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Comparision of DNA extraction methods to detect trace amounts of tree nut allergens in chocolates

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November 1–4, 2011

Prague, Czech Republic

5 International Symposium on

RECENT ADVANCES IN

FOOD ANALYSIS

Jana Pulkrabová and Monika Tomaniová

Editors

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November 1

–4, 2011

Prague, Czech Republic

5 International Symposium on

RECENT ADVANCES IN

FOOD ANALYSIS

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Jana Pulkrabová and Monika Tomaniová

Editors

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ICT Prague Press

Technická 5

166 28 Praha 6

Czech Republic

 Jana Hajšlová and Michel Nielen, 2011

Cover design  2011 by Tomáš Čajka

Edited by Jana Pulkrabová and Monika Tomaniová

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November 1–4, 2011

• Prague • Czech Republic

Clarion Congress Hotel Prague

Organized by

Institute of Chemical Technology, Prague, Czech Republic

&

RIKILT–Institute of Food Safety, Wageningen University,

The Netherlands

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Prof. Jana Hajšlová, Institute of Chemical Technology, Prague, CZ (chair)

Prof. Michel Nielen, RIKILT-Institute of Food Safety, Wageningen, NL (co-chair)

Prof. Damia Barcelo, Institute of Environmental Assessment and Water Studies IDAEA-CSIC,

Barcelona, ES

Prof. Chris Elliott, Queen's University Belfast, Belfast, UK

Prof. Hans-Gerd Janssen, Unilever Research and Development, Vlaardingen, NL

Prof. Henryk Jelen, Poznań University of Life Sciences, PL

Prof. Rudolf Krska, University of Natural Resources and Life Sciences, Vienna, IFA-Tulln, A

Dr. Steve Lehotay, United States Department of Agriculture, Wyndmoor, USA

Dr. Bert Popping, Eurofins Scientific Group, Pocklington, UK

Prof. Peter Schieberle, Technical University of Munich, Garching, D

Dr. Richard Stadler, Nestlé Product Technology Centre, Orbe, CH

Dr. Michele Suman, Barilla Food Research Labs, Parma, I

Prof. Franz Ulberth, JRC, Institute for Reference Materials and Measurements, Geel, B

Dr. Frans Verstraete, European Commission, DG Health and Consumers (DG SANCO), B

Prof. Zhiua Ye, Chinese Academy of Agricultural Sciences, Beijing, CN

Organising committee:

Staff of the Institute of Chemical Technology, Prague

Monika Tomaniová, PhD (chair)

Jana Pulkrabová, PhD

Prof. Vladimír Kocourek

Assoc. Prof. Tomáš Čajka

Assoc. Prof. Jan Poustka

Assoc. Prof. Kateřina Riddellová

Assoc. Prof. Vera Schulzová

Jana Kohoutková, PhD

Petra Hrádková, MSc

Ondřej Lacina, MSc

Mr Michal Žůrek

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CONTENT

5th International Symposium on Recent Advances in Food Analysis, 37

November 1–4, 2011, Prague, Czech Republic CONTENT

SEMINARS AND WORKSHOPS ... 69

SYMPOSIUM WORKSHOPI, NOVEMBER 1, 2011 (14:00–17:30) ... 71

WORKSHOP I „YOUNG SCIENTISTS IN EU RESEARCH ACTIVITIES RESEARCH ACTIVITIES AND OPPORTUNITIES FOR COLLABORATION STRENGTHENING“

SYMPOSIUM WORKSHOP II, NOVEMBER 1, 2011 (14:00–17:00) ... 72

WORKSHOP ON “INFRARED AND RAMAN SPECTROSCOPY FOR MONITORING OF AGRICULTURAL FOOD AND FEED PRODUCTS” SPONSORED BY FOSS AND BRUKER

VENDOR SEMINAR, NOVEMBER 2, 2011 (13:15–14:15) ... 73 INNOVATIVE NOMINAL AND ACCURATE MASS BASED LCMSMS WORKFLOWS AND SOLUTIONS FOR ADVANCED QUALITATIVE AND QUANTITATIVE FOOD ANALYSIS

