L and Lepri L. Thin-layer chromatography

enantioseparations on chiral stationary phases: a review. Analytical & Bioanalytical

Chemistry 2013; 405: 533-554.

132





P 76

ANALYTICAL APPROACH TO VALIDATION OF HPLC METHOD

FOR ANALYSIS AND CHARACTERISATION OF PROTEINS IN

PROTEINACEOUS FOOD SUPPLEMENT

V. Komorowska, M. Hutta

Department of Analytical Chemistry, Faculty of Natural Sciences, Comenius University in

Bratislava, Mlynská Dolina CH-2, SK-84215 Bratislava, Slovakia


Validation of analytical method for analysis of biomacromolecules is more complex

than validation of the method for analysis of lightweight substances, especially because of

biological activity of those substances. Biological activities of substances bring additional

potential variables into the analytical process. The method stability indicates how changes in

the analytical procedure can influence the final results.

Proteolytic enzymes as analytes were a subject of our study. We focused on the

validation of the stability of working protein solution observed before and during separation

process. The main problem of sample solution instability caused by proteolytic enzymes in

the sample is autoproteolytic reaction among molecules of enzymes. This phenomenon

significantly negatively affects the reproducibility of the analytical process. Therefore

different solvents with different pH and additives were used for validation of solution

stability. Influence of solvent temperature used for preparation sample solution was also

evaluated. Moreover a time interval between sample preparation and injection of the sample

solution into chromatographic system was studied.

Separation process was performed in a reversed-phase high-performance liquid

chromatographic (RP-HPLC) mode in a system with Zorbax 300 SB-18 column under the

conditions of gradient elution with buffered eluents using tandem of fast spectrophotometric

and mass detection. Different mobile phases and various column temperatures were evaluated

with respect to basic chromatographic figures-of-merit.

This work was generously supported by the grant of project VEGA 1/1349/12 and the

grant of project APVV-0583-11. This work is partially outcome of the project VVCE-0070.

133





P 77

SYNTHESIS OF MOLECULAR IMPRINTED POLYMERS AS A

FILLING FOR CHROMATOGRAPHY COLUMNS FOR THE

DETERMINATION OF MYCOTOXIN

K. Kwaśniewska, R. Gadzała-Kopciuch

Department of Environmental Chemistry and Bioanalytics, Faculty of Chemistry,

Nicolaus Copernicus University, Gagarin 7, 87-100 Torun, Poland


In the recent years, pathogens of mycological of origin have become a subject of

interest. Particular attention should be given to zearalenone (ZEA). It is a mycotoxin produced

by some fungi of the Fusarium species that mimics the reproductive hormone estrogen. This

compound, along with phytoestrogens, metalloestrogens, certain pharmaceuticals and some

pesticides, belongs to the group of substances that disrupt endocrine functions. Because ZEA

is bioaccumulated by living organisms, it can contaminate every element of the food chain,

from grains, corn and other crops to humans. Due to structural similarities (the presence of

macrocyclic lactone ring), zearalenone shows affinity for estrogen receptors and in natural

metabolic pathway it competes with natural estrogens (e.g. 17β-estradiol) in binding to the

estrogen receptor. This effect of ZEA on animals exposed to contaminated food may lead to

hyperestrogenism, whose symptoms include infertility, pseudopregnancy, swelling of

genitalia and fewer offspring in a litter [1]. Negative influence of zearalenone on human

organisms has also been noted [2,3]. As endocrine disruptor, zearalenone and its metabolites

may be involved in carcinogenesis of tumors related to secondary sex characteristics [4].

The paper presents a synthesis of copolymer methacrilic acid- divinylobenzene with

molecular imprinting dedicated to the selective recognition of zearalonone. The

polymerization process was carried out at 68 °C for 23 hours, a non-imprinted polymer (NIP)

is also synthesised in the same way as the MIP but in absence of the template. The

cyclododecyl-2,4-dihydroxybenzoate (CDHB) was used as a template to produce MIPs.

Spherical polymer particles were obtained a diameter of about 2 μm. The sorbent was packed

to plastic tubing of polyetheretherketone (PEEK) (column size 2.1 x 150 mm). Quantitative

analysis was performed by high performance liquid chromatography with fluorimetric

detection of zearalenone.

Acknowledgements

The work was financially supported by the National Science Centre in the frame of the

project OPUS No. 153119 (2011/01/B/ST4/00543).

References

1 H. Malekinejad, R.F. Maas-Bakker, J. Fink-Gremmels, Arch. Toxicol. 79 (2005) 547.

2 R. Khosrokhavar, N. Rahimifard, S. Shoeibi, M.P. Hamedani, M.J. Hosseini, Toxicol.

Mech. Meth. 19 (2009) 246

3 M.B. Lioi, A. Santoro, R. Barbieri, S. Salzano, M.V. Ursini, Mutat. Res-Gen. Tox En.,

557 (2004) 19

4 S. Ahamed, J.S. Foster, A. Bukovsky, J. Wimalasena, Mol. Carcinog. 30 (2001) 88.

134





P 78

INFLUENCE OF DIFFERENT KINDS OF CYCLODEXTRINS ON

SEPARATION OF PRAVASTATIN AND BOVINE SERUM ALBUMIN-

APPLICATION TO PROTEIN BINDING STUDIES BY CAPILLARY

ELECTROPHORESIS

A. Gonciarz1,2

, A. Kij1, K. Kuś

1,2, J. Suraj

1,2, M. Szafarz

1,2, A. Zakrzewska

1, M.

Walczak1,2


Bobrzynskiego 14, 30-348 Krakow, Poland 2Department of Pharmacokinetics and Physical Pharmacy, Jagiellonian University Collegium

Medicum, Medyczna 9, 30-688 Krakow, Poland


Capillary electrophoresis (CE), one of the separation techniques, allows to perform a

wide range of analyses, among others drug- protein binding studies, what is an important

issue in the drug discovery.

In capillary electrophoresis frontal analysis (CE/FA), which seems to be favorable in

that kind of studies, the premixed protein–ligand sample is injected as a large plug and

separated during electrophoresis into the free ligand and protein/protein–ligand complex

zones, due to the differences in their mobilities. Albumin, the most abundant plasma protein,

binds primarily acidic drugs, which in physiological conditions are negatively charged.

However it can be difficult to apply CE/FA for anionic drugs because the dissimilarities in

mobility between plasma proteins and those drugs are small. Cyclodextrins (CDs), which

possess a hydrophilic exterior and hydrophobic interior cavity, are able to selectively interact

with the guest molecule changing its mobility and successfully repelling the protein and

retaining the drug.

In present study, the effect of different kinds of CDs on the apparent mobility of the

pravastatin was investigated. Negatively charged α-cyclodextrin (α-CD), β-cyclodextrin (β-

CD), neutral- 2-hydroxypropyl- β-cyclodextrin (HP-β-CD) and positively charged quaternary

β-cyclodextrin (QA β-CD) were used as complexation agents and mobility modifiers. The

optimum concentration of CD in the separation solution was chosen and then CE/FA was

performed to determine the ratio of unbound to CD-bound drug. Two kinds of capillaries,

uncoated and polyacrylamide-coated, and increasing concentrations of bovine serum albumin

(BSA) were examined in drug- protein separation.

The strongest binding of pravastatin to QA β-CD was observed, and in presence of this

CD the drug and BSA were completely separated, but only in mixtures with low protein

concentration. Better results were obtained with neutrally-coated capillary due to suppression

of the interaction of the inner wall and protein, however the time of analysis was significantly

longer because of electroosmotic flow suppression.

The addition of CD affected the mobility and apparent solubility of pravastatin as well

as the sensitivity of the method and allowed to implement FA to determine the binding of this

anionic drug to plasma proteins.

135





Acknowledgments

This work was financed by the European Union from the resources of the European


by JCET-UJ, No POIG.01.01.02-00-069/09).

136




P 79

OXIDATIVE METABOLISM OF CARVEDILOL BASED ON

ELECTROCHEMICAL SIMULATION COUPLED TO LIQUID

CHROMATOGRAPHY-MASS SPECTROMETRY (EC-LC/MS/MS)

K. Kuś1,2

, A. Gonciarz1,2

, A. Kij2, J. Suraj

1,2, M. Szafarz

1,2, A. Zakrzewska

2, M.

Walczak1,2


Bobrzyńskiego 14, 30-348 Kraków, Poland 2Department of Pharmacokinetics and Physical Pharmacy, Jagiellonian University Collegium

Medicum, Medyczna 9, 30-688 Kraków, Poland


Carvedilol is a widely prescribed anti-hypertensive and anti-anginal drug, that has β-

adrenergic blocking and vasodilating activities. It is known to be metabolized into various

metabolites both by oxidation and conjugation pathways in the liver.

In the early stages of drug discovery and development, so-called metabolic stability of

drug candidates is commonly assessed, using in vitro models, like subcellular fractions

(microsomes, cytosol, S9) or liver cells. The electrochemical mimicry of oxidative reactions

lately has become a complementary tool in metabolism investigation.

The aim of this study was to simulate the oxidative metabolism of carvedilol using

electrochemistry, separate and structures elucidation of potential metabolites using

LC/MS/MS technique.

The system used for the generation of oxidation products consists of a thin-layer cell

(ReactionCell™, Antec-Leyden) controlled by a ROXY™ potentiostat (Antec-Leyden). The

electrochemical oxidations were conducted on Glassy Carbon (GC) and Magic Diamond™

(MD) working electrodes with HyREF™ as reference electrode. All LC/MS measurements

were performed on the triple quadrupole mass spectrometer QTRAP API 5500 (AB SCIEX)

coupled to UFLC Nexera (Shimadzu).

In order to obtain an overview of the oxidation behavior of carvedilol, the EC unit was

directly coupled to the ESI-QTRAP mass spectrometer and mass voltammograms were

recorded. The protonated signal of carvedilol (m/z 407) decreased considerably with

increasing voltage and simultaneously signals from potential metabolites were observed, what

implies oxidation of the drug. To confirm molecular formulas and structure of oxidation

products MS, MS2 and MS3 spectra of forming molecules were used. Additional information

about the properties and quantities of the generated products has been obtained applying LC

to separate carvedilol and all possible metabolites.

Acknowledgments

This work was financed by the European Union from the resources of the European


by JCET-UJ, No POIG.01.01.02-00-069/09).

137





P 80

METHOD DEVELOPMENT FOR SEPARATION OF ARACHIDONIC

ACID METABOLITES FROM BIOLOGICAL SAMPLES AND THEIR

QUANTIFICATION USING LC-MS/MS TECHNIQUE

A. Kij1, A. Gonciarz

1,2, K. Kuś

1,2, J. Suraj

1,2, M. Szafarz

1,2, A. Zakrzewska

1, M.

Walczak1,2


Bobrzynskiego 14, 30-348 Krakow, Poland 2Department of Pharmacokinetics and Physical Pharmacy, Jagiellonian University Collegium

Medicum, Medyczna 9, 30-688 Krakow, Poland


Introduction: Arachidonic acid can be metabolized by cytochrome 450 (CYP450),

cyclooxygenase (COX) or lipoxygenase (LOX). The CYP450 and LOX pathways generate

the hydroxyeicosatetraenoic acids (HETEs). Cyclooxygenase enzyme is involved in

prostanoids and thromboxanes production, while the LOX delivers leukotrienes (LTs). These

oxidative metabolites of arachidonic acid, known as eicosanoids, play a crucial role in

inflammation [1]. The aim of this study was to separate and quantify 15(S)-HETE, 12(S)-

HETE, 5(S)-HETE, LTB4, LTD4, LTE4, LTC4, EXD4, EXE4, EXC4 using LC-MS/MS

technique.

Methods and Results: The quantitative determination of investigated compounds was

accomplished employing UFLC Nexera (Shimadzu) coupled with the triple quadrupole mass

spectrometer QTRAP API 5500 (AB SCIEX). In the first stage of method development, the

mass transitions for each studied metabolites were estimated with nitrogen used as a collision

gas. The compound-dependent parameters such as the collision energy (CE), declustering

potential (DP), entrance potential (EP) and collision cell exit potential (CXP) were optimized

using a manual tuning process with continuous infusion of standard solution by syringe pump.

