AOAC EUROPE - NMKL - NordVal 7 – 8 May 2012
RAPID METHODS
AOAC EUROPE ‐ NMKL‐ NordVal Welcome to the Interna onal Symposium on Rapid Methods chemical, microbiological and sensory analysis of foods IDA Mee ng Centre, Kalvebod Brygge 31‐33, Copenhagen, Denmark
NMKL, Nordic Commi ee on Food Analysis, www.nmkl.org, Email: nmkl@ve nst.no c/o Norwegian Veterinary Ins tute, P.O.Box 750, Sentrum, N‐0106 Oslo, Norway
1 EUROPE SECTION OF AOAC INTERNATIONAL NordVal performs a third‐party review and AOAC Europe is a sec on of AOAC INTERNATIONAL cer fies alterna ve chemical and and was established in 1989. It is an organiza on of professional scien sts that exchange knowledge microbiological methods for food, and informa on to help each other excel in their feed, water, faeces and profession. environmental tes ng.
The ac vi es of the sec on are of interest to many NordVal offers: stakeholders from industry and trade, consumer a user‐friendly valida on and environmental protec on agencies, public au‐ protocol thori es and regulators in Europe and/or Mediter‐ scien fic confirma on policies ranean countries. specified acceptance criteria www.aoaceurope.com independent and rapid approval procedures guidance in the valida on NMKL offers process
reliable analy cal methods within chemical, microbiological and sensorial methods independent reviews and NordVal cer fica ons of proprietary and other The Nordic Commi ee on Food alterna ve methods Analysis, NMKL, was founded in 1947 and consists of chemists, relevant guidelines microbiologists, sensory analysts courses/workshops/seminars and sta s cians from the five Nordic countries, Denmark Finland, network of analy cal experts Iceland, Norway and Sweden. updated list of contact persons of the na onal reference laboratories within the NMKL is linked to the Nordic Nordic countries Council of Ministers.
www.nmkl.org
2 Contents
Program Monday—Plenary Session 4
Program Tuesday Chemistry 8
Program Tuesday Microbiology 6
Program Tuesday Sensory 7
Presenta on of the Moderators and Speakers at the Plenary Session 8‐13
Presenta on of the Moderator and Speakers at the Chemistry Session 14‐21
Presenta on of the Moderator and Speakers at the Microbiology Session 22‐29 Presenta on of the Moderator and Speakers at the Microbiology Session 30‐35
Poster abstracts 36‐46
List of par cipants 47‐50
Organisa on commi ee Hilde Skår Norli, NMKL c/o Norwegian Veterinary Ins tute, Norway Nina Bakkelund, NMKL c/o Norwegian Veterinary Ins tute, Norway Sune Eriksson, President of AOAC Europe, Sweden Sven Qvist, Chair of NordVal, Denmark Pierre Metra, Merieux Nutrisciences, France Klaus Reif, Phytolab GmbH & Co, Germany Bert Pöpping, Eurofins, United Kingdom Alfredo Montes Nino, Microbio cos, Spain Grethe Hyldig, Danish Technical University, Denmark Mika Tuomola, Finland Charlo a Engdahl Axelsson, Eurofins, Sweden
The organisa on commi ee gives thanks to the Exhibitors / Sponsors: 3M AB SCIEX Deutschland GmbH Agilent Technologies AOAC Europe Biolab A/S BIOTECON Diagnos cs Bruker Daltronics Food Diagnos cs ApS IonSence, Inc Neogen Europe Ltd NMKL / NordVal Oxoid & Remel, Thermo Scien fic, Thermo Fisher R‐Biopharm AG, Global Marke ng Food & Feed Diagnos cs ROMER LABS Division Holding GmbH Statens Serum Ins tut, SSI Diagnos ca
3 Program Monday 7 May
PLENARY
12:30 ‐ 13:00 Registra on
Moderators: Ulla Edberg, Chair of NMKL, Na onal Food Administra on, Sweden Sune Eriksson, President of AOAC Europe, Sweden
13:00 ‐ 13:30 Opening Welcome from the organisa ons, Ulla Edberg, Chair of NMKL Na onal Food Administra on Sune Eriksson, President of AOAC Europe, Sweden
13:30 – 13:45 Defini ons of rapid, proprietary, screening and alterna ve methods, Hilde Skår Norli, NMKL Secretary General, Norway
13:45 ‐ 14:00 Importance of valida on organisa ons in food safety management, Sven Qvist, Chair of NordVal, Denmark
14:00 ‐ 14:30 EU/Codex regula on on the use of analy cal methods (rapid to conven onal methods), Roger Wood, United Kingdom
14.30 ‐ 15:00 The advantages/disadvantages with the use of rapid methods, Charlo a Engdahl Axelsson, Eurofins, Sweden
15.00 ‐ 16:00 Coffee break / Exhibi on
16:00 ‐ 16:30 The value of standardisa on and an independent review, Mika Tuomola, Finland
16:30 ‐ 17:00 Valida on and verifica on of analy cal tools, Russel Flowers, Silliker, USA
17:00 ‐ 17.20 Food safety inspec on and commercial methods valida on in China, Li Zhiyong, Guangdong Entry‐Exit Inspec on and Quaran ne Bureau, China
4 Program Tuesday 8 May
CHEMISTRY
Moderator: Harriet Wallin, Chair of the NMKL Chemical Commi ee, Finnish Food Safety Authority, Evira
09:00 ‐ 09:30 Qualita ve chemistry method valida on guideline, Krystyna McIver, AOAC INTERNATIONAL, USA
09:30 ‐ 10:00 Valida on protocols and Nordval protocol – focus on ELISA and allergens, Ylva Bolin Sjögren, Na onal Food Administra on, Sweden
10:00 ‐ 10:30 Performance criteria for valida on, verifica on and applica on of molecular methods, Arne Holst‐Jensen, Norwegian Veterinary Ins tute
10:30 ‐ 11:00 Coffee break/ Exhibi on / Posters
11.00 ‐ 11:30 Food allergens profiling with an imaging surface plasmon resonance‐based biosensor, Nathalie Smits, Wageningen University and Research Centre, The Nederlands
11.30 ‐ 12:00 ELISA methods for determina on of algal toxins, Ingunn Anita Samdal, Norwegian Veterinary Ins tute
12:00 ‐ 12:30 NIRS Standards EN ISO 12099 and EN 15948 – se ng new performance standards for NIR cali‐ bra ons, Jürgen Möller, Consultant, Sweden
12:30 ‐ 13:30 Lunch / Exhibi on / Posters
13:30 ‐ 14:00 Can LC/MS/MS be used as a rou ne tool for Allergens analysis including Mustard? Stephen Lock, ABSCIEX, UK
14:00 ‐ 14:30 Accurate quan fica on of 11 regulated mycotoxins by UHPLC‐MS/MS using 13C isotope la‐ beled internal standards, John Lee, Agilent, UK
14.30 ‐ 15:00 Coffee break/ Exhibi on / Posters
15:00 ‐ 15:30 Rapid analysis of solid and liquid Samples by direct introduc on with triple quadruple (GC‐ MS/MS), Gordon van 't Slot, Bruker Daltonics, Germany
15:30 ‐ 16:00 Rapid Test Methods versus LC‐MS/MS Technology in Rou ne Mul Mycotox in Analysis, Alois Schiessl, Romer Labs, Austria
16:00 ‐ 16:30 A HACCP based approach for mycotoxin management: RIDA®QUICK tests plus RIDA®QUICK Scan, Ronald Niemeijer, R‐Biopharm AG, Germany
16:30 ‐ 17:00 Gluten detec on with a new genera on of monoclonal an body, Elisabeth Hammer, Romer Labs, Austria
5 Program Tuesday 8 May
MICROBIOLOGY
Moderator: Sven Qvist, Chair of NordVal, Denmark
09:00 ‐ 09:25 Analy cal methods related to the Commission regula on (EC) No 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs, Niels L. Nielsen, Danish Veterinary and Food Administra on, Denmark
09:25 ‐ 09:30 CEN mandate – status on the valida on of the reference methods, Sven Qvist, NordVal, Denmark
09:30 ‐ 10:00 Comparing the different protocols for valida on of proprietary methods, Russ Flowers, Silliker, Past President of AOAC Interna onal , Chair of ISPAM (Interna onal Stakeholder Panel on Alterna ve Methods, AOAC Interna onal), USA
10:00 ‐ 10:30 Development and valida on of molecular methods for the detec on of food‐borne patho‐ gens ‐ current status of the method standardisa on, Dietrich Maede, Landesamt für Verbraucherschutz Sachsen‐Anhalt, Germany 10:30 ‐ 11:00 Coffee break/ Exhibi on / Posters