THE USE OF LC/MS/MS FOR THE ROUTINE SCREENING OF FOOD CONTAMINANTS USING HIGH RESOLUTION MASS SPECTROMETRY SYSTEMS

FIGHTING BACKGROUND USING IMPROVED SELECTIVITY FOR BETTER QUANTITATION LIMITS IN LC/MS/MS EASY ADOPTION OF LC/MS/MS IN ROUTINE FOOD LABORATORY

DETECTION OF ALLERGENS BY LC-MS/MS USING A MULTI-ALLERGEN ASSAY

VENDOR SEMINAR, NOVEMBER 2, 2011 (13:15–14:15) ... 74 RAPID SCREENING FOR FOOD ADULTERATION AND QUALITY BY DART MS

AMBIENT MASS SPECTROMETRY EMPLOYING A DART ION SOURCE FOR METABOLOMIC FINGERPRINTING/PROFILING: A POWERFUL TOOL FOR BEER ORIGIN RECOGNITION

DART MASS SPECTROMETRY AND ITS COUPLING WITH PLANAR CHROMATOGRAPHY: IDENTIFICATION OF FLAVONOIDS AND PHENOLIC COMPOUNDS IN PROPOLIS

ULTRA-FAST DART SCREENING TO THE RESCUE: DETECTING ADULTERATION OF DIETARY SUPPLEMENTS AND IDENTIFYING RESIDUAL PESTICIDES USING DIRECT ANALYSIS IN REAL TIME (DART) HIGH-RESOLUTION ACCURATE MASS ANALYSIS

EVALUATING POROUS MATERIALS FOR SAMPLING PESTICIDES FROM SURFACES USING DIRECT ANALYSIS IN REAL TIME (DART)-MASS SPECTROMETRY

VENDOR SEMINAR, NOVEMBER 2, 2011 (13:15–14:15) ... 76 QUALITY ASSURANCE FOR MYCOTOXIN MONITORING IN A HACCP BASED APPROACH – REFERENCE MATERIALS AND PROFICIENCY TESTING

QUALITY ASSURANCE ASPECTS OF MYCOTOXIN TESTING

VENDOR SEMINAR, NOVEMBER 2, 2011 (13:15–14:15) ... 77 WATERS TODAY. FEATURED: SCIENTIFIC INNOVATION, FOOD AUTHENTICITY, PROFILING & QUANTITATION

CHAIR: DR SANDRA RONTREE, EUROPEAN HEADQUARTERS, WATERS CORPORATION

USE OF THE XEVO TQ-S AS APPLIED TO THE ROUTINE AND NOT SO ROUTINE ANALYSIS OF ANIMAL TISSUES FOR RESIDUES OF VETERINARY MEDICINES

PROFILING OF HIGHLY COMPLEX CITRUS JUICE SAMPLES USING UPLC ION MOBILITY TIME OF FLIGHT MASS SPECTROMETRY

VENDOR SEMINAR, NOVEMBER 3, 2011 (7:30–8:30) ... 78 INNOVATIVE TOOLS FOR FOOD ANALYSIS WITH HYPHENATED TECHNIQUES

INNOVATIVE TOOLS FOR FOOD ANALYSIS WITH HYPHENATED TECHNIQUES

VENDOR SEMINAR, NOVEMBER 3, 2011 (7:30–8:30) ... 79

HOW TO DETECT MULTIPLE ANALYTES FROM ONE SAMPLE, INCLUDING ANTIBIOTIC RESIDUES AND BACTERIAL CONTAMINANTS

PART I: HOW TO USE YOUR LAB RESOURCES MORE EFFICIENTLY: MULTI-ANALYTE SUSPENSION ARRAY AFFINITY ASSAYS!

PART II: ENSURE FOOD SAFETY AND QUALITY WITH UNISENSOR RAPID, MULTI-ANALYTE AND ON-FIELD TESTING.