During the FIA optimization step the ion source-dependent parameters, like spray voltage, ion

source temperature, curtain and nebulizing gases values, were estimated. Electrospray

ionization process was accomplished in the positive ion mode, except for the HETEs and

LTB4, where the negative polarization was performed. The best chromatographic conditions

were achieved applying Acclaim RSCL C18 (2.1x100, 2.2μm, 120Å) analytical column. The

mobile phases consisted of 0.01% acetic acid in water and acetonitrile were delivered in

gradient elution. The next step prior to quantification of eicosanoids was isolation of the

analytes from the biological fluids including rat urine or serum. In this work, the sample

purification process was conducted using solid-phase extraction (SPE), liquid-liquid

extraction (LLE) and protein precipitation. For analytes extraction, the adequate volumes of

ethyl acetate, dichloromethane, methyl tert-butyl ether, hexane and chloroform were

examined. In case of the protein precipitation, the appropriate volumes of methanol,

acetonitrile, acetone and ethanol were investigated. The best linearity range for all studied

eicosanoids was obtained using acetonitrile as a precipitant without evaporation step (100-

1500 pg/mL of urine). In these methods, different organic solvents were used to obtain both

the best extraction efficiency and high sensitivity.

138





References

1. Anthony J. Blewett, Deepi Varma, Tiffany Gilles, Joseph R. Libonati, Susan A. Jansen,

J. Pharm. Bio. Anal., 46 (2008) 653-662.

Acknowledgments This work was supported by the European Union from the resources of the European


by JCET-UJ, No POIG.01.01.02-00-069/09).

139




P 81

FAST GC COMBINED WITH A HIGH-SPEED TRIPLE QUADRUPOLE

MASS SPECTROMETER FOR THE ANALYSIS OF UNKNOWN AND

TARGET CITRUS ESSENTIAL OIL VOLATILES

F.A. Franchina1, P.Q. Tranchida

1, I. Bonaccorsi

1, L. Schipilliti

1,

P. Dugo1,2

, L. Mondello1,2





[email protected], [email protected], [email protected]

The present research description is focused on the evaluation of a high-speed triple

quadrupole mass spectrometer (QqQ MS), carried out under moderately-fast GC conditions.

The MS device is capable of operation under high-speed GC conditions, in both full-scan

(maximum scan speed: 20 000 amu/s) and multiple reaction monitoring (MRM) modalities.

Furthermore, the QqQ system can generate full scan and MRM data at the same time, also in a

very rapid manner.

A fast method was developed for the: (i) qualitative analysis of untargeted essential oil

constitents, and (ii) the quali/quantitative analysis of targeted ones, namely three preservatives

(o-phenylphenol, butylated hydroxytoluene, butylated hydroxyanisole). The MS system

generated a more-than-sufficient number of data points/peak for both identification and

quantification purposes. The level of sensitivity, reached through the MRM mode widely

exceeded the requirements of current legislation. Method validation, related to the targeted

analysis, was also performed.

Acknowlegments




140










P 82

FAST SPME GC COUPLED WITH HIGH-SPEED TRIPLE

QUADRUPOLE MASS SPECTROMETRY FOR THE UNTARGETED

AND TARGETED ANALYSIS OF TEA SAMPLES

F.A. Franchina1, M. Zoccali

1, P.Q. Tranchida

1, R. Costa

1, P. Dugo

1,2, L. Mondello

1,2






The present investigation is focused on the evaluation of a novel high-speed triple

quadrupole mass spectrometer (QqQ MS), under moderately-fast GC conditions. The QqQ

MS system is capable of operation under high speed GC conditions, in both full-scan

(maximum scan speed: 20,000 amu/sec) and multiple reaction monitoring (MRM) modalities.

Furthermore, the QqQ MS system can generate full scan and MRM data simultaneously, also

in a very fast manner. Analytes extraction was performed by using headspace solid-phase

microextraction (SPME), for different blends of tea. A fast GC-MSMS method was developed

for the: I) qualitative analysis of unknown tea constituents, and II) MRM quali/quantitative

analysis of targeted ones, namely thirty phyosanitary analytes. The QqQ MS system generated

a satisfactory number of data points/peak for both qualitative and quantification purposes. The

level of sensitivity, reached through the MRM application, widely satisfied the requirements

of current regulations.

Acknowlegments




141










P 83

DETERMINATION OF BIO-ACTIVE POLYPHENOL COMPONENTS

OF PEPPER FRUIT CAPSICUM ANNUUM L. BY LIQUID

CHROMATOGRAPHY COUPLED TO MASS SPECTROMETRY

DETECTION

M. Mokhtar1, J. Soukup

2, P. Donato

3,4, F. Cacciola

5,6, P. Dugo

3,4, A. Riazi

7, P. Jandera

2,

L. Mondello3,4

1Department of Biology, University of Hassiba Benbouali, 02000 Chlef, Algeria

2Department of Analytical Chemistry, University of Pardubice, Studentská 573, 53210

Pardubice, Czech Republic

E-mail: [email protected] 3Centro Integrato di Ricerca (C.I.R.), University Campus Bio-Medico of Rome, Via Álvaro del

Portillo 21, 00128 Roma, Ital. 4Dipartimento di Scienze del Farmaco e dei Prodotti per la Salute, University of Messina,

Viale Annunziata, 98168 Messina, Italy 5Dipartimento di Scienze dell'Ambiente, della Sicurezza, del Territorio, degli Alimenti e della

Salute, University of Messina, Viale F. Stagno d'Alcontres 31, 98166 Messina, Italy 6Chromaleont s.r.l. A start-up of the University of Messina, c/o Dipartimento di Scienze del

Farmaco e dei Prodotti per la Salute, University of Messina, viale Annunziata,

98168 Messina, Italy

E-mail: [email protected] 7Department of Biotechnology, University of Mostaganem, 27000 Mostaganem, Algeria

In recent years phenolic compounds have attracted the interest of researchers because

they show promise of exerting beneficial effects toward human health, as related e.g. to the

antioxidant and antimicrobial activities. Polyphenols are a group of secondary metabolites,

which are synthesized by plants as a result of adaptation to biotic and abiotic stress

conditions; since they cannot be produced by the human body, they must be taken in mainly

through the daily diet. Knowledge about the nutritional and therapeutic role of dietary

phenolic antioxidants is essential for the development of functional foods, which refers to the

improvement of conventional foods with added health benefits. On the other hand, detailed

chemical composition of foods considered to be functional is needed, and the main goal of the

chemistry of natural compounds is screening for promising biologically active substances of

plant origin.

This research was aimed to achieve the full characterization and quantification of

flavonoids and other phenolic components extracted from Capsicum annuum pepper fruits, to

correlate to the antimicrobial activity tested. An RPLC-DAD-MS system was optimized,

employing a partially porous octadecylsilica column as stationary phase. Determination was

carried out on the basis of the complementary information obtained from their migration

times, diode array spectra, MS ions and MS/MS fragments.

142






P 84

PAH’S CONCENTRATIONS IN PM10, PM2.5 AND PM1 PARTICULATE

FRACTION IN THE AIR

I. Jakovljević, G. Pehnec, V. Vađić

Institute for Medical Research and Occupational Health, Ksaverska cesta 2, Zagreb, Croatia


Polycyclic aromatic hydrocarbons (PAHs) are products of incomplete combustion or

organic matter pyrolysis. Many have been identified as potential carcinogens. Benzo(a)pyrene

(BaP) is the most commonly measured PAH due to its inevitable presence. Chromatographic

methods for PAH analyses in environmental media have been developed and evaluated

extensively over the past few decades, including HPLC and GC analysis. Liquid

chromatography with fluorescence detection is suitable for samples with low concentrations

of anthracene and perylene, due to their selective and sensitive fluorescence.

Investigations of PAHs in the air are mostly focused on those bounded at particles

with an aerodynamic diameter of less than 10 μm (PM10). Nowadays, smaller particles attract

the most interest. This paper presents the results of PAH measurements in particle fractions

PM10, PM2.5, PM1.

Daily samples of particle fractions were collected on a quartz filter during two winter

months (January and February 2011) and two summer months (June and July 2011). The

samples were analyzed with HPLC fluorescence detection and changeable excitation and

emission wavelength. The samples were analyzed for the following PAHs:

benzo(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, and

benzo(a)pyrene. The recoveries of PAHs were determined by adding a known amount of the

PAH to a blank filter. The average recoveries of all of the analyzed PAHs varied from 85% to

92%.

Average mass concentrations of BaP in the winter period were 2.258 ng/m3 in the

PM10 fraction, 2.221 ng/m3

in the PM2.5 fraction, and 1.474 ng/m3

in the PM1 fraction. In the

summer period, BaP concentrations were 0,073 ng/m3 in the PM10 fraction, 0.069 ng/m

3 in the

PM2.5 fraction, and 0,060 ng/m3

in the PM1 fraction. Comparison of BaP concentrations in

PM10 and PM1 showed that more than 60% of BaP concentrations in the winter period and

more than 80% in the summer period were in the PM1 particle fraction. Other PAHs showed a

similar tendency to BaP.

143





P 85

NEW TRITERPENOIDS AND PHYTOSTEROLS IN VEGETABLES

IDENTIFIED BY TLC-MS

K. Naumoska1, M. Puklavec

1, A. Albreht

1, I. Vovk

1,2

1National Institute of Chemistry, Hajdrihova 19, SI-1001, Ljubljana, Slovenia 2EN-FIST Centre of Excellence, Dunajska 156, SI-1000 Ljubljana, Slovenia


Plant cuticular waxes, among other compounds, are composed of two groups of

secondary metabolites, triterpenoids (C30) and phytosterols (C18-C30). The scientific interest

for studying them increased in the last decades, especially with the new evidences of their

anticancer, antiinflamatory, antimicrobial, antiviral, hypocholesterolemic and other activities.

Until now, there are little or no information on the presence of triterpenoids and phytosterols

in certain vegetables. The reason that they have not been extensively studied is their structural

similarity, which makes their identification laborious. So far, the TLC methods described in

the literature do not offer efficient separation among the certain compounds. Triterpenoid

isomers show the same fragmentation patterns in MS2 spectra and differ only by the relative

intensities of some characteristic product ion mass peaks. Therefore, a combination of

powerful chromatographic and mass spectrometric methods is required for their accurate

identification. For that purpose, a new TLC method was developed, which enabled efficient

separation between the compounds of interest, and therefore was suitable for TLC-MS

hyphenation. Moreover, a new MS method for their identification was optimized, to give

stable and reproducible MS and MS2 spectra for the analyzed standard compounds. Wax

extracts obtained from various vegetables such as: salads, cabbage, eggplant, zucchini,

peppers, spinach and mangold were further analyzed on the content of the target triterpenoids

and phytosterols. For the first time, the MS and MS2

spectra, obtained by TLC-MS analysis,

enabled discrimination among the structurally related compounds. As a result, new

triterpenoids and phytosterols in edible vegetables are reported.

144





P 86

URINARY NICOTINE METABOLITES IN PATIENTS WITH

MULTIPLE SCLEROSIS DETERMINED BY

PLANAR CHROMATOGRAPHY

K. Tyrpień, C. Dobosz, A. Damasiewicz-Bodzek

Department of Chemistry, Faculty of Medicine and Division of Dentistry in Zabrze,

Silesian Medical University in Katowice, Jordana 19 street 41-808 Zabrze, Poland


Multiple sclerosis (MS) is a serious, one of the most common disorders of the nervous

system and is still uncharted course and etiology. Modern medicine characterized MS as a

disease of multifactorial, complex pathogenesis. The studies were designed to test some new

dependencies and determine exposure to selected environmental factors, including passive

and active smoking using separation techniques in biological materials levied noninvasively.

The aim of these research was to determine by TLC technique of major nicotine

metabolites in urine samples (earlier described) taken from people with multiple sclerosis.

Urine samples (28) were collected non-invasively from patients with multiple sclerosis

living in the region of Upper Silesia, Poland. The control samples (21) came from healthy

people - family and friends of people with MS. Urine, after alkalinisation were shaken with

Amberlite XAD-2. Next, after centrifugation, the fractions containing nicotine and its

metabolites were desorbed with a mixture of acetone and dichloromethane (1 +3, v/v) and the

obtained extracts were separated on the plates coated with a stationary phase C18 (5x10 cm,

Macharey Nagel)). The mobile phase consisted of acetonitrile and water (88:12 v/v) with the

addition of sodium octasulphonate. Visualization of the chromatograms was performed at a

wavelength = 254nm, but using densitometer for quantitative analysis at = 260nm at

linear scan type.

Including active and passive smoking 42.86% of the respondents were exposed to

ingredients of tobacco smoke. The concentration of cotinine in the urine samples analyzed,

however no statistically significant in comparison with control group, ranged from 16.1 ng

/mL to 453.15 ng /mL, while the concentration of trans-3'-hydroxycotinine was relatively

higher 25.5 - 1011ng/mL. Ratio of main nicotine metabolites was assessed.

These results were supported by KNW-1-070/P/2/0 project.