11:00 ‐ 11:30 Rapid methods in the meat industry, Flemming Hansen, Danish Technological Ins tute, Denmark
11:30 ‐ 12:00 The use of rapid methods for the microbiological quality control in the food chain, Adrianne Klijn, Rdls, NestleResarch Centre, Switzerland
12:00 ‐ 12:30 Why and how – RAPID’ Salmonella, an example from a test‐kit producer, Frederic Mar nez, Bio‐Rad, France
12:30 ‐ 13:30 Lunch / Exhibi on / Posters 13:30 ‐ 14:00 Current developments and future trends in rapid methods technology, Jeffrey Hoorfar, Technical University of Denmark
14:00 ‐ 14:30 An easy, fast and accurate method for the detec on of the TOP 7 Shiga Toxigenic E.coli (STEC), including E.coli O157:H7, Jane e Handley, BioControl Systems, United Kingdom
14.30 ‐ 15:00 Coffee break/ Exhibi on / Posters 15:00 ‐ 15:30 Performance of a new molecular pla orm for the detec on of Salmonella and E.coli O157, Julie Yang, 3M, USA
15:30 ‐ 16:00 Oxygen‐Deple on method for the enumera on of aerobic bacteria‐ current status and fur‐ ther work, Alan Traylor of Mocon Inc., USA
16:00 ‐ 16:30 Listeria Precis™: a rapid, culture‐based method, validated to ISO 16140, for the detec on of Listeria monocytogenes and other Listeria species from food and environmental samples , Cheryl Mooney, Thermo Fisher Scien fic 6 Program Tuesday 8 May
SENSORY
Moderator: Grethe Hyldig, Technical University of Denmark
09:00 ‐ 09:30 Sensory analysis: Measurement uncertainty and presenta on of results, Per Lea, Nofima, Norway
09:30 ‐ 10:00 Rapid sensory descrip ve methodologies – Scope and applica ons, Chris an Dehlholm, University of Copenhagen, Department of Food Science, Denmark
10:00 ‐ 10:30 Quality index method – an objec ve rapid tool for determina on of sensory quality of fish, Grethe Hyldig, DTU Food – Na onal Food Ins tute, Denmark
10:30 ‐ 11:00 Coffee break/ Exhibi on / Posters
11.00 ‐ 11:30 Sensory characters of Cabernet Sauvignon dry red wine from Changli County (China), Ninino Federico, University of Udine, Italy
11:30 ‐ 12:00 Rapid and simultaneous analysis of xanthines and polyphenols as bi er taste markers in bakery products by FT‐NIR spectroscopy, Michele Suman, Barilla SpA, Parma, Italy
12:00 ‐ 12:30 Holis c approach for consumer surveys, Lene Meinert, DMRI, Danish Technological Ins tute, Denmark 12:30 ‐ 13:30 Lunch / Exhibi on / Posters
13:30 ‐ 14:00 State of the art of the ar ficial nose—What we are up against, can do and expect, Thomas Lindblad, KTH – Physics Department and NoseLabs AB, Sweden
14: 00 ‐ 14:30 Quality control test for drinking water (NMKL 183), Urd Bente Andersen, Chair of the Norwegian Na onal Commi ee of NMKL, Norway
14.30 ‐ 15:00 Coffee break/ Exhibi on / Posters
15:00 ‐ 15:15 Use of PanelCheck—drinking water, Urd Bente Andersen, NMKL, Norway
15:15 ‐ 15:30 The so ware PanelCheck – quality control of the sensory analysis, Grethe Hyldig, DTU Food – Na onal Food Ins tute, Denmark
15:30 ‐ 16:00 Discussions
7 Dr. Ulla Edberg, Chairman of NMKL, Na onal Food Administra on, Sweden E‐mail: [email protected]
Ulla Edberg is the Chairman of NMKL and the Swedish Na onal Commi ee. Edberg is Ass. Professor, Head of Chemical Division 2 at the Na onal Food Agency , Sweden. The Agency has the task of protec ng the interests of the consumers by working for safe food, fair prac ces in the food trade, and healthy ea ng. Chemical Division 2 works with method development and analysis of e.g. metals, allergens, GMO , organic persistent pollutants. Ulla Edberg is the Swedish representa ve in CEN/TC 275 Food Analysis‐ Horizontal Methods and in Codex Commi ee on Method of Analysis and Sampling.
Welcome from NMKL NMKL, Nordic Commi ee on Food Analysis, is an important network for chemists, microbiologists and sensory analysts working in food laboratories (private and governmental), food industry, research ins tu ons and in food control authori es. NMKL was established in 1947. The members of NMKL are appointed expert from the five Nordic countries; Denmark, Finland, Iceland, Norway and Sweden. NMKL cooperates with several interna onal organisa ons and has interested par es from more than 40 countries worldwide. NMKL elaborates and collabora vely validate analy cal methods, elaborates guidelines on quality assurance (a list is given at the penul mate page) and arranges courses and workshops. NMKL also deals with rapid methods, and holds the secretariat of NordVal. NordVal gives an independent review of alterna ve methods. NordVal now also offers an independent review of chemical test‐kits.
Dr. Sune Eriksson, President of AOAC Europe Sec on 2011‐2012 Er DevCo Consultants AB, Sweden E‐mail: [email protected]
Educa on: B Sc, Uppsala University, 1981, Ph D, Stockholm University, 2005. Work experience: More than 30 years in contract laboratory, mainly as R&D manager. Today: Own consultancy busi‐ ness, Er DevCo Consultants AB, working mainly in Sweden, Africa and China including 25% project employment at Stockholm University.
Welcome from AOAC Europe Quicker analyze me, lowered detec on limit, more robust technique, validated methods are always requested from offi‐ cials, commercial laboratories, research ins tutes and industry for all type of analysis. This is seminar number 14 since year 1990 with AOAC Europe in different topics. Many mes the seminar has been in collabora on with different organiza ons. This me with NMKL. Our mother organiza on, AOAC Interna onal (The Scien fic associa on dedicated to analy cal excel‐ lence), is based in Gaithersburg, about 40 km from Washington, DC. AOACI is an old organiza on, which has its 126th annual conference this year in Las Vegas. AOAC Europe has a price for best poster at this conference. The price is free a endance (including travel and accommoda‐ on) at next AOAC Europe conference (Paris 2013).
8 PLENARY Hilde Skår Norli, NMKL Secretary General, Norwegian Veterinary Ins tute E‐mail: nmkl@ve nst.no
Since 1997, Hilde Skår Norli has been the Secretary General of the Nordic Commi ee on Food Analysis, NMKL. The office of the NMKL Secretary General is hosted by the Norwegian Na onal Veterinary Ins tute, where Norli has been employed since 1995. Hilde Skår Norli holds a Cand.Scient degree in analy c organic chemistry from the University in Oslo, Norway, and has been employed at a couple of private laboratories and at the Norwegian Food Safety Authority. Norli has been a Director of the AOAC Interna onal Board of Directors and is involved in interna onal organiza ons like CEN, ISO and in Codex Commi ee on Methods of Analysis and Sampling.
Defini ons of rapid, proprietary, screening and alterna ve methods What is a rapid method? The term “rapid method” usually refers to a method much faster than respec ve reference methods. For chemical and sensory analyses, “rapid” is in terms of minutes rather than hours, for microbiological analysis it is o en in terms of hours rather than days. Everything is rela ve. However, rapid methods should have some common features: the methods should be simple and easy‐to‐use, the methods should be rela vely fast, be fit for their purpose, and mean reduc ons of costs for the laboratories. Rapid methods could then be an alterna ve to the reference method. Proprietary methods (test‐kits) are o en referred to as rapid methods. Codex Commi ee on Methods of Analysis and Sampling has just agreed upon a defini on of proprietary methods.