VENDOR SEMINAR: NOVEMBER 3, 2011 (13:15–14:15) ... 80

BRUKER – INNOVATION AND TRADITION IN FOOD ANALYSIS

A NEW COMPLETE SOLUTION FOR AUTOMATED, COMPREHENSIVE ESI-(Q-)TOF FULL SCAN ACCURATE MASS SCREENING OF PESTICIDES IN FOOD WITH HIGH CONFIDENCE

MATRIX MATCHED STANDARDS REVEAL MATRIX MRM INTERFERENCES AND MINIMISE FALSE RESULTS IN PESTICIDE RESIDUE ANALYSIS OF GRAINS AND PULSES

USE AND QUALIFICATION OF TXRF FOR TRACE ELEMENT ANALYSIS OF DIETARY SUPPLEMENTS AND NUTRIENTS NMR-BASED FOOD QUALITY SCREENING

VENDOR SEMINAR, NOVEMBER 3, 2011 (13:15–14:15) ... 82 HIGH-END SOLUTIONS FOR YOUR FOOD ANALYSIS CHALLENGES: SAMPLE PREP – SEPARATION – MS DETECTION

NEW SUPERFAST AND HIGH-RESOLUTION LECO TOF MS INSTRUMENT LINE: NO COMPROMISE ANYMORE

HIGH QUALITY ANALYSIS OF PESTICIDES IN MARIJUANA FOR MEDICINE USING QUECHERS, CARTRIDGE SPE CLEANUP, AND GCXGC-TOFMS

DYNAMIC HEADSPACE - A POWERFUL TOOL FOR FLAVOUR AND FRAGRANCE ANALYSIS

VENDOR SEMINAR, NOVEMBER 3, 2011 (13:15–14:15) ... 83 NEW INNOVATIVE CHROMATOGRAPHY COLUMNS AND METHOD OPTIMIZATION FOR FOOD APPLICATIONS

USE OF IONIC LIQUID STATIONARY PHASES IN THE GC ANALYSIS OF FOOD VOLATILES

DETERMINATION OF HERBICIDES AT LOW TRACE LEVEL (PPT), USING WATER SAMPLE DIRECT INJECTION IN UHPLC/MS/MS COUPLE WITH RP AMIDE AND F5 ASCENTIS EXPRESS FUSED CORE HPLC COLUMN

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5th International Symposium on Recent Advances in Food Analysis, 55

November 1–4, 2011, Prague, Czech Republic

G-67

LEVELS OF BENZOIC AND SORBIC ACID PRESERVATIVES IN PROCESSED FOOD IN TURKEY (2008–2011)

Pelin Ulca1*, Beril Atamer2, Yeliz Ozturk3 ... 266

G-68

MONITORING THE ILLEGAL USE OF DYES IN CHILLI POWDERS IN TURKEY (2008–2011)

Pelin Ulca1*, Yeliz Ozturk2, Beril Atamer3 ... 266

G-69

CALIBRATION OF LOW COST ON-LINE VISIBLE-NEAR INFRARED SENSOR FOR THE MONITORING OF THE FERMENTATION PROCESS AND THE QUALITY OF THE CIDER

Alberto Villar1*, Eneko Gorritxategi2, Deitze Otaduy3, Jose Ignacio Santos4, Luis Angel Fernández5 ... 267

G-70

LEAST MEDIAN OF SQUARES CALIBRATION USING EXCEL

Panagiotis Steliopoulos1* ... 267

G-71

DEVELOPMENT, VALIDATION AND APPLICATION OF A METHODOLOGY FOR THE DETERMINATION OF Α-, Β – UNSATURATED HYDROXY ALDEHYDES IN SAMPLES OF EDIBLE SOYBEAN OIL