145





P 87

ASSESSMENT OF EXPOSURE TO PAHs PEOPLE WITH MULTIPLE

SCLEROSIS

C. Dobosz, K. Tyrpień, M. Szumska

Department of Chemistry, School of Medicine and Division of Dentistry,

Medical University of Silesia in Katowice, Jordana 19, 41-808 ZABRZE (Poland)


Polycyclic aromatic hydrocarbons (PAHs) present one of the most significant

pollutants. They originate from both natural and anthropogenic sources. The primary

environmental exposure to PAHs is caused by the inhalation of cigarette smoke, automobile

exhaust and consumption of grilled, barbecued or smoked food. Many epidemiological studies

have demonstrated that exposure to PAHs is associated with increasing rates of various

diseases such as cardiovascular, cancer or respiratory diseases. A cross-sectional study was

conducted with the use of a questionnaire regarding patients’ life style and concerning

exposure to certain xenobiotics.

The aim of study was the assessment of exposure to PAHs and determination of 1-

hydroxypyrene as a biomarker in urine of smokers and non-smokers patients with multiple

sclerosis from Upper Silesia region (Poland).

HPLC method in reversed phase system, using Luna C18 (250 x 3 mm; 5, 100A;

Phenomenex) column and gradient elution (methanol with water) as well as fluorescence

detection was used for the determination of 1- hydroxypyrene in case of MS patients’ urine,

after enzymatic hydrolysis followed by solid phase extraction (C18 columns).

Statistical analysis was performed by the use of the procedures available in the

Statistica 8.0 software. Daily calibration curves for 1- hydroxypyrene determined by HPLC

with fluorescence detection were performed, because the detection was changeable with time.

That is why the determination of hydroxy- PAHs needs to be optimised. Obtained results for

MS patients were compared with those for control group - healthy relatives and friends from

the same region as well as the evaluation of life style was also performed based on the survey

data.

These results were supported by KNW-1-070/P/2/0 project.

146





P 88

DEVELOPMENT OF A NEW LC/DAD/MS/MS METHOD FOR

SIMULTANEOUS QUANTITATION OF 11 ACTIVE INGREDIENTS IN

VARIOUS DIETARY SUPPLEMENT MATERIALS AND

FORMULATED PRODUCTS FOR MENOPAUSAL SYMPTOMS

T. Buhač1, A. Mornar

2, M. Sertić

2, B. Nigović

2

1Centar Ars Pharmae, Kesterčanekova 2b, 10 000 Zagreb

2Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovačića 1, 10 000 Zagreb


Approximately two-thirds of women that reach menopause develop menopausal

symptoms, primarily vascular instability, hot flashes, insomnia and rapid heartbeat. Hormone

therapy was long considered the first line of treatment for vasomotor symptoms. However,

given the results of the Women’s Health Initiative, many women are reluctant use exogenous

hormones for symptomatic treatment and are turning to dietary supplement products for relief.

Therefore, an increased number of dietary supplement products used for treating women in

menopause have appeared in the European market. This growth highlights the need for a

systematic evaluation of quality of these products.

The aim of our work was to propose a new method for simultaneous determination of

11 active ingredients (genistein, daidzein, formononetin, biochanin A, 8-prenylnaringenin,

xanthohumol, isoxanthohumol, secoisolariciresinol, matairesinol, apigenin and casticin) in

various botanical dietary supplements for menopausal symptoms using LC/DAD/MS/MS

technique.

All experiments were done using Agilent 1100 Series LC/MSD Trap system. A

Symmetry C18 (150 x 4.6mm, 3.5 μm) analytical column was used for the chromatographic

separation. The mobile phase consisted of 0.1% formic acid in ultrapure water and of 0.1%

formic acid in acetonitrile. A gradient separation was performed at 25.0 ± 0.1 °C with the

flow rate set at 1 mL/min. After UV detection the split (1:2) to mass spectrometric (MS)

detector was used. In order to maximize method sensitivity the electrospray ionization source

temperature was optimized at 350 °C while the capillary voltage was set at 3.5 kV. Nitrogen

was used as drying gas and nebulizer gas at a flow fate of 10 L/min and a pressure of 20 psi.

The full scan mass spectra were acquired over a range of m/z 100-600. Helium was used as

collision gas and the MSn studies of analytes were carried out.

The proposed method was successfully applied for quantitative determination of 11 active

ingredients in various dietary supplement materials as well as formulated dietary supplements

products supplied from dietary supplement manufactures, local health food stores and

community pharmacies.

147





P 89

FAST ALLICIN SCREENING IN GARLIC DIETARY SUPPLEMENTS

BY DIRECT INJECTION TANDEM MASS SPECTROMETRY

M. Sertić, A. Mornar, B. Nigović

Department of analytics and control of medicines, Faculty of Pharmacy and Biochemistry

University of Zagreb, Ante Kovačića 1, Zagreb, Croatia


Allium sativum, commonly known as garlic, is well known for its culinary use, as well

as medicinal purposes. Many health claims are contributed to garlic, such as reducing

atherosclerosis and high cholesterol levels, inhibition of vascular calcification, antibacterial,

antiviral, antifungal and even anticancer activity. The main active ingredient, allycin, is

produced during the crushing of the garlic by the interaction of alliin with an enzyme,

aliinase.

Due to its numerous health benefits, garlic dietary supplements are among top three

best-selling herbal dietary supplements. Unfortunately, not all products give their intended

value for money. This is due to many quality control problems dietary supplements have,

especially in terms of having non or to little of the active ingredient labelled on the product or

having undeclared active ingredients, registered as drugs. When it comes to garlic the problem

is even more interesting because of the fact that the intact plant does not contain allicin and

the technological production procedure plays a key role. Many reports have been made about

garlic dietary supplements that did not contain allicin.

Therefore the aim of this work was to develop a sensitive and rapid method to

determine the presence of allicin in dietary supplements.

The samples were injected directly into a mass spectrometer using a syringe pump.

The MS conditions were: capillary voltage 3.5 kV, source temperature 350 °C, nebulizer gas

(nitrogen) at 15 psi and 5 L/h. The MSn studies were carried out by keeping the collision

energy at 30%.

The allicin showed the pseudomolecular ion at m/z 163. The ESI-MS/MS spectrum

showed the presence of the two major fragment ions at m/z 121 and 73. Ion at m/z 121 is

assumed to be formed due to the loss of propylene from protonated allicin, while ion at m/z 73

is assigned to the allyl sulfide cation.

The method was applied for the analysis of garlic dietary supplements commercially

available on the market. In all three examined samples, allicin was found. The method has

proven to be fast, reliable, selective and sensitive and can be used by the manufacturers as

well as the regulatory agencies for screening of these popular dietary supplements.

148





P 90

ANALYSIS OF PAHs IN STORMWATERS COLLECTED FROM

AIRPORT AREA

A.M. Sulej, Ż. Polkowska, J. Namieśnik

Gdansk University of Technology, Faculty of Chemistry, Department of Analytical Chemistry,

Narutowicza 11/12 Str. 80-233 Gdańsk, Poland


Increases in aviation developments have serious consequences on the all parts of

environment. Significant problems in this respect are the stormwaters that form when

precipitation or atmospheric deposits flush the airport surface during its operation. A variety

of chemical agents get into the environment with airport runoff waters [1-4].

The present work deals with the determination of polycyclic aromatic hydrocarbons

(PAHs) found in airport runoff waters from four international airports. This work presents the

investigation and optimization of the conditions under which the PAHs, can be isolated using

different type of solid-phase extraction (SPE) and finally determination by gas

chromatography coupled with mass spectrometry (GC-MS). The data presented here provide

a useful baseline for airports—a source of information essential for assessing the hazard to

surface and ground waters in their vicinity. It is therefore crucial that samples of runoff waters

from airports should be monitored and analysed for the largest possible number of

contaminants. Only monitoring of this kind can supply data that can be used to assess the

intensity of the effects of airport operations on the biotic and abiotic environment.

Keywords: airport runoff water, stormwater, PAHs, SPE, GC-MS

References:

1. A.M. Sulej, Ż. Polkowska, J. Namieśnik, Crıt. Rev. Env. Sci. Tec. 42 (2012) 1691-

1734.

2. A.M. Sulej, Ż. Polkowska, J. Namieśnik, Sensors 11 (2011) 11901- 11920.

3. A.M. Sulej, Ż. Polkowska, J. Namieśnik, Crit. Rev. Anal. Chem. 41 (2011) 190-213.

4. A.M. Sulej, Ż. Polkowska, J. Namieśnik, Pol. J. Environ. Stud. Vol.3 (2012), 725-739.

149





P 91

SAMPLE PREPARATION PROCEDURES FOR DETERMINATION OF

ANTI-COROSIVE AGENTS IN AIRPORT RUNOFF WATERS

A.M. Sulej, Ż. Polkowska, J. Namieśnik

Gdansk University of Technology, Faculty of Chemistry, Department of Analytical Chemistry,

Narutowicza 11/12 Str. 80-233 Gdańsk, Poland


Despite the many postives emerging from the intensification of air traffic, all airport

activities pollute the environment. One of the most important problems in this respect are the

runoff waters (stormwater) that form when precipitation or atmospheric deposits flush the

airport surface. Airport runoff can contain high concentrations of various pollutants like

benzotriazoles, the environmental levels of which have to be monitored.

The subject of this research are sample preparation and chromatographic procedures of

determination of anticorrosive compound found in airport runoff waters from international

airports in Europe. The aromatic fractions were separated by solid phase extraction (SPE) and

solid phase microextraction (SPME) and analysed by gas chromatography with mass

spectrometry (GC-MS) and two-dimensional gas chromatography coupled to time-of-flight

mass spectrometry (GCxGC-TOF-MS). Target analytes concentrations are discussed in terms

of sampling location, seasonal variation, origin. The results obtained during the

implementation of this research programme will constitute a source of information essential

for assessing the extent of the threat to surface and ground waters.

Keywords: airport runoff water, benzotriazols, SPE, SPME, GC-MS, GCxGC-TOF-MS

References

1. A.M. Sulej, Ż. Polkowska, J. Namieśnik, Crıt. Rev. Env. Scı. Tec. 42 (2012) 1691-

1734.

2. A.M. Sulej, Ż. Polkowska, J. Namieśnik, Sensors 11 (2011) 11901-11920.

3. A.M. Sulej, Ż. Polkowska, J. Namieśnik, Crit. Rev. Anal. Chem. 41 (2011) 190-213.

4. A.M. Sulej, Ż. Polkowska, J. Namieśnik, Pol. J. Environ. Stud. Vol.3 (2012), 725-739.

150





P 92

PARTIAL-FILLING AFFINITY CAPILLARY ELECTROPHORESIS

IN STUDY OF DNA-INTECALATOR INTERACTIONS

M. Ruzicka1,2

, M. Jirasek2, M. Cizkova

2, F. Teply

2, D. Koval

2, V. Kasicka

2

1Faculty of Science, Charles University in Prague,

Albertov 6, 128 43 Praha 2 2Institute of Organic Chemistry and Biochemistry, v.v.i., Academy of Sciences of the Czech

Republic, Flemingovo nám. 2., 166 10 Praha 6, Czech Republic E-mail: [email protected]

The understanding of biomolecular interactions is important in drug research. In this

work, we would like to demonstrate application of Partial-Filling Affinity Capillary

Electrophoresis (PF-ACE) in the study of DNA-intercalator interactions.

Recently synthesized compounds from the group of helquats (helical extended diquats)

[1] were proposed to have a significant ability to intercalate into DNA. Rough affinity

estimation indicated that their binding constants are comparable to those of ethidium bromide

and thus considered to be carcinogenic agents. On the other hand, if successfully targeted onto

tumor cells, intercalating compounds are widely used as anticancer drugs.

In this work, using the PF-ACE technique, dissociation constant of the analyte-ligand

complex is calculated from migration time changes of analyte depending on the length of the

ligand zone in the capillary [2]. Hydroxypropyl-cellulose coated capillary with internal

diameter 50 μm and 30/40 cm effective/total length was used. Studied intercalating

compounds were injected as a ligand plug of various length (0 – 3 min at 20 mbar) and

positioned hydrodynamically in thermostated part of capillary. DNA oligonucleotide was

injected as an analyte. During the electrophoresis, studied compounds migrate in opposite

direction and interact in temperature controlled region. To suppress observed drifts in

separation system during the measurements, we developed suitable experimental sequence.

We investigated interactions between DNA oligonucleotides and well characterized

intercalator ethidium bromide as a model compound. Behavior of double stranded DNA

molecule during electrophoresis and its impact on the stability of DNA-intercalator complex

was thoroughly examined. Double stranded structure of short DNA oligonucteotides (12 – 20

mer) showed tendency to dissociate while passing through non-cooled part of capillary and to

re-associate when reached thermostated region. We also focused on possible ligand adsorption

on capillary wall due to degradation of coating with resulting increment in electroosmotic

flow (EOF) velocity. Positively charged ethidium bromide manifested observable interactions

with capillary wall, thus partially introducing electrochromatography effect into experiment.