Dr. Sven Qvist, Chair of NordVal, E‐mail: [email protected]
Sven Qvist holds a degree of Veterinary Medicine and a postgraduate course in food microbiology and hygiene from the Royal Veterinary and Agricultural University in Copenhagen. Qvist has been head of the microbiological department at the Danish Meat Products Laboratory under the Ministry of Agriculture working with microbiological projects and quality programs for meat products for the home market and for export. Sven Qvist has for many years been working with classical microbiological methods within the Nordic Commi ee on Food Analyses (NMKL), the Interna onal Dairy Federa on (IDF) and the Interna onal Standard Organiza on (ISO). Sven Qvist has during the last decades followed closely the development and marke ng of alterna ve microbiological methods and was ini ator of the Danish valida on system (DanVal) and in 1999 ini ator of the transi on of this system into the Nordic valida on system (NordVal). Sven Qvist has func oned as chairman for both DanVal and NordVal. Importance of valida on organiza ons in food safety management Food borne diseases are of concern for consumers, the food industry, retail shops, restaurants and the authori es. Therefore food safety management systems and regula ons have been adopted both on the na onal level and in the framework of interna onal organiza ons, such as EU, CODEX , WTO and WHO. Although strong emphasis has correctly been focused on preven on systems such as HACCP, microbiological tes ng for safety has maintained an important role in food produc on and food trade. In fact this role has been increasing as a consequence of mass produc on of food items distributed on the global market. Thus failures in producing safe food can have drama c health risks and lead to big economic losses. The rising need for microbiological tes ng of foods in na onal and interna onal trade has been followed by the need for rapid results avoiding long me storage of especially perishable foods. Since classical microbiological methods cannot cover
9 this need, research ins tutes and commercial companies have during the last couple of decades developed and made available an increasing number test kits fulfilling the requirements of providing rapid results. This development has been followed by regulatory requirements for proof, that the rapid methods produce results equivalent to the accepted standard reference methods. Interna onal valida on organiza ons have been established to provide the necessary documenta on to the authori es on the acceptability of the use of rapid methods in tes ng for food safety. Thus such organiza ons have become important links within the whole chain of food safety management systems.
Great efforts are at present carried out to elaborate one global accepted valida on protocol to be followed by all valida on organiza ons. This will benefit a worldwide recogni on of valida ons carried out irrespec ve of the organiza on providing the valida on results. The advantage of having several organiza ons opera ng in the market is avoidance of monopolies as a guarantee for fair valida on prices.
Dr. Roger Wood, Food Standards Agency, UK – re red Email: roger.shirley@b nternet.com
Roger Wood obtained a doctorate in analy cal chemistry at Imperial College of Science and Technology, London and then qualified to be a UK food control analyst. He took responsibility for the Food Analysis Sec on of a public analyst's (food control) laboratory. He moved to the Food Standards Agency (previously the Ministry of Agriculture, Fisheries and Food) to be responsible for methods of analysis and sampling for foods, especially with respect to their introduc on into legisla on. He has represented the UK at numerous European Union Methods of Analysis and Sampling for Foods Working Groups, the Codex Commi ee on Methods of Analysis and Sampling as well as various na onal Commi ees, and has served with various Interna onal Organisa ons (e.g. AOAC INTERNATIONAL, ICUMSA, CEN etc.). He is currently Chairman of the Inter‐Agency Mee ng at which such organisa ons meet to discuss common problems in this area. His par cular interests, besides methods of analysis and sampling, now lie with the quality of analysis, accredita on and proficiency tes ng of food analysis laboratories. Of par cular interest is that he is co‐author of the Interna onal Harmonised Protocol/Guidelines of Proficiency Tes ng, for Internal Quality Control, Recovery Factors and Single Laboratory Valida on and these have influenced many laboratories in his sector. He was awarded the OBE in 2003 for his work in the area. He re red from the Agency in March 2010.
EU/Codex regula on on the use of analy cal methods (rapid to conven onal methods) The talk will: Emphasise that for many of the ini a ves, par cularly as it affects food and feed control, there is a strong emphasis on third‐part assessment, e.g. for laboratories that includes requirements regarding accredita on to the ISO/IEC Standard 17025 and of proficiency tes ng. Briefly re‐state the “quality assurance requirements” of the food analysis laboratory which are now well defined as the result of EU and Codex Alimentarius Commission requirements, these now giving confidence to introduce the criteria approach. But it will also comment on the benefits, or otherwise, of the accredita on requirements that have been adopted by these organisa ons
Describe the ra onale for the introduc on of the “criteria” or “performance‐based” approach to methods of analysis, and in par cular that the approach means greater flexibility than the tradi onal procedure adopted by organisa ons such as Codex and the EU. Describe how the adop on by some organisa ons of “proprietary methods” has lead to the development of Codex text on “Proprietary Methods” and the adop on of such text will affect the work of Standardisa on Organisa ons such as NMKL/AOAC/CEN etc.
10 PLENARY Dr. Charlo a Engdahl Axelsson, Eurofins Food and Agro Tes ng Sweden
E‐mail: Charlo aEngdahlAxelsson@eurofins.se
Charlo a Engdahl Axelsson studied chemistry and biology at the University of Uppsala and obtained a PhD in microbiology at the University of Agricultural Sciences in Uppsala in 1993. She has been working as microbiologist and a Quality Manager for the Food Industry and as a R & D Manager for micro‐ and molecularbiology for AnalyCen Nordic AB. Her present posi on is as group Quality Manager for Eurofins Food and Agro Tes ng Sweden. Charlo a is a microbiological expert of NMKL and involved in standardisa on of microbiological methods at CEN and ISO levels, a member of CEN WG 12 for allergens, a member of MicroVal technical commi ee and a board member of Eurachem/Eurolab in Sweden.
The advantages/disadvantages with the use of rapid methods Rapid alterna ves to reference standard methods or conven onal methods are commonly used for microbiological and chemical analysis of samples from different parts of the food chain. The poten al for using a rapid method depends on performance characteris cs, legisla on, customer acceptance, official valida on status, ease of use, throughput, costs etc. Experiences of using rapid methods for analysis of samples from primary produc on and foods are presented with examples from analysis of plant pathogens, mycotoxins, nutri onal value of feed, quality control of milk, food associated microorganisms and food allergens.
The use of rapid methods to screen soil for plant pathogens gives an opportunity for an effec ve crop management. An appropriate valida on and understanding of the biology of the pathogen are necessary for a method to be fit for purpose. ELISA tests are used as rapid tests for screening of mycotoxin in grain at harvest. This has been par cularly useful during last year when high levels of DON were found in grain from Sweden and Norway. Some cross‐reac vi es were found for similar compounds when comparing results to LC MS MS.
NIR technology is widely used for rapid evalua on of the nutri onal value of feed. Advantages are speed, no need for sample prepara on and low costs. The disadvantages are that a very thorough calibra on for each matrix is required and a con nuous extensive monitoring of the performance of the calibra on.
Automa c system based on flow cytometry and IR/FTIR is used for quality control of milk. The system allows efficient and very cost‐effec ve analysis of a large nr of samples (400‐500 samples/h) for fat, protein, cells etc. Calibra on is comparably easy to perform. Recently also rapid real‐ me PCR methods for analysis of bacteria causing mas s in milk are rou nely used. During the last decade alterna ve methods for analysis of food associated microorganism have been widely accepted in Sweden also for official control of foods. New legisla on and official protocols for valida on of alterna ve methods have enhanced the development of rapid alterna ves. Great advantage using PCR methods has been experienced in rela on to outbreaks of Salmonella and EHEC.
For detec on and quan fica on of food allergens ELISA tests are most commonly used. Valida on data from suppliers has improved during recent years but there are s ll major differences in how kits from different suppliers are calibrated and validated. There is a need for alterna ve tests to confirm ELISA results.
11
Dr. Mika Tuomola, Adjunct Professor, Finland
E‐mail: [email protected]
Dr. Mika Tuomola is an independent consultant on food and environmental diagnos cs. He has a PhD in molecular biotechnology with specialisa on in vitro diagnos cs and MSc in food chemistry. Dr. Tuomola has 20 years of experience working on the development and applied research of ad‐ vanced diagnos c methods in the field of chemical and microbiological food safety. This includes execu ve posi ons in industrial diagnos cs companies and academic R&D experience.
The value of standardisa on and an independent review Food laboratories can enjoy today from an increasing number of alterna ve analy cal methods. These novel methods offer poten ally several benefits over tradi onal procedures, including for example shorter me to answer, ease of use, lower total cost of analysis, possibility for automa on, and improved analy cal characteris cs such as be er selec vity or lower limit of detec on. End‐users can not however just jump in and start to use a new method assuming that manufacturer’s claims con‐ cerning the analy cal performance are automa cally valid for their purposes. Obtaining a value for a measurement is not suffi‐ cient; the objec ve is to be able to report the true result. Valida on process is used to inves gate whether the analy cal purpose of the method is achieved; that analy cal results are produced with an acceptable uncertainty level. A full method valida on is however only possible for a limited number of labor‐ atories because it requires a significant effort. The solu on is to use an independent external valida on and cer fica on pro‐ cess, which provides impar al data with regards to method performance and ensures that the characteris cs of the method have been found to be in compliance with its claims. Once a validated analysis method has been selected for use, the laborato‐ ry only needs to verify the correct implementa on by tes ng and documen ng its competence in using the new method.