Pedro Pereira1*, Luciane Bastos2, Hortênsia Rocha3 ... 268

G-72

TWO NEW MODIFIED ACTIVATED CABONS BY HISTIDINE AND ARGININE FOR THE SOLID PHASE EXTRACTION OF TRACE LEAD IN WATER SAMPLES AND SOME OF FOOD SAMPLES

Rostam Shabani1*, Maryam Majidi2, Fatemeh Abedi3 ... 268

G-73

THE APPLICATION OF HYPHENATED SEPARATION TECHNIQUES FOR RESEARCHING OF LUNG CANCER BIOMARKERS

Agnieszka Ulanowska1*, Grzegorz Strączyński2, Bogusław Buszewski3 ... 269

G-74

QUANTITATIVE LATERAL FLOW STRIPS FOR MULTI-ANALYTE ASSAYS OF FOOD CONTAMINANTS

Boris Dzantiev1*, Yuri Venerov2, Anatoly Zherdev3, Nadezhda Byzova4 ... 269

G-75

COMPARISON OF DNA EXTRACTION METHODS TO DETECT TRACE AMOUNTS OF TREE NUT ALLERGENS IN CHOCOLATES

Joana Costa1, Vitor S. Melo2, Cristina G. Santos3, Isabel Mafra4*, Joana S. Amaral5, Letícia Estevinho6, M.B.P.P. Oliveira7 ... 270

MYCOTOXINS, MARINE AND PLANT TOXINS ... 271

H-1

THE UPTAKE OF 14C-ATROPINE FROM SOIL BY WHEAT AND ITS TRANSLOCATION TO SHOOTS

Zora Jandrić1*, Nasir Rathor2, Joseph Adu-Gyamfi3, Jaroslova Švarc-Gajić4, Leo Mayr5, Britt Maestroni6, Sorivan Chhem-Kieth7, Andrew

Cannavan8 ... 273

H-2

DETERMINATION OF SELECTED MYCOTOXINS IN CEREAL PRODUCTS

Marcin Bryła1, Mieczysław Obiedziński2*, Renata Jedrzejczak3 ... 273

H-3

ELISA AND SAMPLE CLEAN-UP METHODS FOR DETERMINATION OF OCHRATOXIN B

Fernando Rubio1*, Thomas Glaze2, Alexandra Heussner3, Daniel Dietrich4 ... 274

H-4

EFFECT OF FUSARIUM CULMORUM ON QUALITY OF SIX WHEAT VARIETIES

Ondřej Jirsa1*, Ivana Polišenská2 ... 274

H-5

CRITICAL ASSESSMENT OF DETERMINATION OF T-2 AND HT-2 TOXINS IN CEREALS: RESULTS OBTAINED BY UPLC–MS/MS AND ELISA METHODS

Marta Vaclavikova1*, Dania Al-Balaa2, Zdenka Veprikova3, Jana Hajslova4 ... 275

H-6

PHOMOPSIN A IN FOOD SAMPLES FROM RETAIL IN THE NETHERLANDS

Monique de Nijs1, Diana Pereboom-de Fauw2, Theo de Rijk3, Hans van Egmond4, Hans Mol5* ... 275

H-7

FT–NIR SPECTROSCOPY AS A SCREENING TOOL FOR DEOXYNIVALENOL CONTAMINATION IN WHEAT

Annalisa De Girolamo1*, Barbara Giussani2, Michelangelo Pascale3, Salvatore Cervellieri4, Vincenzo Lippolis5, Angelo Visconti6 ... 276

H-8

DETECTION OF PYRROLIZIDINE ALKALOIDS IN HONEY, MILK AND CHEESE USING LC–MS TECHNOLOGIES

Caroline T. Griffin1*, Brid Brosnan2, Martin Danaher3, John O'Mahony4, Steven Crooks5, D. Glenn Kennedy6, Ambrose Furey7 ... 276

H-9

THE ADVANTAGE OF FULLY STABLE 13C-LABELED INTERNAL STANDARDS IN LC–MS/MS MYCOTOXIN ANALYSIS

Alois Schiessl1* ... 277

H-10

ANALYSIS OF MYCOTOXINS IN POPPY SEEDS

Zdenka Veprikova1*, Milena Zachariasova2, Marta Vaclavikova3, Zbynek Dzuman4, Jana Hajslova5 ... 277