Measured complex dissociation constants were partially EOF velocity dependent.

Subsequently, we used PF-ACE technique to investigate affinity of several newly

synthesized helquates to DNA oligonucleotides and calculate dissociation constants of the

complexes.

This work was supported by grant 6294/2012 of Grant Agency of the Charles University,

grants, P206/12/0453, 13-17224S and 13-32974S, of Czech Science Foundation, and by

research project RVO 61388963 of the Academy of Sciences of the Czech Republic.

151





References

1. L. Severa, D. Koval, P. Novotna, M. Oncak, P. Sazelova, D. Saman, P. Slavicek, M.

Urbanova, V. Kasicka, F. Teply, New J. Chem., 34, 1063-1067 (2010).

2. M. Nilsson, V. Harang, M. Bergstrom, S. Ohlson, R. Isaksson, G. Johansson,

Electrophoresis, 25, 1829-1836 (2004).

152




P 93

EVALUATION OF AN INTEGRATED PROTOCOL FOR NON-

TARGET ANALYSIS OF HYDROPHOBIC AND POLAR

CONTAMINANTS IN AQUEOUS SAMPLES USING GAS

CHROMATOGRAPHY/MASS SPECTROMETRY AND LIQUID

CHROMATOGRAPHY/TIME-OF-FLIGHT MASS SPECTROMETRY

S. Koprivica, I. Krizman, I. Mikac, I. Senta, S. Terzić, M. Ahel

Division of Marine and Environmental Research, Rudjer Boskovic Institute,

Bijenicka c. 54, 10000 Zagreb, Croatia


The assessment of hazardous chemical contamination in the aquatic environment is

a challenging task. Wastewater and surface water samples contain extremely complex

mixtures of thousands of individual contaminants having different chemical properties and

ecotoxicological characteristics. Nevertheless, the prevailing approach, applied in the

assessment of the chemical status of a water body, is the determination of relatively

limited number of target contaminants using specific methods, optimized for the pre-

selected priority chemicals of concern. Recently, a more comprehensive approach, based

on effect-directed analysis (EDA), which combines the complementary advantages of

chemical and biological tools, has been proposed to address the problem of water quality

and pollutant prioritisation. In contrast to target analyses, applied in the monitoring of

mandatory priority contaminants, the EDA approach comprises application of protocols

suitable for a broad-spectrum analysis of non-target contaminants. Such protocols must

fulfill two prerequisites: exhaustive extraction of a wide range of physico-chemically

different contaminants and efficient fractionation scheme allowing combination of two

complementary techniques: gas chromatography/mass spectrometry (GC/MS) for

hydrophobic nonpolar contaminants and liquid chromatography/mass spectrometry

(LC/MS) for the polar contaminants. The application of such complex protocols for

qualitative work is relatively common in the literature, however their application in

quantitative manner is an extremely challenging task. The aim of this paper is therefore the

evaluation of an integrated protocol for the analysis of a wide range of organic

contaminants, including classic contaminants such as polycyclic aromatic hydrocarbons

and pesticides as well as several classes of emerging contaminants (e.g. pharmaceuticals,

surfactants and their transformation products).

153





P 94

MOLECULARLY IMPRINTED SILICA MICROPARTICLES FOR

SELECTIVE SPE CONCENTRATION AND SEPARATION OF CHOSEN

PHYTOESTROGENS

A. Chrzanowska, A. Poliwoda, P.P. Wieczorek

Opole University, Faculty of Chemistry, Pl. Kopernika 11a, 45-040 Opole, Poland


Phytoestrogens are defined as non-steroidal compounds and the members of

polyphenolic secondary plant metabolites. Because of their structural similarity to natural

endogenous hormones, they can have a serious influence on their proper functioning

(transport, absorption, etc.) what can lead to disruptions in organism’s hormonal management.

In order to monitor the presence and the concentration of phytoestrogens in the

environment and in living organisms it is necessary to consider the fact that in analyzed

samples these compounds occur in very low concentrations, in the presence of many

interferents and their direct analysis with available chromatographic and/or electrophoretic

techniques is rather impossible. According to this fact, there is a need to apply pre-treatment

methods that would allow for easy and effective isolation, concentration and determination of

phytoestrogens.

One of the potentially useful methods that can be applied is using molecularly

imprinted silica microparticles (MISMs) as the sorbents in solid phase extraction (SPE). Their

main advantages are: easy synthesis, the ability of a selective recognition of particular

compounds or chemical, thermal and mechanical stability.

Our goal was to develop the method of MISMs synthesis and to evaluate the

effectiveness of such synthesized sorbents when urine samples were analyzed. The

experiments that were carried out on the standard solutions revealed that such sorbents gave a

desirable selectiveness and efficiency. The preliminary investigation of urine samples showed

as well that it was possible to clean-up the matrix in an effective way and to obtain satisfying

limits of detection and quantification.

Anna Chrzanowska is a recipient of a Ph.D. scholarship under a project funded by the

European Social Fund.

154





P 95

DEVELOPMENT AND VALIDATION OF AN HPTLC METHOD FOR

THE QUANTIFICATION OF APIGENIN-7-O-GLUCOSIDE IN

GERMAN CHAMOMILE FLOWERS (MATRICARIA RECUTITA L.)

AND ITS APPLICATION FOR DETECTION OF ADULTERANTS

E. Ariburnu1, I. Vovk

2,3, E. Yesilada

1

1Yeditepe University, Faculty of Pharmacy, Department of Pharmacognosy and

Phytotherapy, Kayısdagı Cad., Atasehir, 34755, Istanbul, Turkey 2National Institute of Chemisty, Labotratory

for Food Chemistry, Hajdrihova 19. SI-1000

Ljubljana, Slovenia 3EN-FIST Centre of Excellence, Dunajska 156, SI-1000 Ljubljana, Slovenia


German Chamomile (Matricaria chamomilla L.), one of the most popular and well-known

medicinal plant, has been consumed for centuries as a safe remedy to alleviate the symptoms

of gastro-intestinal complaints, inflammatory and nervous problems etc. However, lay people

collect different kinds of flowers with similar appearance as chamomile believing that it is M.

recutita L. According to European Pharmacopoeia (Ph.Eur.) only M. recutita L. can be used

for medical purposes and apigenin-7-O-glucoside is addressed as a biomarker component for

the quantitative analysis of German chamomile samples by HPLC. As an alternative analysis

method a new validated HPTLC densitometric method was developed for quantification of

the apigenin-7-O-glucoside in German chamomile samples. Separations were performed on

HPTLC silica gel 60 NH2 F254 plates using a developing solvent EtOAc-HCO2H-AcH-H2O

(30:1.5:1.5:3, v/v/v/v). Quantitative evaluation was performed by densitometry in absorption

reflectance mode at 340 nm. The developed HPTLC method was validated for linearity,

specificity, precision, accuracy, limits of detection and quantification, stability and robustness

and was finally applied to the analyses of the real samples and the detection of adulterants.

The obtained results were compared to those obtained by the HPLC method described in Ph.

Eur., with a separation on a C18 ODC column (5.0 µm, 250 mm x 4.6 mm, i.d.) and a gradient

elution by phosphoric acid-water (0.5:99.5 v/v) as mobile phase A and phosphoric acid-

acetonitrile (0.5:99.5 v/v) as mobile phase B. The developed HPTLC method was found to be

useful for the quantitative analysis of apigenin-7-O-glucoside in German chamomile flowers

and can be used as an alternative method to the HPLC method in Ph. Eur.

155





P 96

GAS CHROMATOGRAPHY METHOD FOR DETERMINATION OF

SULFUR COMPOUNDS IN NATURAL GAS

I. Belamarić, J. Jelić-Balta, M. Brusić

INA d.d. SD Istraživanje i proizvodnja nafte i plina, Služba laboratorijskih istraživanja


Determination of sulfur compounds in natural represents important analysis for

production and distribution of hydrocarbons as well as the control of transportation systems.

Natural gas processing requires constant monitoring of the quantity of sulfur compounds due

to protection of producing wells equipment, construction of gathering and transportation

system and environmental protection. It is known that sulfur compounds are aggressive,

corrosive, and most importantly above certain concentrations (>100 ppm) endanger human

life.

Natural gas that INA produce for distribution and sale needs to satisfy the quality

requirements specified in the internal standard “INA N-52-003: Natural Gas”. INA’s

specification defines limit values of hydrogen sulfide and mercaptanes as well as test methods

for determination of these compounds.

Until now, the sulfur compounds were determined by indirect iodometric titration

method: ASTM D2385-81(1990): Test Method for Hydrogen Sulfide and Mercaptan Sulfur in

Natural Gas (Cadmium Sulfate-Iodometric Titration Method) (Withdrawn 1995) or ISO 6326-

3:1989: Natural gas -- Determination of sulfur compounds -- Part 3: Determination of

hydrogen sulfide, mercaptan sulfur and carbonyl sulfide sulfur by potentiometry. Those

methods are not suitable and are abandoned in the European Union because of harmful

chemicals that are used in the methods.

Generally accepted method for determination of sulfur compounds in natural gas is gas

chromatography (direct identification). Method for gas-chromatography analysis of sulfur

compounds adopted in INA’s laboratory is ISO 19739:2004: Natural gas -- Determination of

sulfur compounds using gas chromatography.

This poster presents a detailed development of gas chromatography method for

determination of sulfur compounds in natural gas on Agilent 7890 system equipped with

capillary column, pulsed flame photometric detector for low level concentrations and thermal

conductivity detector for higher lever concentrations of sulfur compounds.

156





P 97

EVOLUTION OF AMINO ACIDS AND BIOGENIC AMINES IN

CANNONAU WINE FERMENTATION PROCESSES

F. Congiu, C. Tuberoso

Department of Life and Environmental Sciences, University of Cagliari, Via Ospedale, 72,

09124 Cagliari (CA)


The aim of this work was to develop and validate an accurate, precise and sensitive

analytical method for the determination of amino acids (AA) and biogenic amines (BA) and

to apply it to quantify them during the fermentation processes of a typical Sardinian red wine

(Cannonau). BA are compounds of different nature produced in food and beverages by

several microorganisms during the fermentation and can have direct or indirect effects on the

human vascular and nervous systems. AA and BA are difficult to be analyzed simultaneously

because of their structural diversity. A LC-Fluo analysis on derivatized AA and AB was

developed. The reaction mixture, consisting of pH 9.3 borate buffer, sample (wine or

standards), norvaline (internal standard) and dansyl chloride (derivatization agent) was left for

30 min at 40 °C in ultrasonic-bath and later centrifuged 12000 rpm for 10 min. The analyses

were carried out by a LC system fitted with Luna C18 (100x4.60mm, 3µm) column, using

acetonitrile and pH 4.0 buffer acetate as mobile phase with a gradient elution program.

Fluorimetric detector wavelengths were set up at 293 nm (Ex) and 492 nm (Em). 34

compounds were separated and dosed using calibration curves. Method was validated with

coefficient of correlation, linearity range, recovery, repeatability, reproducibility, LOD, and

LOQ. The developed LC-Fluo method allowed to easily determine various amines and in the

analyzed grape and wine, 23 compounds were identified. AA as Ala, Phe, Gln, Arg, Glu and

Pro were found in high quantities. Phe was the most predominant AA reaching 468 mg/L and

3132 mg/L in grape and wine, respectively. BA were detected in concentration < 30 mg/L,

except GABA that showed concentration of ca. 168 mg/L in grape and 243 mg/L in wine. The

results of this work demonstrated a low level production of BA due to proper winemaking

processes.

157






P 98

OIL AND BITUMEN HYDROCARBON DISTRIBUTION FROM

WELL A, SAVA DEPRESSION, CROATIA, ANALYZED BY GAS

CHROMATOGRAPHY

D. Keškić, N. Trkulja

INA-Industrija nafte d.d, Exploration & Production Business Department, Exploration &

Production Research Laboratory Department, Lovinčićeva b.b., Zagreb, Croatia


Chromatography is a widely spread analitical technique in the oil industry, and has

great significance as a separation, quantitative and qualitative technique for analysis of

complex hydrocarbon mixtures as oils, bitumens and their fractions.

„Whole“ oil is a concept in gas chromatography that displays the entire hydrocarbon

distribution as it includes n-alkanes, iso-alkanes, naphthanes and aromatics. That kind of „oil

fingerprint“ is an indication of bulk organic matter type. Molecular range of hydrocarbons in

oil varies from structures with two carbon atoms (C2) to 45 carbon atoms (C45), dependent of

the origin of organic matter, oil maturity and influences of degradation.