Dr. Russell S. Flowers, Ph.D., Chairman and Chief Scien fic Officer, Merieux NutriSciences (formerly Silliker Group), Chicago, USA E‐mail: [email protected] Dr. Flowers received his BS and MS degrees from North Carolina State University, and his Ph.D. for the University of Illinois, and was an Assistant Professor at the University of Arizo‐ na, prior to joining Silliker in 1979. He began his career at Silliker as the Laboratory Director at the Illinois laboratory and advanced to become President in 1990, the posi on he held un l 2007. During this me, Silliker expanded from a small collec on of laboratories in North America to more a global network with more than 45 loca ons offering a full range of analy cal and advisory services related to food safety and quality. In January of 2007, Flow‐ ers moved to the posi on of Chief Scien fic Officer and became Chairman of the Board of Directors. His current responsibili es are to spearhead strategic growth opportuni es, and assure that Silliker remains on the forefront of science and technology related to food safe‐ ty and quality.
In addi on to his management responsibili es for Silliker, Flowers has been an ac ve researcher, author and speaker in the field of food microbiology, with par cular emphasis on development of rapid analy cal methods, and valida on of methods and laboratory performance. Flowers was the study director for the valida on of the first Enzyme Immuno‐Assay and Nucleic Acid Hybridiza on Assay approved by AOAC, and many subsequent studies that have lead to industry‐wide implementa on of these methods for detec on of pathogens in foods and food environments. He also, chaired the Food Laboratory Accredita‐ on Working Group that developed specific accredita on criteria for food tes ng laboratories that were eventually adopted by AOAC and A2LA. Silliker became the first food tes ng laboratories to become ISO accredited in North America.
12 PLENARY
The recipient of numerous industry awards and honors, Dr. Flowers is an ac ve member of the several professional organ‐ iza ons and socie es including; Ins tute of Food Technologists (board member and fellow), AOAC Interna onal (past pres‐
ident, board member and fellow), American Meat Ins tute (board member), Interna onal Commission on Microbiological Specifica ons for Foods (ICMSF), and Interna onal Associa on for Food Protec on (IAFP).
Valida on and verifica on of analy cal tools
There are many conflic ng defini ons of valida on and verifica on of methods. Applica on of these defini ons into prac‐ ce can be difficult, especially when mul ple disciplines are involved; e.g., microbiology, chemistry and sensory. The ISO, AOAC and some examples of regulatory defini ons for valida on and verifica on will be discussed. In addi on, some prac cal examples of how Silliker defines valida on and verifica on in selec ng and pu ng analy cal into prac ce. The role of proficiency tes ng for verifica on and valida on will be discussed.
Dr. Li Zhiyong, Guangdong Entry‐Exit Inspec on and Quaran ne Bureau, China Email: [email protected]
Li Zhiyong, professor of Guangdong Entry‐Exit inspec on and quaran ne Bureau, obtained his Ph.D degree in college of bioengineering, South China University of Technology. Now he is a council member of Guangdong Society of Microbiology. He has been engaged in rapid inspec on technology such as microbiological inspec on, GMO tes ng, immunological in‐ spec on and rapid screening of heavy metal and veterinary drug in food.
Food safety inspec on and commercial methods valida on in China
Following the globaliza on of the food trade, the popularity of processed foods, and the change in ea ng habits, food safe‐ ty is star ng to affect people’s lives more and more in China. From farm to fork, food safety is separately responsible by several departments. Individual ministries in each territory and local governments implement relevant suppor ng systems. Food safety legisla on system gradually becomes more and more perfect, and the inspec on organiza ons and standards are increasing day by day in China. SN standard is officially recognized standard adopted by Imp＆Exp commodity inspec‐ on and quaran ne authority of China. Now all SN standards using commercial methods should be validated before issues. Cer fica on and Accredita on Administra on of P.R.C (CNCA) established the guidelines, technical requirements and vali‐ da on procedure for cer fying analy cal performances of commercial methods.
13
Chemistry
Moderator: Harriet Wallin, Chair of the NMKL Chemical Commi ee, Finland
E‐mail: Harriet.Wallin@evira.fi
Harriet Wallin graduated in 1974 from Helsinki university with organic chemistry as the main subjects. She first worked at the university with research and teaching tasks and passed a licen ate exam in organic and analy cal chemistry in 1976. From the beginning of the 80’s she was employed as a research scien st at VTT Food Research Laboratory and through that was introduced to the work of NMKL. In the period 1985‐ 1997 she was Secretary General of the Commi ee and later the Chairman of NMKL’s Finnish Na onal Commi ee and Chairman of the NMKL Chemical Commi ee. She now works as a senior officer for food control for the Finnish Food Safety Authority Evira.
14 Krystyna McIver, Sr. Director, Stakeholder Communi‐ ca ons at AOAC Interna onal E‐mail: [email protected] CHEMISTRY
Qualita ve chemistry method valida on guideline AOAC ini ated a project in 2011 to develop a valida on guideline for qualita ve chemistry methods. Few guide‐ lines existed prior to this ini a ve so method developers and conformity assessment organiza ons like AOAC de‐ veloped valida on protocols for qualita ve chemistry methods on a case‐by‐case basis. Exis ng and developing guidance documents were reviewed and considered. The scope of the project, development process, key con‐ cepts, and future plans will be discussed. This project funded by the AOAC Research Ins tute.
Ylva Sjögren Bolin, Na onal Food Administra on, Sweden E‐mail: [email protected] Ylva Sjögren Bolin is a Senior Chemist at the Science department at the Na onal Food Agency in Sweden. She is responsible for analyses of food allergens, which are mainly conducted with ELISA methods. She is a member of CEN TC 275/WG 12 that develops standard methods/technical reports for analyses of food allergens. She is also a mem‐ ber of the chemical steering group of Nordval. Ylva has a PhD in immunology from Stockholm University.
Valida on protocols and Nordval protocol – focus on ELISA and allergens An body‐based methods can be used for analysis of a wide range of substances in foods. Enzyme‐linked immunosorbent assays (ELISAs) are one example of an body‐based methods which are o en used for analyses of food allergens. Standard methods and cer fied reference materials regarding analysis of food allergens are scarce. In addi on, ELISAs are o en based on commercial test kits in which a proprietor owns the full informa on of the test kit. For the end‐user, a cer fica‐ on of the test kit is valuable since it shows that an independent assessment of a kit’s performance characteris cs has been performed and thus that the test kit show compliance with its claims. Nordval is an independent third‐party that reviews and cer fies test kit. During the talk, Nordval’s guidelines and other protocols for valida on of chemical test kits will be pre‐ sented.
15 Dr. Arne Holst‐Jensen, Norwegian Veterinary Ins tute E‐mail: arne.holst‐jensen@ve nst.no Arne Holst‐Jensen, born 1963. Dr.scient. at University of Oslo, Norway in 1995 (fungal molecular evolu on and systema cs), postdoc at UofO 1996‐1998, senior scien st and head of the GMOs unit at the Norwegian Veterinary Ins tute since 1998. Coordinator/par cipant in several EU‐ funded research projects on GMO and toxigenic fungi. Member of the Steering Commi ee of the European Network of GMO Laboratories (ENGL). Chairman/member of several ENGL ad hoc working groups. Ac ve in the development of the first CEN/ISO standards on GMO analysis. Published more than 50 peer review papers.
Performance criteria for valida on, verifica on and applica on of molecular methods Molecular methods are widely applied and yet there is li le harmonisa on of performance criteria and acceptance values both within and across sectors. The European control laboratories for gene cally modified organisms (GMOs) in food and feed were officially organised in the European Network of GMO Laboratories (ENGL) in 2002. GMO detec on in Europe is performed almost exclusively with deriva ves of the polymerase chain reac on (PCR) and this is also reflected in the EUs GMO legisla on. This led the ENGL to publish “Method acceptance criteria and method performance requirements” in 2004, while at the same me largely adop ng the so called “modular approach”. A revision of the ENGL guidance document was published in 2008 (Defini on of minimum performance requirements for analy cal methods of GMO tes ng) and a new revision is currently in prepara on. The listed criteria and acceptance values have proven to be both func onal and realis c for individual and combined PCR modules within certain limita ons. However, cri cs have argued that matrix effect influence on analy cal reliability has been largely ignored. Including performance criteria for valida on of DNA extrac on modules is necessary to close the gap. Interes ngly, that could also allow for verifica on of the fitness for purpose of individual DNA extracts and improved quality assurance when applying molecular methods. In its widest consequence this would make the modular approach both more flexible and reliable than the more tradi onal “global approach”. The associated performance criteria and the modular dis nc on between a) the sample prepara on; b) the analyte extrac on and purifica on; and c) the analyte detec on steps may apply also outside molecular GMO detec on.