H-11

CO-OCCURENCE OF MYCOTOXINS IN FEEDS PRODUCED IN THE CZECH REPUBLIC

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GENERAL FOOD ANALYSIS

270 5th International Symposium on Recent Advances in Food Analysis,

November 1–4, 2011, Prague, Czech Republic

G-75

COMPARISON OF DNA EXTRACTION

METHODS TO DETECT TRACE AMOUNTS OF

TREE NUT ALLERGENS IN CHOCOLATES

Joana Costa1, Vitor S. Melo2, Cristina G. Santos3, Isabel

Mafra4*, Joana S. Amaral5, Letícia Estevinho6, M.B.P.P.

Oliveira7

147 Requimte, Faculty of Pharmacy, University of Porto, Portugal 2356 Polytechnic Institute of Bragança, Portugal

*Corresponding author - E-mail: [email protected], Phone: +351 222078902

Food-induced allergies represent an emerging problem of food safety. Thus, to safeguard the health of sensitised consumers, food ingredients that may cause allergic reactions should be properly labelled and possible cross-contamination should be avoided. Among food allergies, abnormal immunological responses towards tree nuts are pointed as a frequent source of serious atypical reactions, in which the hypersensitivities associated to almond and hazelnut ingestion are considered dangerous due to their incidence and severity [1,2]. Although immunological methods have been used for the direct detection of the almond and hazelnut allergens with high sensitivity, these assays are susceptible to cross-reactivity with other tree nuts. More recently, alternative approaches based on polymerase chain reaction (PCR) have been developed for the detection of almond and hazelnut allergens in foods [3-5]. However, highly processed food matrices such as chocolate, are very rich in polyphenols, carbohydrates and aromatic compounds that can interfere and inhibit DNA amplification. Since molecular methods are extremely dependent on the DNA extraction and purification procedures, adequate recovery of nucleic acids and removal of PCR inhibitors are required. Presently, several extraction methods are commercially available for the isolation of DNA from foods although only a few can provide extracts with suitable quality and purity for PCR amplifications. In these work we intend to compare different DNA extraction protocols (CTAB, Wizard, Wizard Magnetic and Nucleospin methods) with and without the addition of polyvinylpyrrolidone (PVP) and/or the presence of RNAse. For this purpose, model chocolates containing known amounts of almond or hazelnut (10-0.01%) were prepared and the DNA isolated with the selected protocols. DNA amplification was tested by qualitative PCR and real-time PCR with the use of specific fluorescent probes for almond and hazelnut. From the tested protocols, Nucleospin Food Kit evidenced the best results for almond and hazelnut isolation and amplification. This method showed the highest reproducibility of PCR fragments for almond and hazelnut chocolates until a relative limit of detection (LOD) of 0.01%, which was confirmed by real-time PCR. CTAB-PVP and Wizard methods exhibited the second best results for almond and hazelnut extraction, respectively. However, both methods were less reproducible since the DNA extracts presented major variations in PCR amplifications. To our knowledge this was the first attempt to compare different extraction methods for the specific detection of nut allergens in chocolates.

[1] Chen L et al. (2008) BMC Genomics 9:543 [2] Bettazzi F et al. (2008) Anal Chim Acta 614:93-102 [3] Köppel R et al. (2010) Eur Food Res Technol 230:367-374 [4] Piknová L, Pangallo D, Kuchta T (2008) Eur Food Res

Technol, 226:1155

[5] D’Andrea M et al. (2009) JAFC 57:11201-11208

Keywords: Food allergens, almond detection, hazelnut detection, PCR, chocolate

Acknowledgement: J. Costa is grateful to FCT PhD grant (SFRH/BD/64523/2009) financed by POPH-QREN (subsidised by FSE and MCTES).

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