Gas chromatography analyses are carried out on Varian 3900, on fused silica column

50 m x 0,25 mm x 0.4μm, CP-SIL 5CB. Three oil samples from two different depths, and five

bitumen samples, gained by rock extraction in chloroform, were examined. Hydrocarbon

distribution of analyzed oil is from C2 to C36, while for bitumen is from C13.

Complete alkane distribution and molecular ratio are used for oil and bitumen

correlation (CPI, iso-alkanes/n-alkanes, Pr/n-C17, Ph/n-C18, Pr/Ph). Pristane (Pr) i phytane

(Ph) are both iso-alkanes originated from chlorophyll which in their mutual relationship and

in regard to their belonging n-alkanes serve to determine sedimentation environment

conditions as well as maturity and type of original organic matter. Carbon preference index

(CPI) can be used to help determine origin, depositional environment and thermal maturity of

precursor.

Parameters like heptane, isoheptane index, aromaticity and LER (light end ratio) used

to characterize oil can be obtained by detailed whole oil analyses. Heptane values are used to

differentiate paraffinic from nonparaffinic oil, while isoheptane values indicate the degree of

sedimentation microbiological activity. LER points to mature or degraded oils.

According to hydrocarbon relationships in oils from well A, located in central Croatia,

the oils originate from algal organic matter deposited in suboxic to anoxic conditions,

preserved in sandstones. Heptane values place them as normal to mature oils, but on the other

hand isoheptane values indicate some characteristics of biodegraded oils. Light aromatics are

mostly removed from the samples shown by low aromaticity index what can be interpreted as

water rinsing. Bitumens extracted from marl were deposited in oxic conditions determined

from Pr/Ph ratio = 3.09. Meanwhile, in sandstone extracts phytane dominates over pristane

(Pr<Ph), meaning that original organic matter was deposited in suboxic to anoxic conditions

correlating to analyzed oil. Dominance of even over odd n-alkanes in the area C23-C33 (CPI =

1.71) can be seen in bitumen extracted from marls meaning that it comes from higher plant

material which is not common for oils found in central Croatia.

158





References

1. Barić, G. (2006): Naftna geokemija, INA-Industrija nafte d.d., 228 p, Zagreb.

2. Hunt, J.M. (1996): Petroleum Geochemistry and Geology. W.H. Freeman and

Company, San Francisco, 743 p.

3. Thompson, K.F.M. (1983): Classification and thermal history of petroleum based on

light hydrocarbons, Geochim. Cosmochim. Acta, 47, 303-316p

159




P 99

DEVELOPMENT OF RP-HPLC METHOD FOR DETERMINATION OF

SIMETHICONE IN ORAL SUSPENSION

K. Klepac, D. Žgela

PLIVA CROATIA Ltd., Generics Research and Development,

Prilaz baruna Filipovića 29, 10000 Zagreb, Croatia


Although excipients are pharmacologically inactive substances, they could be

pharmaceutically active and therefore can affect all aspects of drug product performance. The

identification and quantitation of excipients in pharmaceutical products can be a great

challenge due to the chemical diversity and complexity of the sample matrix.

Simethicone is a complex mixture of silicon-based polymer polydimethylsiloxane

(PDMS, dimethicone) and hydrated silica gel. It is often used as an excipient in

pharmaceutical formulations, with a role as an anti-foaming agent.

A new, gradient, RP-HPLC method has been developed for the determination of the

polydimethylsiloxane (PDMS) component of Simethicone, an anti-foaming agent in oral

suspension. Due to the absence of a chromophore for UV detection, an evaporative light

scattering detector (ELSD) was used for detection and quantification of PDMS.

Gradient RP-HPLC method was developed using C18 column (5 um particles size),

using gradient of acetonitrile and chloroform as a mobile phase. The flow rate was set at 1.0

mL/min. For ELSD detection, gas pressure was set to 60.0 psi and the drift tube temperature

to 40°C. The retention time of PDMS was 13.7 min.

The sample solution for determination of PDMS in oral suspension was obtained by

extraction of simethicone with dichloromethane from suspension and subsequent drying of the

extract with sodium sulfate.

The RP-HPLC method was found to be suitable for quantifying PDMS component of

simethicone in an oral suspension with detection by the ELSD. PDMS was efficiently

extracted with dichloromethane from oral suspension by a single extraction. A specific

method in the presence of other excipients and active substance was developed.

160





P 100


CHROMATOGRAPHY –

PART I: CONVENTIONAL QSRR APPROACH

P. Žuvela, M. Novak, Š. Ukić, T. Bolanča


University of Zagreb, Marulićev trg 20, 10000 Zagreb, Croatia


In this work, quantitative structure-retention relationships (QSRR) methodology was

applied in ion chromatography of sugars. Models obtained with QSRR can be applied to

predict the retention times of unknown compounds, identify the most meaningful structural

descriptors or gain insight into the separation mechanism operating in the chromatographic

system.

Till now, QSRR model were generally applied for description of components’

retentions under highly predefined conditions (i.e. isocratic elutions or different gradients with

highly limited variability). In this specific case, partial least square QSRR models were

developed for 10 different isocratic elutions. The predicted retention times were used as input

to gradient elution model based on isocratic elution data, which provided retention prediction

for unlimited number of different gradient profiles. The developed synergy of models was

tested through retention prediction of new set of sugars and the good predictive results were

obtained (relative error < 25%).

The shown characteristics indicate the applied methodology as excellent choice for

preliminary prediction when new unknown components need to be separated in ion

chromatographic system.

161





P 101


CHROMATOGRAPHY –

PART II: ARTIFICIAL INTELIGENCE QSRR APPROACH

A. Vlahović, M. Novak, Š. Ukić, T. Bolanča




Quantitative Structure-Retention Relationships, QSRR, is a common name for

methodology that is predicting chromatographic retention time explicitly on basis of analytes’

molecular-structure. Application of these models can generally short the selection-time for

propriate method when dealing with other similar analytes, or can replace the time-consuming

“trial and error” approach in method optimization.

In this work, artificial intelligence was applied in order to develop good QSRR model.

The genetic algorithm was used for selection of the most appropriate molecular descriptors,

i.e. descriptors with the highest content of useful information. Artificial neural networks,

which are generally known as universal approximators, were taken as QSRR models.

The QSRR models were developed for several isocratic elutions, all indicating good

prediction ability.

The results of QSRR prediction were used for development of isocratic retention

model, providing prediction over whole domain of isocratic elutions.

In order to enable prediction for gradient elutions, a gradient model based on isocratic

data was applied. Although the results indicated slight systematic error, the prediction

remained satisfactory good.

162





P 102

DEVELOPMENT OF UPLC METHOD FOR DETERMINATION

OF IMPURITIES IN DRUG PRODUCT USING

QUALITY BY DESIGN (QbD) APPROACH

S. Mutka

PLIVA CROATIA Ltd., Generics Research and Development,

Prilaz baruna Filipovića 29, 10000 Zagreb, Croatia


Analytical method development is very important part of drug substance or drug

product development. In most cases method development is a time-consuming process since

methods are usually developed by changing only one variable at time.

Quality by design (QbD) approach to method development uses a systematic screening

to evaluate more variables at time, such as different stationary phases, different organic

modifiers or pH of mobile phase. QbD approach uses statistical design of experiments (DoE)

to develop a robust method design space, which defines the experimental region in which

changes to method parameters will not significantly affect the results.

This approach to analytical method development has two major advantages over

conventional method development. It brings considerable time savings for scientists. Also,

robustness is being checked out as the method is being developed.

A new, gradient UPLC method has been developed for the determination of impurities

in injectable suspension. Method development was performed using the Empower software

and Fusion AE Method Development software. The sample used for method development was

selectivity solution. The initial screening varied BEH Phenyl, BEH C18 and CSH C18

columns and acetonitrile and methanol as organic modifiers. From obtained results it was

concluded that BEH C18 column and methanol will be used for further method development.

The initial method was optimized in terms of column temperature, gradient slope and

flow rate. The defined criteria were number of peaks and resolution between them. After

analyzing obtained data the method that showed the best separation was chosen for further

routine analysis of impurities in drug product. Final method uses C18 column with 1.7 μm

particle size, with a mobile phase consisting of water and methanol. The flow rate is set to 0.3

mL/min, column temperature to 20 °C and UV detection is carried out using DAD detector.

Above described analytical UPLC method was developed faster than conventional

analytical method. Robustness parameters were tested during development. The method was

found to be suitable for routine quantitative determination of impurities in drug product.

163





P 103

HPLC-DAD-FLD DETERMINATION OF VETERINARY

PHARMACEUTICALS IN WASTEWATERS OF PHARMACEUTICAL

INDUSTRY WITH AND WITHOUT PRECOLUMN DERIVATIZATION

BY FLUORESCAMINE

D. Ašperger, V. Tišler, D. Mutavdžić Pavlović, S. Babić,

A.J.M. Horvat, M. Kaštelan-Macan




Many veterinary pharmaceutical industries use the surface water as a recipient for

wastewater effluent after treatment. Those wastewaters are usually loaded with very high

concentration of pharmaceuticals, especially with veterinary antibiotics. Classical wastewater

treatment plants are not suitable for removing pharmaceuticals from wastewaters of

pharmaceutical industry, so, they can get into the receiving surface waters with the effluents.

From these reasons it is necessary to monitor pharmaceuticals in wastewaters before and after

treatment. In this purpose it was developed a robust, simple, realistic and practical analytical

method capable of simultaneous determination of target compounds [sulfonamides

(sulfaguanidine, sulfadiazine, and sulfadimidine), a sulfonamide synergist (trimethoprim), a

tetracycline (oxytetracycline), fluoroquinolones (ciprofloxacin, enrofloksacin, norfloksacin)

and local anesthetic procaine], by HPLC with diode array (DAD) and fluorescence (FLD)

detectors.

A liquid chromatographic method with fluorimetric detection is proposed for the

determination of pharmaceuticals in wastewater samples because of highly fluorescent

fluoroquinolones (FQ). Limit of detection for FQ is almost ten times lower with HPLC-FLD

then with HPLC-DAD. Other investigated pharmaceuticals need to be derivatized with a

fluorescamine solution in acetonitrile (5 mg/10 mL acetonitrile) and acetic buffer for

determination on HPLC-FLD. The fluorescent compounds are detected at different λex/λem

(nm/nm) values: sulfonamides, trimethoprim, oxytetracycline and procaine at 405/495 and

fluoroquinolones at 277/445.

The analytes are preconcentrated by solid phase extraction (SPE) using Strata-X

cartridges and acetonitrile as eluent. The chromatographic separation is performed on a

Synergy Fusion-RP18 80 Å, 150 mm × 4.6 mm, particle size 4 m column with a gradient

elution program based on binary mixtures of solvents A (0.1% acetic acid in water) and B

(0.1% acetic acid in acetonitrile). The flow rate was 1.0 mL/min.

The performance characteristics of the SPE-HPLC-FLD method were established by

validation procedure. Selectivity, linearity, limits of detection (LOD) and quantification

(LOQ), precision and recovery were studied.

The described method is applied to the determination of the investigated

pharmaceuticals in pharmaceutical industry wastewater.

Acknowledgements

This work has been supported by Croatian Ministry of Science, Education and Sports

Project: 125-1253008-1350 Development of advanced analytical methods for

164





pharmaceuticals determination in the environment and Bilateral project HR-SLO:

Determination of toxicity and physico-chemical properties of pharmaceuticals.

165




P 104

SEDIMENT SAMPLE PREPARATION FOR CHROMATOGRAPHIC

DETERMINATION OF VETERINARY PHARMACEUTICALS

D. Ašperger1, S. Prašnički

1, M. Gavranić

1, D. Drljača

1,

D. Mutavdžić Pavlović1, S. Babić

1, I. Mikac

2, M. Ahel

2

1Department of Analytical Chemistry, Faculty of Chemical Engineering and Technology,


E-mail: [email protected] 2Division for Marine and Environmental Research, Rudjer Boskovic Institute,

Bijenička cesta 54, 10000 Zagreb, Croatia

Pharmaceuticals used in human and animal medicine are very heterogeneous class of

emerging organic pollutants and they have been determined in several matrices such as

sewage, ground water or sediments. Sediments act as a potential receiver for many hazardous

chemicals, including the pharmaceuticals, which have been emitted to surface waters. Due to

their physico-chemical properties, many of these pharmaceuticals sorbed to sediment. These

sediments may become a secondary source of pollution when they are eroded and transported

further downstream. From these reasons it is necessary to monitor the pharmaceuticals in

sediment with adequate analytical techniques, in most cases that are chromatographic

methods. Chromatographic methods demand good and delicate sample preparation procedure

because of complex sediment matrix.