Nathalie Smits, RIKILT‐Ins tute of Food Safety, Wageningen UR, The Netherlands
E‐mail: [email protected]
Nathalie Smits is a research assistant at RIKILT‐Ins tute of Food Safety. Her exper se is development of immunologically based screening assays for proteins, in which she is primarily focussed on allergens and growth hormones. Nathalie Smits has a B.Sc. in Clinical Chemistry and B.Sc. in Environmental Sciences.
Imaging SPR-based biosensor for food allergens profiling
Here we present how direct on‐chip allergen screening using imaging Surface Plasmon Resonance (iSPR) can be applied to food profiling, offering a powerful analy cal alterna ve to exis ng methods. Mul ple allergen detec on is achieved in a single reagent format using on‐chip direct iSPR‐based immunoassay, omi ng the need in labelling, signal amplifica on, and washing steps. The use of iSPR technology provides a complete allergen profile within short measurement me and with adequate sensi vity. The applicability of this approach was tested by analyzing cookies and dark chocolate from different manufacturers. Hazelnut content of the tested food products was also determined by commercially available enzyme linked immunosorbent assay and was found to correlate well with the hazelnut content determined by iSPR. This newly developed method opens the door to automated and high throughput allergen analysis, ul mately providing the consumer with safer food.
16
Dr. Ingunn Anita Samdal, Norwegian Veterinary Ins tute E‐mail: ingunn.samdal@ve nst.no Ingunn Anita Samdal is working as a scien st at the Department of Chemistry and Toxicology at the Norwegian Veterinary Ins tute in Norway. She completed her doctoral degree in the year 2005, working on an bodies and use and development of the YTX‐ELISA and defended her thesis “Yessotoxins in algae and mussels – studies on its sources, disposi on, and levels.” She has spent in total nine months as working on various aspects of CHEMISTRY ELISA development with Lyn Briggs at AgResearch, New Zealand. She par cipated in the EU‐ project BIOTOX with early stages of developing an azaspiracid ELISA. Recently she has been working on developing a microcys n ELISA as a cost‐effec ve, reliable and easy‐to‐use diagnos c tool for cyanotoxins in water and wildlife, and an important aim is transfer the technology and know‐how to partner ins tu ons in Southern Africa.
ELISA methods for determina on of algal toxins Algal toxins can cause diarrhoea, vomi ng, paralysis and other effects in humans, mammals or fish. Algal toxins are produced by various algae and can be retained in shellfish or contaminate drinking water. A series of such marine algal toxins are known, including okadaic acid and the dinophysistoxins, saxitoxins, brevetoxins, domoic acid, azaspiracids, pinnatoxins, yessotoxins, pectenotoxins and cyclic imines, and most of them are found all over the world. Algal toxins are responsible for many incidents of human intoxica on every year, some of them fatal.
Dr. Jürgen Möller, Höganäs, Sweden Email: [email protected]
Jürgen Möller is Dr. rer. Nat. (Ph.D.) in analy cal chemistry from the Technical University of Berlin in 1977 on trace elements by neutron ac va on analysis. He has been a Laboratory Man‐ ager and has held several other management posi ons within Tecator AB, Perstorp Analy cal AB and Foss Analy cal AB. He has par cipated in several dozen standardiza on projects within AOAC, ISO and CEN, not at least as a project leader for the two projects he is going to talk about. Möller has long experience in analy cal chemistry, laboratory & quality management as well as interna onal standardiza on of analy cal methods in the Food/Feed/Agri/ Environmental area as well as in business development. Since 2011 he has been working as independent consultant.
NIRS Standards EN ISO 12099 and EN 15948 – se ng new performance standards for NIR calibra ons? Today there are maybe 50 million analyses annually performed using indirect spectroscopic methods like NIRs, but un l a few years back there were no applicable interna onal standards that would form a basis for the communica on of NIRs results and allow for an accredita on of users of NIR spectrometry. This is the background for the ISO 12099 project “Guidelines for the applica on of near infrared spectroscopy”. This Interna onal Standard gives guide lines for the determi‐ na on by near infrared spectroscopy of cons tuents such as moisture, fat, protein, starch and crude fibre as well as param‐ eters such as the diges bility in animal feeding stuffs, cereals and milled cereal products. ISO12099 is a general standard that focuses on the valida on of calibra on models with independent test sets. It forms the basis for quality systems or ac‐ credita on schemes when using NIR. The main elements of the standard will be discussed. A first specific standard, developed under the umbrella of EN ISO 12099 is presented, EN 15948:2012 “Cereals ‐ Determina‐ on of moisture and protein ‐ Method using Near‐Infrared‐Spectroscopy in whole kernels” and finally some points to con‐ sider when selec ng NIR solu ons, with focus on the quality of reference data and traceability are discussed.
17 Dr. Stephen Lock, Applica on Manager, AB SCIEX, UK E‐mail: [email protected] Dr. Stephen Lock obtained his PhD in Physical Organic Chemistry from the University College of Swansea in 1993. Previously a lecturer of Chemistry at the University of Hull and a senior scien st at CCFRA he has worked for ABSCIEX™ in various technical support roles for over 14 years and is presently the Applica on Manager for Europe and European managed territories. He has over 20 years’ experience in analy cal chemistry with over 15 in the development of LCMS methods for the analysis of bio‐ logical, environmental and food extracts. Steve is currently a member of the Royal Society of Chemistry and is a Chartered Chemist. He is also a member of the AOAC and sits on the Expert Review Panel for Strategic Food Analy cal Methods. Stephen has presented at well over 25 interna onal mee ngs around the world. He is author of numerous technical publica ons, training programs and journal ar cles.
Can LC/MS/MS be used as a routine tool for Allergens analysis including the detection of Mustard? The prevalence of food allergies in the United States is es mated at around 6% for children and reports suggest that the number of allergies is rising. Screening for allergens is tradi onally performed using enzyme‐linked immunosorbent assays (ELISAs). ELISA can generate variable results, and false‐posi ve as well as false‐nega ve results occur especially. Addi onally each allergen requires a separate kit so a method that could unambiguously confirm the iden fica on of individual allergens in a mul ple allergen screen would be invaluable.
Here we present data acquired by LC/MS/MS using a method ini ally incorpora ng egg and milk which proved the feasibility of such an approach. Food samples were extracted and then the allergic proteins were reduced, alkylated and digested us‐ ing trypsin. The pep des from the digested proteins were purified using solid phase extrac on and these extracts analysed by LC/MS/MS and reverse phase chromatography using posi ve mode electrospray ionisa on. The mass spectrometry methods u lises scheduled MRM™ and informa on dependant acquisi on so that not only are mul ple pep des detected for each allergen but full scan product ion data is collected at the same me for each pep de so that presence of allergen can unambiguously be confirmed.
In this presenta on we will discuss how this approach developed using milk and egg in a single lab verifica on compares against the tradi onal ELISA based assays on incurred samples. We will then show how it can be expanded to detect over 15 allergens in one analysis by applying this methodology to several tree nut species as well as mustard, peanut and gluten. These recent developments will be able to demonstrate how this new type of approach is capable of a true mul allergen screen producing results with a higher level of confidence than current techniques in a ma er of hours.
John Lee, Agilent Email: [email protected] John Lee (BSc Hons in Applied Chemistry) has been with Agilent for 12 years (previously with Dionex Corpora on for 8 years). During much of his me at Agilent he was a specialist in the UK and Ireland for LCMS in all markets. However since 2010 he has managed a ‘food team’ in Europe focusing on collabora on with food labs across Europe and further afield, to generate new or improved approaches to food analysis in areas of LCMS, GCMS and Molecular Biology. The collabora ons from the food group and been very successful & varied. During this presenta on John will present on data generated by one such collaborator; The Laboratory of Rudolf Krska at the Center for Analy cal Chemistry, IFA‐Tulln, University of Natural Resources and Life Sciences ,Vienna, Austria with specific credit to Franz Berthiller and Elisabeth Varga.