So, the purpose of this research is optimization and comparison of different extraction

methods [extraction by agitation (EA), microwave assisted extraction (MAE), ultrasound

solvent extraction (USE), pressurized liquid extraction (PLE) and matrix solid-phase

dispersion (MSPD)] of target analytes from sediment. Investigated chemicals are veterinary

pharmaceuticals: antihelmintics (albendazole, febantel and levamisole), local anesthetics

(lidocaine and procaine), macrolide antibiotic tylosin, and glucocorticosteroids

(dexamethasone and hydrocortisone). Actually, the main aim of this work was to extract

mentioned pharmaceuticals from sediment samples with high efficiency and less matrix

effect. The extraction of listed compounds from sediments has been traditionally performed

using solvent extraction (Soxhlet) or steam distillation techniques. Traditional methods of

extraction are labour-intensive, time consuming and require large volumes of solvents. So,

alternative methods like EA, MAE, USE, PLE and MSPD offers quick and simple extraction,

but also require less volumes of solvents. All extraction methods are optimized. The goal of

optimization process was to find the optimal composition of elution solvent, solvent volume,

suitable sorbent (for MSPD), extraction duration, speed and revolutions per minute (for EA),

temperature, pressure (for MAE and PLE).

Finally results showed that EA was the least effective than other methods, highest

matrix effect was observed for EA and USE, the most effective methods were PLE and MAE,

and the least matrix effect was observed for MSPD on C18 sorbent for investigated

pharmaceuticals. Extraction efficiency was determined by high performance liquid

chromatography with diode array detector (HPLC-DAD) on the column InterSustain™

250x4.6 mm, 5 µm; GL Sciences, Japan at 30 oC and injection volume was 30 μL. The

analysis was conducted using eluent A (0.01 % formic acid in MilliQ water) and eluent B

(0.01 % formic acid in acetonitrile) in gradient elution mode. The flow rate was 0.5 mL/min.

166





Acknowledgements

This work has been supported by Croatian Ministry of Science, Education and Sports

Project: 125-1253008-1350 Development of advanced analytical methods for

pharmaceuticals determination in the environment and Bilateral project HR-SLO:

Determination of toxicity and physico-chemical properties of pharmaceuticals.

167




P 105

NANOSILVER PARTICLES VERSUS IONIC SILVER –

CHROMATOGRAPHIC DIFFERENTIATION AND QUANTIFICATION

I. Vinković Vrček1, F. Mlynek

2, W. Goessler

2

1Institute for Medical Research and Occupational Health,

Ksaverska cesta 2, 10 001 Zagreb, Croatia

E-mail: [email protected] 2Karl-Franzens University Graz, Institute for Chemistry - Analytical Chemistry,

Stremayrgasse 16/ III, A-8010 Graz, Austria


Research and development in the area of new nanomaterials containing silver

nanoparticles (AgNPs) are seen as having an enormous economic potential for new drugs and

medical treatments, electronics, environmental remediation, surface treatments, etc. owing to

their excellent antibacterial properties s. To date, little is understood concerning the

distribution, accumulation, and target organs of AgNPs in organisms. Moreover, there have

been some reports indicating that AgNPs move into the brain by traversing the blood-brain

barrier. The size of a particle and its differentiation from ionic metal form are key parameters

to be determined when attempting to predict the fate, and potential toxicological impact, of

silver NPs released into the environment. Analytical techniques based on mass spectrometry

(MS) can provide both elemental and molecular information. Recently, ICP-MS has been

demonstrated to be a highly valuable tool for ultrasensitive detection and characterization of

metalloid-containing NPs. Size distribution analysis of NPs and differentiation of

nanoparticulate and ionic form in polydispersed samples could be achieved with liquid

chromatography. Several recent studies presented the separation and quantitative

determination of Ag species using high-performance liquid chromatography (HPLC) coupled

interfaced to a multi-element detector (ICPMS). This could be a novel bioanalytical tool that

enables investigations into the behaviour and fate of a range of inorganic silver NP's in ‘real-

world’ situations. We present a validation and evaluation of the proposed methods for

differentiation and quantification of ionic and nano form of silver. Reproducibility and

robustness of the LC-ICPMS methodology were evaluated for a range of silver ions and

AgNPs in different sample matrices in order to test the suitability for routine applications.

168






P 106

THE USE OF MICROFLOW UHPLC AS A WAY TO SOLVENT USAGE

IN PESTICIDE SCREENING OF FOOD SAMPLES BY LC-MS/MS

S. Lock, E. Loge

AB SCIEX UK, Pheonix House

Centre Park, Warrington

WA1 1RX

United Kingdom


Traditionally in pesticide screening of food samples, sample are prepared using

generic extraction procedures, like QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and

Safe) and then analyzed by LC/MS/MS or GC/MS/MS. Usually when analyzing samples, LC

flow rates are in excess of 500 µl/min and are used in combination with high pressures with

smaller particle size HPLC columns to maintain sharp peaks and fast chromatography. These

flow rates produce fast speeds and excellent peak shapes and results, but have a draw back in

that they require higher volumes of organic solvent. The consumption of HPLC organic

solvents, such as acetonitrile and methanol, is a growing cost of analysis and its disposal has

an environmental impact. Therefore, ways to reduce solvent consumption in pesticide residue

testing will be beneficial to the environment and reduce running costs of a testing lab.

Here we present new data using microflow LC (using an ExpressHT™-Ultra running

at 10 µL/min) in combination with a LC-MS/MS method developed on a AB SCIEX

QTRAP® 4500 system utilizing the Scheduled MRM™ algorithm in combination with fast

polarity switching and acquisition of MS/MS spectra for compound identification. Initially

this approach has been applied to a screen of over 100 pesticides in QuEChERS food extracts

and we will present robustness and sensitivity data on several matrices to show its

applicability in food analysis.

169






P 107

CAN LCMSMS BE USED IN HORSE MEAT DETECTION?

S. Lock, E. Loge



WA1 1RX

United Kingdom


Following the UK Food Standards Agency (FSA)’s announcement in January that

horse and pig DNA had been identified in beef products sold by several supermarket chains,

further testing across Europe and beyond has revealed widespread incidences of such food

contamination. However, most testing methods are based on detection of species-specific

DNA in meat, using the polymerase chain reaction (PCR) – which does not detect or identify

proteins. This is a concern because DNA can be easily disrupted or removed during standard

meat processing and food manufacturing. As a result, horse tissue or other contaminants

remain undetected in food samples, despite strong presence of the contaminating proteins. An

alternative protein-based method, ELISA, can be used to complement DNA testing, but this

method has limitations, including that it detects only one part of the protein and not multiple

protein markers.

The LC-MS/MS based method presented offers a more accurate and reliable approach

to meat speciation than PCR or ELISA-based techniques or other indirect methods, and also

allows for the detection of veterinary drug residues in the same analysis, which is not possible

by ELISA or PCR. The method is developed using an Eksigent micro LC system coupled

with an AB SCIEX QTRAP® 5500 LC/MS/MS system and uses multiple reaction monitoring

(MRM) to detect peptide markers for horse and is capable of providing sequence information

by acquiring a product ion scan for each triggered MRM which can be used to further confirm

the peptide’s / proteins and therefore the species identity. Using the same extraction and

LCMSMS method it is also capable of simultaneously of detecting veterinary drug residues

by adding additional MRM experiments. The method has been shown to be capable of

simultaneously detected phenylbutazone below 10 µg/kg as well as a 1% contamination of

horse meat in beef.

170






P 108

EXPLORING THE SENSITIVITY DIFFERENCES FOR TARGETED

PEPTIDE QUANTIFICATION IN THE LOW FLOW RATE REGIME

S. Lock, E. Loge, C. Hunter, R. van Soest, X. Zhu



WA1 1RX

United Kingdom


[email protected], [email protected]

In this study, sensitivity & throughput differences between nano and micro flow rates

for targeted quantitation are explored. Separations were performed on an Eksigent ekspert™

nanoLC 425 system and cHiPLC® system (Eksigent, USA) using four different flow regimes.

Column diameters and flow rates used were: 75 μm cHiPLC® column at 300 nL/min, 200 μm

cHiPLC® column at 1 μL/min, 300 μm microflow LC column at 4 μL/min, 500 μm

microflow LC column at 10 μL/min. Concentration curves on a set of ten tryptic peptides

were analyzed and the lower limits of quantification (LLOQ) for each peptide at each column

size was measured using MRM acquisition.

The LLOQs for a set of ten tryptic peptides were measured using the 4 different

columns and flow rates. A nanoflow source with glass capillary tips was used for the 75 and

200 µm ID column experiments and the high flow source with a low dead volume 25 µm ID

hybrid electrode was used for the 300 and 500 µm ID column experiments. The average

increase in LLOQ relative to running a 75 µm ID column at 300 nL/min was measured for

each peptide at each column diameter. For the 200 µm ID column running at 1 µL/min, a 2.5

fold decrease in sensitivity was observed. Moving to the 300 µm ID column at 4 µL/min, a 3x

difference in sensitivity was seen. Finally, 4x difference in sensitivity was observed when the

500 µm ID column at 10 µL/min was compared to the same experiment on a 75 µm ID

column. However the separation was performed in less than half the time with the 500 µm ID

column compared to the 75 µm ID column (7 minutes at 10 µL/min vs. 18 minutes at 300

nL/min). Therefore, as flow rate increases, increased robustness and throughput can be

obtained with just a small decrease in sensitivity.

171









P 109

GAS CHROMATOGRAPHIC/MASS SPECTROMETRIC TECHNIQUES

FOR THE ANALYSIS OF DECOMPOSITION PRODUCTS AND GASES

EMITTED FROM LITHIUM-ION CELLS UNDER EXTREME

CONDITIONS OF USE

C. Kanakaki1, E. Rosenberg

1, A. Trifonova

2, J.-H. Han

2

1Vienna University of Technology, Institute of Chemical Technologies and Analytics,

Getreidemarkt 9, 1060 Vienna, Austria 2AIT Austrian Institute of Technology GmbH, Mobility Department, Giefinggasse 2, 1210 Vienna,

Austria


In order to reduce CO2 emissions and fossil fuel dependence, the development and use

of electric vehicles play a key role. Achieving performances acceptable for a wide range of

users, and not only for the early adapters, requires the development of innovative energy storage

with higher energy density, higher safety and lower environmental impact. To serve this purpose,

larger lithium-ion batteries are developed, increasing the necessity of the risk assessment for

unexpected chemical hazards, due to misuse or extreme operating conditions. The aim of this

research work is to develop a standardized test method for the characterization of the

thermally and electrochemically driven degradation products of the carbonate-based

electrolytes and particularly the gaseous emissions of the lithium-ion cells, focusing on the

improvement of safety and environmental issues.

The qualitative and quantitative analysis of the chemical compounds generated during

cycling operation and misuse conditions, will be performed by gas chromatography/mass

spectrometry. Apart from conventional GC/MS analysis, two different approaches of GC/MS

will also be used, in order to enhance the time resolution. The vacuum outlet GC/MS

technique [1], utilizing short, mega-bore columns, will be applied first, and will be followed

by the Multiplex-GC/MS [2] technique, enabling also on-line measuring of the decomposition

products of Lithium-ion cells.

Precise analysis of compounds produced in cases of misuse or accidents can be

achieved with the implementation of the aforementioned GC/MS techniques. This issue is of

great importance, particularly since the potential hazards of gas emissions from lithium-ion

cells used under extreme conditions have not yet been studied systematically. Consequently,

the results of this work can be used for future optimizations on the components of the

batteries, contributing to the development of highly efficient, safe and environmentally

friendly Li-ion batteries.

Financial support of this study, provided by the Austrian Research Promotion Agency

(FFG, Project ‘‘SiLithium’’, Project Number 835790), is gratefully acknowledged.

References

1. van Deursen M., Janssen H. G., Beens J., Lipman P., Reinierkens R., Rutten G.,

Cramers C.: J. Microcol. Sep. 12 (12) (2000) 613-622.

2. Phillips J. B.: Anal. Chem. 52 (4) (1980) 468-478.

172





P 110

LIQUID MEMBRANE EXTRACTION TECHNIQUES AS A SAMPLE

PRETREATMENT PROCEDURE IN ANALYSIS OF ENDOCRINE

DISRUPTING COMPOUNDS FROM FOOD SAMPLE MATRICES

A. Poliwoda, K. Orłowska, K. Bury, P.P. Wieczorek

Opole University, Faculty of Chemistry, Pl, Kopernika 11a, 45-040 OPOLE (Poland)


Endocrine disrupting compounds (EDCs) are synthetic chemicals that when absorbed

into the body, either mimic or blocks hormones and disrupts the body’s normal functions.

Recently, many plant and animal species (especially aquatic organisms) are showing signs of

ill health due to exposure to endocrine disrupting chemicals. Therefore the necessity to

determine the presence and concentration level of those compounds in various complex

sample matrices is required. The complexity of those samples forces the need of pretreatment

stage proceeding. It means that isolation of the target substance and/or removing of the

interfering molecules before the final analysis have to be proceed. This step must also bring

the analyte of interest to a suitable concentration (detection) level, because in most cases they

are present in the samples in trace amounts.

Therefore in this work the developed in our laboratory sample cleanup methodologies,

based on liquid membrane extraction, will be presented. The presentation will focused on

analysis of alkylphenolic endocrine disrupters (i.e.: bisphenol A, 2-phenylphenol) from

various food complex matrices like honey, juices, etc.. The description will include the

comparison of applied sample pretreatment methods, taking into account both selectivity and

efficiency of enrichment procedure, the influence of selected extraction factors (composition

of membrane, donor and acceptor phase), level of detection and quantification, and

occurrence of studied analytes in analysed materials.

Katarzyna Orłowska is a recipient of a Ph.D. scholarship under a project funded by the

European Social Fund.

173





P 111

STUDY OF A ZINC BASED GELLAN GUM CHROMATOGRAPHIC

STATIONARY PHASE USING EXPERIMENTAL DESIGN

A.I.C. Gonçalves1, L.A. Rocha

1, J.M. Dias

2, A. Sousa

1, L.A. Passarinha

1


6201-506 Covilhã, Portugal 2University of Cambridge, Cambridge System Biology Centre, Sanger Building, 80

Tennis Court Road, Cambridge CB2 1GA, England


To improve the purification efficiency and in order to decrease the use of resources in

chromatographic processes the development of new matrices is increasingly important. To

accomplish this aim, different types of materials are used in the preparation of

chromatographic matrices namely natural or synthetic polymers. Thus, gellan gum is a natural

anionic polysaccharide with potential to be a chromatographic matrix, since it shares some

properties with common chromatographic stationary phases. These properties are its porosity

and hydrophilicity, and since this polymer is negatively charged it enables interaction with

specific biomolecules in anionic exchange conditions. So, with the present work it is intended

to take advantage of these properties of gellan in order to prepare an anionic chromatographic

gel. To find the best gel formulation to be applied in the chromatographic assays, stability

experiments were made. In order to facilitate the optimization of this process, experimental

design tool was applied, allowing a better understanding of the relative importance of all the

components in the matrix. Subsequently, to test the applicability of the matrix, several

chromatographic assays with three model proteins were made (bovine seric albumin (BSA),

α-chymotrypsin and lysozyme). The results showed that the retention occurred in function of

the net charge of each protein in buffer pH 6.2 and the elution was performed by increasing

the ionic strength. Finally, to better characterize and to compare this matrix with commercial

resins, the dynamic binding capacity was studied. The obtained values for the gellan

stationary phase were 3.9 mg/mL and 17.4 mg/mL, at 10% and 50% of breakthrough,

respectively. Concluding, this research work shows that gellan gum is a promissory

chromatographic matrix, exploring ionic interactions, being applied in different purification

strategies and getting the best benefit from its use at low cost, since this polymer may be

bacterially produced.

174





P 112

DETERMINATION OF BIOGENIC AMINE, HISTAMINE, IN SAMPLES

OF CANNED FISH USING HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY

M. Bevardi, J. Bošnir, G. Horvat, S. Serdar, D. Brkić

Institute of Public Health: „Dr. A. Štampar“, Mirogojska cesta 16, Zagreb



Aim and purpose: To validate a method for the determination of histamine in samples of

canned fish and to calculate the amount of histamine in selected samples. And also obtained

results compared with current regulations.

Materials and method: Samples were analyzed by liquid chromatography with DA detector.

The method was validated on the following parameters: limit of quantification, recovery,

precision, linearity, ruggedness. We analyzed 258 samples of canned fish during 2012/13.

year.

Results: All validation parameters have satisfied the required criteria. Of the total number of

analyzed samples of canned fish, 30% were positive for the presence of histamine, and in 30%

of samples concentration exceeded the maximum levels prescribed by Commission

Regulation (EC) No 2073/2005 on microbiological criteria for foodstuffs. The highest

recorded concentration was 223 mg kg-1

which was two times higher than maximum levels set

by EC Regulation. The remaining 70% of the analyzed samples were below the limit of

quantification specified for this method.

Conclusion: Histamine is biogenic amine that is mostly found in fish, wine, beer, sauerkraut,

cheese, fermented meat products, soy products. Histamine is characteristic for oily fish: tuna,

macherel, sardines and anchovies. In humans, histamine can cause allergic reactions. About

95% of histamine entered into the human body through the digestive system and intestinal

were acetylated and degraded by bacteria whereby after that histamine become inactivated.

Concentration from 60 to 100 mgkg-1

cause poisoning. As canned fish is relatively common

foods in the diet it is very important to performe regular controls on the presence of this toxin.

175









P 113

DETERMINATION OF SPECIFIED POLYCHLORINATED

BIPHENYLS IN PAPER AND BOARD

S. Jelušić, A. Galić, L. Barušić, D. Brkić, J. Bošnir

Institute of Public Health: „Dr. A. Štampar“, Mirogojska cesta 16, Zagreb



Aim and purpose: To determine the mass fraction of specific polychlorinated biphenyls in

selected samples of paper and board and on the basis of the results obtained to determine their

safety in accordance with existing laws and regulations.

Materials and methods: The analysis used samples of paper and cardboard. Samples are

extracted with boiling ethanolic potassium hydroxide solution. An aliquot of the extract

obtained is mixed with water and subjected to liquid-solid partitioning on a disposable C18

solid phase extraction cartridge followed by elution with hexane. Identification and

quantification of polychlorinated biphenyls each was determined by gas chromatography

using an electron capture detector. We analyzed 20 samples of paper and 5 samples of board.

Samples were analyzed for the presence of seven specific polychlorinated biphenyls:

- 2,2',5-Trichlorobiphenyla (PCB 18); 2,4,4

'-Trichlorobiphenyl (PCB 28)

- 2,2',5,5'-Tetrachlorobiphenyl (PCB 52); 2,2',4,5,5'-Pentachlorobiphenyl (PCB 101)

- 2,2',3,4,4',5'-Hexachlorobiphenyl (PCB 138); 2,2',4,4',5,5'-Hexachlorobiphenyl (PCB

153)

- 2,2',3,4,4',5,5'-Heptachlorobiphenyl (180) according to HRN EN ISO 15318:2001

Results: In all samples of paper and board was determined the presence of PCB 101

(2,2',4,5,5'-Pentachlorobiphenyl) with an average value of 0.48 μgkg-1

, 40% of the samples

contained PCB 153 (2,2',4,4',5,5'-Hexachlorobiphenyl), and 20% of the samples contained

traces of other specific PCBs.

Conclusion: PCBs can potentially be found in the tissues, toilet paper, napkins, diapers.

Cardboard was mainly analyzed in food packaging. PCBs are potentially carcinogenic and

require careful control and analysis. They can be found in the items for everyday use as in

children and adults that is why PCBs must be subjected to specifically control in the items

that come in contact with skin or food. In this study in all 25 analyzed samples was found that

the sum of PCBs has a lower value than those prescribed by Regulations, and thereby the

analyzed samples were found safe for human consumption.

176









P 114

INFLUENCE OF LATERAL DODECYLOXY GROUPS ON THERMAL

AND ANALYTICAL PROPERTIES OF A NEW HPLC BONDED

LIQUID CRYSTAL STATIONARY PHASE

O. Ferroukhi1, S. Guermouche

1, M.H. Guermouche

1, J.P. Bayle

2

1 Laboratoire de chromatographie, faculté de Chimie, USTHB,

Bp 32 El-Allia BEZ, Alger ALGERIA

E-mail: [email protected] 2 Laboratoire de Chimie structurale, ICMO, Bt 410, Université de Paris-Sud,

91405 Orsay-Cedex, France

A new bonded liquid crystal stationary phase (BLCSP) noted Si-3OC12 for high

performance liquid chromatography was studied. In precedent work [1,2], thermal and

analytical properties of two other bonded stationary phases noted Si-OC12 and Si-2OC12,

possessing respectively one and two lateral dodecyloxy group and differing from Si-3OC12

by the number and the position of dodecyloxy groups OC12, give satisfying results.

Si-3OC12 is obtained by coupling between Lichrospher Si 100 NH2 and mesogenic

carboxylic acids 3OC12. Characterization of LC was made with proton NMR, and the nematic

states were determined by DSC. BLCSP were characterized by solid state 13

C NMR and

elemental analysis. Surface areas were determined by the BET method.

Thermal and analytical chromatographic behaviors were investigated in normal and reversed

phases. In normal phase, separation of polyaromatic hydrocarbons (PAHs) is described using

isooctane. Using acetonitrile/water (60/40), reversed phase data of aromatic hydrocarbons are

described. Comparison study of chromatographic behaviors of the three liquid crystal bonded

phases is described to show influence of number and position of dodecyloxy groups.

Keywords: bonded liquid crystal stationary phases, transition temperatures, polynuclear

aromatic hydrocarbons

References

1. O. Ferroukhi, S. Guermouche, M.H. Guermouche, J. Courtieu, J.P. Bayle,

Chromatographia 52 (2000) 564-568.

2. O. Ferroukhi, S. Guermouche, S. Sebih, M.H. Guermouche, P. Berdague, J.P. Bayle,

J.Chromatogr.A, 971 (2002) 87-94.