18 Accurate quan fica on of 11 regulated mycotoxins by UHPLC‐MS/MS using 13C isotope labelled internal standards About 300‐400 substances are recognized as mycotoxins – toxic secondary metabolites of fungi, which are o en found as contaminants in food. For mul ‐mycotoxin analysis of food, unified LC‐MS based methods can save costs and me in the long run. Matrix effects caused by suppression or enhancement of the analyte signal must however be addressed. We present a novel method for the quan fica on of 11 regulated (or soon to be) mycotoxins. Acidified shake extrac on with subsequent addi on of 11 fully 13C‐labelled internal standards was followed by UHPLC‐MS/MS analysis using an Agilent CHEMISTRY 6490 QQQ mass spectrometer. Spiking at mul ple levels on blank samples of maize allowed recoveries of extrac on to be validated as fit for purpose and the sensi vity, accuracy and precision of the method was seen to be commensurate with MRL’s associated with all food analysis including that of baby food as outlined by European Commission Regula on No. 1881/2006.
Dr. Gordon van’t Slot, Bruker Daltonics GmbH, Germany E‐mail: [email protected] Gordon van ’t Slot holds a PhD in Food Chemistry from the “Wes älische Wilhelms‐Universität”, Münster, Germany. He is now working as an Applica on Chemist at “Bruker Daltonik GmbH” for GC and GC‐MS based analyses. Dr. Gordon van ’t Slot has also worked as Laboratory Manager of QS – accredited pes cide residue analysis at “Labor Dr. Lippert GmbH”, and has been a lecturer in dietary biochemistry at the “Schule für Gesundheitsberufe” at the “St. Franziskus Hospital” in Münster. He has also worked at the “Landwirtscha liche Untersuchungs‐ und Forschungsanstalt NRW” in Münster with priority on food‐analy cs and residue analysis.
Rapid Analysis of Solid and Liquid Samples by Direct Introduc on with Triple Quadrupole (GC)‐MS/MS
A direct introduc on of samples can save a lot of me during various analyses. Common direct inlet probes into ioniza on chamber are without so ware control and bear the risk of a source contamina on. The ChromatoProbe introduced into a PTV (programmable temperature vaporiza on) injector can be coupled directly to the ion source with 2 m of uncoated fused silica capillary or via a usual analy cal column, with respect to complexity of the sample.
Samples may range from liquids over slurries to solids. For dirty samples the benefit is the usage of single use microvials, which keep non‐vola les, high boilers and thermally degraded components inside. This decreases run me per sample and maintenance. Column bake out is usually also not necessary to allow running more samples a day and preserves the column performance.
Street drugs can be easily inves gated as a solid sample or even directly from the introduc on of a single hair. Plas cs, dyes and drugs may contain residual solvents which can be analyzed with SPME. An alterna ve is a direct inser on in pieces in a microvial. This sample introduc on is less discrimina ve compared to others. Plant ssues that normally are not considered amenable to GC MS/MS analysis can be easily inves gated with the ChromatoProbe.
The injector s ll keeps all its benefits as split flow of the sample and a stepwise increase of the temperature to mimic a dis lla on process. All this lowers the risk of contamina ng the ion source in comparison to a direct inlet into the ion source. In synthesis control and in combinatory chemistry approaches so ware features like automa c MS/MS breakdown and the signal stability are a benefit. All changed se ngs are automa cally recorded and can be reviewed during data analysis and allow a convenient workflow.
19 19 Alois Schiessl, Romer Labs Division Holding GmbH, Austria E‐mail: [email protected] Alois Schiessl, born in Salzburg, Austria, has a BSc degree in Food Science and Biotechnology from the University of Natural Resources and Applied Life Sciences Vienna, Austria and a BSc (Hons) degree in Biotechnology from Murdoch University Perth, Australia. Alois Schiessl joined Romer Labs Division Holding GmbH, Tulln, Austria, in January 2010 as Product Manager and is responsible for rapid and reference tes ng solu ons in the Mycotoxin and GMO fields for food and feed tes ng. He holds memberships in AOAC Interna onal and the IAESTE Austria (Interna onal Associa on for the Exchange of Students for Technical Experience). Furthermore, he is listed as an expert in the Food Analysis Commi ee of the Austrian Standards Ins tute.
Rapid Test Methods versus LC‐MS/MS Technology in Rou ne Mul Mycotoxin Analysis A current trend in rou ne mycotoxin analyses is the need for mul analyte detec on. At present rapid test methods are of importance as a fast screening tool for single analyte detec on. In contrast the popularity of the LC‐MS/MS technology, which is related to mul analyte detec on, is constantly increasing. More and more laboratories are now using LC‐MS/MS for mul mycotoxin rou ne analysis. Nevertheless, a problem with LC‐MS/MS can be interferences from matrix components leading to differences in analyte ioniza on. To overcome this ioniza on effect 13C stable isotope labelled internal standards can be used. However these are o en associated with high prices but a correct applica on of these standards will reduce the costs significantly. A set of 12 mycotoxin 13C stable isotope labelled internal standards applied in an op mized mul mycotoxin LC‐MS/MS method accounts for less than 3% of the total analysis cost.
Rapid test methods are more affordable because no expensive equipment and no expensive consumables are necessary. By using enzyme linked immunosorbant assays (ELISA) or the lateral flow device (LFD) technology fast quan ta ve results are produced that allow a quick es ma on on present mycotoxin contamina on. In general accuracy and precision of such rapid tests are comparable to reference methods. Typical recoveries determined by ELISA are within 80 to 100% and the precision lies below 15% rela ve standard devia on. However, when mul mycotoxin analyses are required, different test kits for each single analyte have to be used. This is a disadvantage compared to the LC‐MS/MS technology that can detect various analytes at once. Also their well known advantages of speed and low cost have to be compared to the latest LC‐MS/ MS developments, such as high sensi vity, and the resul ng methods that have been op mized for rou ne test laboratories.
This presenta on will demonstrate a comparison of analy cal method parameters and economical factors of rapid test methods versus state‐of‐the‐art mul toxin reference methods, such as LC‐MS/MS. Furthermore, it will illustrate the importance of correctly applying 13C stable isotope labelled internal standards when implemen ng quan ta ve mycotoxin analyses on an LC‐MS/MS system. Finally, it will show advantages and disadvantages in mul mycotoxin analysis when using rapid test methods versus the LC‐MS/MS reference method in rou ne mycotoxin analysis.
Ronald Niemeijer, R‐Biopharm AG, Darmstadt, Germany E‐mail: r.niemeijer@r‐biopharm.de Ronald Niemeijer is global marke ng manager food & feed diagnos cs at R‐Biopharm AG in Darmstadt, Germany. He graduated at the Vrije Universiteit of Amsterdam and obtained his masters degree in biochemistry. He has more than 20 years of experience in the sales and marke ng of food & feed diagnos cs, food analysis and food produc on and has held several posi ons at companies like ALcontrol, Unilever and now R‐Biopharm. His main professional topics of interest are mycotoxin analysis, food microbiology and allergen tes ng using immunological and molecular biological methods. Currently Ronald Niemeijer is working on development and marke ng of reference materials, check sample programs and analy cal services. He is also responsible for global marke ng for Trilogy Analy cal Laboratory in Washington, Missouri (US), a R‐Biopharm subsidiary.
20 A HACCP based approach for mycotoxin management: IDA®QUICK tests plus RIDA®QUICK Scan of food and feed product imposes a risk to human and animal health and has serious economic impact. Since mycoMycotoxins are secondary metabolites formed in agricultural products, such as cereals, nuts and (dried) fruits. Mycotoxin contamina on toxins are natural occurring toxins exposure can not be 100% controlled. To meet interna onal regula ons and guidelines products are tested for the amount of mycotoxins. Yet, instead of tes ng large numbers of end‐ products, a more pro‐ac ve approach would have many benefits. In order to assure safe food an feed various quality
assurance tools can be applied as GAP and GMP. CHEMISTRY During the produc on process of food and feed cri cal steps can be iden fied where it is possible to minimise the risk of unacceptable mycotoxin concentra ons in the end product: This means a HACCP based approach for mycotoxin management. The RIDA®QUICK lateral flow tests for mycotoxin analysis in combina on with the RIDA®QUICK SCAN, enabling on‐site quan ta ve analysis for Aflatoxin, DON, Fumonisin and now introducing Zearalenone, have proven to be a very valuable tool in this HACCP approach. With minimal laboratory equipment samples can be analysed within 10 – 20 minutes. Results show an excellent correla on with HPLC for many commodi es in a wide measurement range.
Elisabet Hammer, Romerlabs, Austria Email: [email protected]
Elisabeth Hammer is Product Manager for the product line of rapid tests for Food Allergen Tes ng at Romer Labs – a provider of diagnos c solu ons with 30 years of experience. Her scien fic background are academic studies in Food Science, Food Technology and Biotech‐ nology at the University of Natural Resources and Life Sciences (BOKU) in Vienna, Austria.
Gluten detec on with a new genera on of monoclonal an body Gluten is the main group of proteins in grains such as wheat, rye, barley and, to a lesser extent,oat. Gluten consists of prola‐ mins and glutelins, called gliadin and glutenin in wheat, respec vely. Coeliac disease is an immune‐mediated enteropathy caused by the inges on of gluten. Recent discussions about coeliac disease have moved from the concept of gluten detec on to detec on of the cereal protein frac ons that are toxic to persons intolerant to gluten which is closer to the provisions of the interna onally agreed Codex Standard 118:1979. In this work the monoclonal an body G12, raised against the QPQLPY pep de from a toxic fragment called 33‐mer of the gliadin, was used to develop a sandwich enzyme linked immunosorbant assay (ELISA),AgraQuant(R) Gluten G12 and a lateral flow device (LFD), AgraStrip(R) Gluten G12. The an body’s specificity was determined by cross reac vity studies on various grains, nuts, oils and starches. Processed samples were tested and recovery of the ELISA was determined by spiking experiments on common food matrices as well as on problema c matrices. The limit of detec on of the LFDwas determined in several spiked commodi es, and rinse water tes ng as well as swabbing recovery experiments from stainless steel and plas c were conducted. The limit of detec on of the AgraQuant(R) Gluten G12 was deter‐ mined to be 2 ppm gluten, with a quan ta on range of 4 to 200 ppm gluten. The results obtained for spiked samples and pro‐ cessed food samples showed comparable performance with a Mendez R5 ELISA method. AgraStrip(R) Gluten G12 allows for the on‐site detec on of gluten within 10 minutes. The limit of detec on was determined to be 5 ppm gluten in spiked com‐ modi es. By varying the amount of dilu on buffer it is possible to adjust to cut off levels of 5, 10 and 20 ppm gluten. When tes ng rinse water, varia on in pH from 5 to 9 does not affect the results. During valida on of AgraQuant(R) Gluten G12 and AgraStrip(R) Gluten G12, posi ve and nega ve responses to oat varie es were obtained. However, the posi ve results appear to be a specific reac on of the an body with the toxic fragment, rather than a non‐specific posi ve signal. This suggests that the G12 an body may shed light on the debate concerning poten al immunotoxicity of oats. Results obtained from immuno‐ chemical test systems based on the G12 an body should be considered to be closer to the ideal of a food safety test by estab‐ lishing the important link between coeliac disease and detec on of the immunotoxic pep des.
21 Microbiology
Moderator: Dr. Sven Qvist, Chair of NordVal, E‐mail: [email protected] Sven Qvist holds a degree of Veterinary Medicine and a postgraduate course in food microbiology and hygiene from the Royal Veterinary and Agricultural University in Copenhagen. Qvist has been head of the microbiological department at the Danish Meat Products Laboratory under the Ministry of Agriculture working with microbiological projects and quality programs for meat products for the home market and for export. Sven Qvist has for many years been working with classical microbiological methods within the Nordic Commi ee on Food Analyses (NMKL), the Interna onal Dairy Federa on (IDF) and the Interna onal Standard Organiza on (ISO). Sven Qvist has during the last decades followed closely the development and marke ng of alterna ve microbiological methods and was ini ator of the Danish valida on system (DanVal) and in 1999 ini ator of the transi on of this system into the Nordic valida on system (NordVal). Sven Qvist has func oned as chairman for both DanVal and NordVal.
Dr. Niels Ladefoged Nielsen, Danish Veterinary and Food Administra on E.mail: [email protected]
The current posi on for Niels Ladefoged Nielsen is in the Danish Veterinary and Food Administra on (DVFA), dept. for feed and food safety with assignments primarily related to general food microbiology, strategy for sampling and analysis, na onal surveillance projects etc. Former employments include Scandinavian Airlines System (Hygiene Officer) and DVFA (na onal reference laboratory and development and coordina on of official microbiological food control).
Analy cal methods related to the Commission regula on (EC) No 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs From the point of view of EU food control authori es as well as the food business operators it is very important that analy cal results are widely accepted. For this purpose methods validated according to a well recognized protocol and by a well recognized organisa on are of course of the outmost importance. Within the majority of EU legisla on in the area of food safety and veterinary issues demands to the analy cal methods used are stated. This is especially important related to official control and the company in house control requested by the legisla on. The main set of legisla on comprising rules for microbiological tes ng is: Regula on (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 on official controls performed to ensure the verifica on of compliance with feed and food law, animal health and animal welfare rules. Guidance document on official controls, 2006 under Regula on (EC) No 882/2004, concerning microbiological sampling and tes ng of foodstuffs. Regula on (EC) No 852/2004 of the European Parliament and of the Council of 29 April 2004 on the hygiene of foodstuffs. Commission Regula on (EC) No 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs
22 Regula on (EC) No 2160/2003 of the European Parliament and of the Council of 17 November 2003 on the control of salmonella and other specified food‐borne zoono c agents. Commission Regula on (EC) No 1688/2005 of 14 October 2005 implemen ng Regula on (EC) No 853/2004 of the European Parliament and of the Council as regards special guarantees concerning salmonella for consignments to Finland and Sweden of certain meat and eggs
For microbiological in house tes ng the Guidance document on official controls states that: “The use of rapid methods is acceptable provided they are validated against the reference method according to certain valida on protocols. For commercial rapid methods (proprietary methods) an addi onal requirement of cer fica on has been set down in the above‐men oned Regula on. If other methods are used for in‐house control purposes, the methods shall be validated according to interna onally accepted protocols and their use authorised by the competent authority. As regards the valida on of microbiological methods, the procedure in EN ISO 16140, including an intra‐laboratory and an inter‐laboratory study (collabora ve study) is highly recommended.” Worth no cing for the companies is that the methods used for In house tes ng, not requested by the authori es, are not comprised by the demand for valida on. The interpreta on of the rules related to the use of analy cal methods varies between the EU countries. Some countries request that alterna ve methods shall be validated according to the ISO 16140 protocol while other countries – e.g. Denmark – allow methods validated against other “similar interna onally recognized protocols”. So far, the Danish food authori es allow alterna ve methods validated by Nordval, AFNOR, Microval and AOAC. In general, the na onal food authori es as well as the companies would benefit from a somewhat more harmonized approach to the interpreta on of the exis ng demands to the valida on of alterna ve methods. Further, discussion on topics concerning common acceptance of one valida on protocol and related acceptance criteria along with the performance of laboratories performing in‐house controls could be of interest. Finally, the use of analy cal methods that does not necessarily MICROBIOLOGY provide an isolate raises the discussion regarding how to perform further characteriza on of the target organism – serotyping, pa ern of an bio c resistance etc.
Dr. Russell S. Flowers, Silliker, Chair of ISPAM (Interna onal Stakeholder Panel on Alterna ve Methods, AOAC Interna onal) E‐mail: [email protected] (biography, see page 12)
Comparing the different protocols for valida on of proprietary methods The objec ve of ISPAM is to develop harmonized, interna onally accepted standard vali‐ da ons guidelines for alterna ve methods. ISPAM consists of 60+ scien fic and technical experts from mul ple countries, including industry, academia, government and organiza‐ ons. This presenta on will be limited to discussing the ac vi es and objec ves rela ve to microbiological methodology. The ISPAM ac vi es and progress to date will be dis‐ cussed.