177





AUTHOR

INDEX




Name Page

Acedo-Valenzuela M.I. ..................... 66, 67

Aharoni A. ................................................. 5

Ahel M. .......................................... 153, 166

Aich U. ...................................................... 9

Albreht A. .................................. 15, 17, 144

Almeida A. ............................................ 102

Almeida P. ............................................... 58

Amelin V.G. .................................. 107, 111

Ancillotti C. ........................................... 132

Ašperger D. ................................... 164, 166

Avdalović N. ............................................. 9

Babić S. ............................. 31, 35, 164, 166

Bączek T. ....................................... 126, 129

Bakry R. .................................................. 92

Balotnik T.A. ........................................... 59

Barceló D. ................................................ 27

Barušić L. .............................................. 176

Bayle J.P. ............................................... 177

Beccaria M. ............................................. 16

Beissmann S. ........................................... 25

Belal F. .................................................... 69

Belamarić I. ........................................... 156

Belka M. ........................................ 126, 129

Bendryshev A.A. ..................................... 60

Benko B. .................................................. 75

Bensebaa M. .......................................... 109

Berek D. ............................................ 23, 73

Bevardi M. ............................................. 175

Bicho D. .................................................. 57

Bielčíková N. ......................................... 128

Bocheńska A. .......................................... 98

Bognar A. .............................................. 119

Bolanča T. ..................................... 161, 162

Bonaccorsi I. .......................................... 140

Bonn G.K. ......................................... 19, 92

Boras M. .................................................... 3

Bošnir J. ......................................... 175, 176

Bouanani S. ........................................... 109

Brkić D. ......................................... 175, 176

Brulc L. .................................................... 15

Brusić M. ............................................... 156

Bruzzoniti M.C. ....................................... 36

Buchberger W. ........................................ 25

Name Page

Buczkowska K. ....................................... 76

Buhač T. ................................................ 147

Bujak R. .................................................. 87

Buratović M. ........................................... 94

Bury K. ................................................. 173

Buszewska-Forajta M. ................ 86, 87, 91

Buszewski B. ........ 8, 12, 62, 63, 68, 70, 87

Cacciola F. ...................................... 16, 142

Caramelo-Nunes C. ................................. 58

Čaušević A. ....................................... 64, 65

Ćavar S. ............................................. 64, 65

Checchini L. .................................... 29, 132

Chiuminatto U. ....................................... 29

Choma I. ................................................. 90

Chrzanowska A. .................................... 154

Ciechomska M. ....................................... 99

Cieszyńska M. ....................................... 113

Cindrić M. ............................................... 22

Ciofi L. ............................................ 29, 132

Cizkova M. ........................................... 151

Clementi L.A. ......................................... 26

Coman V. ................................................ 28

Congiu F. .............................................. 157

Copaciu F. ............................................... 28

Copolovici L. .......................................... 28

Coppini E. ............................................... 29

Corradini D. ............................................ 20

Correia I.J. .............................. 84, 117, 118

Costa J.M. ............................................. 102

Costa R.................................................. 141

Cotroneo A. ............................................. 16

Cruz C. .................................................... 46

Čurmová S. ........................................... 108

Daghir E. ................................................. 87

Damasiewicz-Bodzek A. ...................... 145

De Carlo R.M. ......................................... 36

Debeljak Ž. ............................................. 21

Del Bubba M. .................................. 29, 132

Delerue-Matos C. .................................. 102

Dias J.M. ............................................... 174




Name Page

Dobosz C. ...................................... 145, 146

Dobrzyńska E. ............................... 100, 101

Domingues V.F. .................................... 102

Donato P. ......................................... 16, 142

Drevenkar V. ......................................... 105

Drljača D. .............................................. 166

Dugo P. ...................... 16, 38, 140, 141, 142

Dvoršćak M. .......................................... 105

Dziubakiewicz E. .................................... 70

Eid M.I. ................................................... 69

El-Gamal R.M. ........................................ 69

Fabek S. ................................................... 75

Farkaš H. ............................................... 119

Ferroukhi O. .................................. 109, 177

Figueiras A. ................................... 117, 118

Fischnaller M. .......................................... 92

Franchina F.A. ......................... 38, 140, 141

Furlanetto S. .......................................... 132

Gadzała-Kopciuch R. ...................... 87, 134

Galeano-Díaz T. ................................ 66, 67

Gałęzowska G. ................................ 89, 113

Galić A. ................................................. 176

Gavranić M. ........................................... 166

Gawdzik B. ................................................ 8

Gertsiuk M. .............................................. 32

Glavnik V. ............................................... 15

Godoy-Caballero M.P. ...................... 66, 67

Goessler W. ..................................... 11, 168

Goga A. ............................................. 64, 65

Gonçalves A.I.C. ................................... 174

Gonciarz A. ................... 131, 135, 137, 138

Góra R. .................................................. 128

Gorga M. ................................................. 27

Grochowicz M. .......................................... 8

Guermouche M.H. ................................. 177

Guermouche S. ...................................... 177

Gugić M. .................................................. 83

Name Page

Hájek T. .................................................. 13

Halko R. .......................................... 95, 108

Hamdi A. ............................................... 106

Han J.-H. ............................................... 172

Hasić S. ............................................. 64, 65

Heath E. ............................................ 34, 71

Hermann U. ............................................. 14

Hewelt-Belka W. .......... 122, 123, 124, 125

Hintersteiner I. ........................................ 25

Hinz M. ................................................... 41

Horvat A.J.M. ....................................... 164

Horvat G. .............................................. 175

Hrynkiewicz K. ....................................... 70

Hukelová I. ............................................. 95

Hunter C. ............................................... 171

Hutta M. ........................................ 128, 133

Jaćkowska M. ........................................... 8

Jackowski M. .................................... 62, 63

Jackson P. ............................................... 61

Jakimska A. ................................... 124, 125

Jakovljević I. ......................................... 143

Jandera P. .................................. 13, 79, 142

Jelić-Balta J. .......................................... 156

Jelušić S. ............................................... 176

Jerković I. ............................. 44, 80, 81, 83

Jesionek W. ....................................... 78, 90

Jirasek M. .............................................. 151

Jurišić A. ................................................. 75

Kaliszan R. .......................... 86, 87, 91, 103

Kamber S. ............................................. 104

Kanakaki C. .......................................... 172

Karaseva N.M. ...................................... 107

Kasicka V. ............................................. 151

Kaštelan-Macan M. ......................... 35, 164

Kauzlarić Z. .......................................... 114

Keškić D. .............................................. 158

Kežić M. ........................................... 64, 65

Kij A. ............................ 131, 135, 137, 138

Klebovich I. .............................................. 7

Klepac K. .............................................. 160




Name Page

Koeck R. .................................................. 92

Komorowska V. .................................... 133

Konieczna L. ......................................... 129

Koprivica S. ........................................... 153

Koralewicz G. ........................................ 131

Kościelniak P. .................................... 98, 99

Kosjek T. ........................................... 34, 71

Kośliński P. ............................................. 87

Kostromskikh A.A. ................................. 55

Kot-Wasik A. ................ 122, 123, 124, 125

Koval D. ................................................ 151

Krizman I. .............................................. 153

Krzeminski R. .................................... 62, 63

Kubica P. ............................................... 123

Kuś K. ............................ 131, 135, 137, 138

Kuś P.M. ............................................ 80, 81

Kwaśniewska K. .................................... 134

Lahouel M. ............................................ 106

Lauc G. .................................................. 110

Lee S.H. ................................................... 72

Lee Y.M. ................................................. 72

Lock S. .................................. 169, 170, 171

Loge E. .................................. 169, 170, 171

Lopez-Serna R. ........................................ 27

Lysychenko G. ........................................ 32

Macová E. ................................................ 73

Maia C. .................................................... 45

Maina R. .................................................. 36

Majer-Dziedzic B. ................................... 90

Malinowska I. .................................. 78, 115

Malinowski H. ......................................... 78

Malitsky S. ................................................ 5

Mansilha C. ........................................... 102

Marcos J.C. .............................................. 58

Marijanović Z. ................................... 81, 83

Markovic L. ............................................. 61

Markuszewski M. ...................... 87, 91, 103

Markuszewski M.J. ......................... 91, 103

Marošanović B. ............................. 119, 121

Martins R. ................................................ 45

Name Page

Maslov L. ................................................ 85

Mavrinac M. ......................................... 104

Mebarki H. ............................................ 109

Medić-Šarić M. ....................................... 85

Meira G.R. .............................................. 26

Mendaš G. ............................................. 105

Mesić M. ................................................. 75

Mihaljević K. ........................................ 104

Mikac I. ......................................... 153, 166

Miliša Gregurić M. ............................... 114

Mitrevski M. ........................................... 31

Mlynek F. .............................................. 168

Mokhtar M. ........................................... 142

Mondello L. .............. 16, 38, 140, 141, 142

Moos A. .................................................. 98

Mornar A. ..................................... 147, 148

Mota E. ................................................... 46

Mutavdžić Pavlović D. ................. 164, 166

Mutka S. ................................................ 163

Nakonieczna J. ...................................... 122

Namieśnik J. . 122, 123, 124, 125, 149, 150

Naumoska K. ........................................ 144

Nicoletti I. ............................................... 20

Niedźwiecki M. ..................................... 129

Nigović B. ..................................... 147, 148

Novak B. ................................................. 75

Novak M. ...................................... 161, 162

Novokmet M. ........................................ 110

Nowaczyk A. .......................................... 87

Nowak J. ................................................. 99

Obradović M. .......................................... 80

Okenicová L. ......................................... 108

Okrągła E. ............................................... 89

Orłowska K. .......................................... 173

Oskonbaeva J. ......................................... 54

Ostrowska J. ............................................ 76




Name Page

Pandurević Todorović M. ...................... 121

Pashkova E.B. ......................................... 53

Passarinha L.A. ..................................... 174

Patejko M. ............................................... 86

Pereira P. ....................................... 117, 118

Periša M. .................................................. 31

Perko S. ............................................. 34, 71

Petrović M. .............................................. 27

Pietrzyńska M. ......................................... 43

Pirogov A.V. ................... 49, 50, 53, 55, 60

Platiša O. ................................................. 39

Poliwoda A. ................................... 154, 173

Polkowska Ż. ................................. 149, 150

Pośniak M. ..................................... 100, 101

Prašnički S. ............................................ 166

Pučić Baković M. .................................. 110

Puklavec M. ........................................... 144

Queiroz J.A. ......................... 45, 46, 57, 116

Raczak-Gutknecht J. .......................... 86, 87

Rechak H. .............................................. 106

Repec S. ................................................... 33

Riazi A. .................................................. 142

Rocha L.R. ............................................. 174

Rodić Z. ................................................... 15

Rogachev I. ................................................ 5

Rogozik M. .............................................. 41

Rohárik P. .............................................. 128

Rosenberg E. ......................................... 172

Ruzicka M. ............................................ 151

Rykowska I. ............................................. 48

Sabo K. .................................................. 121

Salkar R.A. .............................................. 40

Sample R.M. .............................................. 6

Sandra K. ................................................. 18

Sandra P. .................................................. 18

Šarolić M. .................................... 80, 81, 83

Sarvin B.A. .............................................. 60

Name Page

Sarzanini C. ............................................. 36

Schipilliti L. .......................................... 140

Senta I. .................................................. 153

Seo C.H. .................................................. 72

Serdar S. ................................................ 175

Sertić M. ....................................... 147, 148

Šestak I. ................................................... 75

Sharaf El-Din M.K. ................................. 69

Shchukina O.I. ........................................ 52

Shin K.O. ................................................ 72

Shpigun O.A. ............ 49, 50, 52, 55, 59, 60

Siluk D. ........................................... 86, 103

Simedru D. .............................................. 28

Simonovska B. .................................. 15, 17

Širac S. .................................................... 33

Šišková A. ............................................... 73

Škeříková V. ........................................... 77

Sławiński J. ........................................... 126

Smirnov R.S. ........................................... 59

Smolenkov A.D. ............................... 52, 59

Soares A. ............................................... 116

Sokolova L.S......................... 49, 50, 53, 55

Sosnowiec K. ........................................ 113

Soukup J.......................................... 79, 142

Sousa A. ............ 46, 57, 116, 117, 118, 174

Sousa F................ 46, 57, 84, 116, 117, 118

Sousa J.A. ............................................... 45

Špicnagel A.M. ....................................... 75

Štambuk J.............................................. 110

Štanfel D. ........................................ 94, 104

Stankov V. ............................................ 119

Staňková M. ............................................ 13

Sternbauer L. ........................................... 25

Stipaničev D. ........................................... 33

Stipičević S. .......................................... 105

Struck-Lewicka W. ......................... 91, 103

Strzemiecka B. ........................................ 41

Studzińska S...................................... 12, 68

Studziński M. .......................................... 78

Sulej A.M. ..................................... 149, 150

Suraj J. .......................... 131, 135, 137, 138

Šuste M. .................................................. 83

Švarc-Gajić J. .......................................... 42

Szafarz M. ..................... 131, 135, 137, 138

Szczesny D. ............................................. 91

Szeliga J. ................................................. 63




Name Page

Szewczyńska M. ............................ 100, 101

Szultka M. ......................................... 62, 63

Szumska M. ........................................... 146

Tariba B. ................................................ 105

Telen S. .................................................... 39

Teply F. ................................................. 151

Terzić S. ................................................ 153

Tessadri R. ............................................... 92

Timofeev A.A. ....................................... 111

Tishbee A. ................................................. 5

Tišler V. ................................................. 164

Tomaz C.T. ........................................ 57, 58

Tomaz I. .................................................. 85

Tóth S.B. ........................................... 93, 96

Tranchida P.Q. ......................... 38, 140, 141

Trbojević Akmačić I. ............................. 110

Tretaykov A.V. .............................. 107, 111

Trifonova A. .......................................... 172

Trkulja N. .............................................. 158

Tuberoso C. ........................................... 157

Tumiatti V. .............................................. 36

Tyrpień K. ..................................... 145, 146

Ukić Š. ........................................... 161, 162

Urban J. ................................................... 77

Vađić V. ................................................ 143

Valente J.F.P. .......................................... 84

Valentić I. ........................................ 94, 104

van Soest R. ........................................... 171

Vega J.R. ................................................. 26

Vera J.L. ................................................ 102

Vinković Vrček I. .................................. 168

Vlahović A. ........................................... 162

Voelkel A. ......................................... 41, 43

Volkova N.M. ........................................ 111

Vovk I. ....................................... 15, 17, 144

Vučković F. ........................................... 110

Name Page

Walczak M. ................... 131, 135, 137, 138

Walczyk M. ............................................. 70

Walkowiak K. ......................................... 86

Wasiak W. ......................................... 48, 76

Wawrzyniak R. ....................................... 76

Weiss J. ............................................... 2, 10

Welsch T. ................................................ 14

Wieczorek P. ......................................... 154

Wieczorek P.P. ...................................... 173

Wietecha-Posłuszny R. ........................... 98

Wilczewska K. ...................................... 123

Wolska L. ........................................ 89, 113

Woźniakiewicz M. .................................. 99

Wronka A. ............................................. 115

Yumba Mpanga A. .......................... 91, 103

Zakrzewska A. .............. 131, 135, 137, 138

Zatirakha A.V. ........................................ 52

Žgela D. ................................................ 160

Zgorelec Ž. .............................................. 75

Zhu X. ................................................... 171

Žigon D. .................................................. 34

Zoccali M. ....................................... 38, 141

Zrnčić M. ................................................ 35

Žuvela P. ............................................... 161

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19th International Symposium on Separation Sciences · HIGH PERFORMANCE LIQUID CHROMATOGRAPHY ......

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