23
Dietrich Mäde, State Office for Consumer Protec on of Saxony‐Anhalt, Halle, Germany E‐Mail: [email protected]‐anahlt.de
Dietrich Mäde, born in 1966, was granted Doctor of Veterinary Medicine in 1994 at the University of Leipzig, Germany. In April 1993, he was employed by the State Office for Consumer Protec on, where he s ll works. At the beginning, he was ac ve in the official service for Animal Health and Animal Welfare. With the need for the development of molecular detec on methods in official food tes ng, he changed into the department of Food Hygiene and started working in the field of PCR in 1994. The 1st methods applied targeted Gene cally Modified Organisms (GMO) in Food and Shiga Toxin producing E. coli. The laboratory grew con nuously during the last 17 years, at present there are methods in place for the relevant authorized and non‐authorized GMO, for animal and plant species detec on, and for bacterial and viral food pathogens. Today, Dietrich Mäde is the deputy head of the Department of Food of Animal Origin and Laboratory and as the head of the molecular laboratory responsible for gene cally modified food and special microbiology. In 1998, he was granted Special Veterinarian of Food Hygiene according to the Veterinary Associa on; and in 2009, he acquired the special veterinary professional qualifica on „Molecular Biology“. Since 2005, Dietrich Mäde is Lecturer at the University of Applied Sciences Anhalt Köthen/Bernburg, where he was appointed Honorary Professor in 2011. Besides the rou ne work, Dietrich Mäde is ac ve in several na onal and interna onal working groups for development and standardisa on on molecular methods. He is Chairman of the Official Na onal working group “Molecular Methods – Microbiology” and Chairman of the Official Na onal working group “Detec on of Viruses in Food”. In addi on, the is member of ISO/TC 34 SC16 “Molecular Biomarker Analysis”, CEN/TC 275/WG 11 "Gene cally modified foodstuffs", CEN/TC 275/WG 6/TAG 3 "PCR for the detec on of food‐borne pathogens in food and animal feeding stuffs" CEN/TC 275/WG 6/ TAG 4 “Detec on of viruses in food”, and the Na onal working group “Development of Methods for Detec on of Gene cally Modified Organisms”. Within the TAG3, Dietrich Mäde was project leader of the EN ISO 22118:2011 “Performance Characteris cs” and EN ISO 22119:2011 (Real‐ me polymerase chain reac on (PCR) for the detec on of foodborne pathogens — General requirements and defini ons). Dietrich Mäde published 40 scien fic papers and one book chapter. He s ll lives in Halle (Saale) and has three nearly grown up children.
Development and Valida on of Molecular Methods for the Detec on of Food‐Borne Pathogenes ‐ Current Status of the Method Standardisa on The need for standardisa on of molecular methods can be dated back to the early 90ies. Besides GMO detec on, priori es were set on molecular microbiology.One of the 1st methods worldwide was the NMKL standard No. 163 for the detec on of pathogenicYersinia(Y.)enterocoli cain 1998. In Germany, a company moved forward the standardiza on of the detec on of Salmonella which became an official na onal standard in 1999 a er a successful interlaboratory valida on study. The Salmonella standard was preceded by a standard laying down general requirements for PCR in food microbiology. Later on, methods for the detec on of thermophilic Campylobacter, Listeria monocytogenes, and STEC by conven onal PCR and Salmonella, Norovirus, and Rotavirus by real‐ me PCR were published so far.
In the European context, the CEN/TC 275/WG6/TAG3 was formed in the early 2000s. The first standards were published in 2005 and 2006. In 2005, “Microbiology of food and animal feeding stuffs ‐ Polymerase chain reac on (PCR) for the detec on of food‐borne pathogens ‐ General requirements and defini ons” (ISO 22174:2005)and the technical specifica on “Performance tes ng of thermal cyclers” (CEN ISO/TS 20836:2005) were developed. In 2006, “Requirements for sample prepara on for qualita ve detec on” (EN ISO 20837:2006) and “Requirements for amplifica on and detec on for qualita ve methods” (EN ISO 20838:2006) followed. The policy of CEN that me was the preven on of conflic ng results with conven onal methods. This is being solved by focusing on molecular methods for food pathogens for which no conven onal methods exist like Clostridium botulinum, STEC, and foodborne viruses.
Since real‐ me PCR became state if the art for molecular detec on methods, EN ISO 22119:2011“Microbiology of food and animal feeding stuffs — Real‐ me polymerase chain reac on (PCR) for the detec on of foodborne pathogens — General requirements and defini ons” was published. As major principle, it was fixed in this standard that probe based real‐ me PCR systems shall be used for the detec on of food pathogens. The second pillar of this standard is the interpreta on of the amplifica on curves. Within the same context as the real‐ me PCR standard was developed, EN ISO 22118:2011 “Performance Characteris cs” was issued. This standard specifies basic requirements for the performance of molecular
24 methods with regard of sensi vity and specificity. The two standards should build a common ground on which other meth‐ ods are being developed in validated, regardless if as in‐house method or by an interlaboratory study. At present, several technical specifica ons are on the way. This comprises a technical specifica on for the detec on of Y. enterocoli ca and Y. pseudotuberculosis, Cl. botulinum, STEC, Norovirus and Hepa s A Virus from several food matrices. On the na onal level, interlaboratory studies for thermophilic Campylobacter and L. monocytogenes were finished. The next ring trial will be a real‐ me PCR for the detec on of STEC in food of plant origin to address the fatal outbreak in Germany in 2011.
Flemming Hansen, Senior Scien st, Technological Ins tute ‐ Danish Meat Research Ins tute E‐mail: @teknologisk.dk Flemming Hansen completed his Master of Science 1987 in Food Microbiology. A erwards he joined “Foss Electric”, a leading diagnos c company in Denmark, being responsible for develop‐ ment of a semi automated ELISA test for Salmonella in food and feed. This par cular Salmonella test was actually the first test capable of detec ng Salmonella in food in less than 24 hours. Sub‐ sequently Flemming was responsible for the development of related immune assays for E. coli O157, Campylobacter and L. monocytogenes. MICROBIOLOGY
In 1997 he transferred to the Danish Meat Research Ins tute, as a project manager and deputy manager for the micro‐ biological laboratory. As a project manager he par cipated in the EU several projects i.e. Food PCR I, Food PCR II, Bio‐ Tracer and Vital.
Working at Foss Electric and DMRI have given Flemming a large experience in pathogen tes ng in food, especially re‐ garding Salmonella, VTEC, Listeria monocytogenes and Campylobacter, as well as experience in developing and evalu‐ a ng rapid methods. As an employee at the DTI, Danish Meat Research Ins tute he has close contact to the meat sector and thereby a great understanding of the needs for diagnos c methods in the meat sector. Also the research at DMRI is carried out in close coopera on with the Danish meat sector securing that both the scien fic and prac cal needs of the sector are met.
Flemming Hansen is member of the Microbiological group in the Nordic Commi ee for Food Analysis (NMKL) since 2002, and appointed chairman of the group for 5 years. He was also a member of the Danish Microbiology group in the ISO organisa‐ on from 1994 ‐ 2010.
Rapid Methods in the Danish Meat Industry
Analyzing for pathogenic bacteria in fresh meat has been carried out for many decades in the Danish Meat Industry, either as an in‐house control or as a request by the Food Authori es.
The major problem was, and s ll are, the long me to obtain a result, meaning that the meat has been disposed and maybe consumed at the me the screening result for pathogens is due. In the 90’es, this ini ated a development of many different rapid methods especially for Salmonella, but also targe ng several other pathogenic bacteria. At the beginning most of the methods were based on the ELISA technology or other an body‐based technologies. One major breakthrough came in 1994 with the launch of the first approved Salmonella ELISA method giving a result within 24 hours. For the next decade, several 24 hours methods were developed and the search for even faster methods based on PCR technology began.
This presenta on will focus on the PCR methods that have been developed on request of the Danish Meat Industry by DMRI in close coopera on with the Technical University of Copenhagen and Statens Serum Ins tut, DK. These methods include the PCR for detec ng Salmonella within 14 hours (the Salmonella 12 hour method), PCR for MDR S. Typhimurium DT104 and a PCR for S. Dublin.
25 Dr. Adrianne Klijn, Nestec Ltdm, Nestle Research Centre, Switzerland E‐mail: [email protected] Adrianne started her educa on in the Netherlands; at Larenstein Interna onal Agricultural College, where she obtained a B.Sc in Food Science and Technology. At University College Cork in Ireland she obtained an M.Sc in Food Microbiology. In Switzerland at the Nestlé Research Centre she obtained her Ph.D working on physiological and molecular characterisa on of stress responses in Bifidobacterium longum NCC2705. A er her Ph.D, Adrianne became head of the microbiology lab at the Nestlé Quality Assurance Centre in York, the UK. Since 2010 she is back at the Nestlé Research Center, working in the Microbiological and Molecular Analy cs group. This group is responsible for maintaining the por olio of laboratory instruc ons in use by Nestlé laboratories.
The use of rapid methods for microbiology quality control in the food chain. Since the introduc on of HACCP there is less emphasis on end point tes ng of products to ensure the microbiology quality control in the food chain. However, there are s ll cases where a reduc on in the me to result is beneficial, for example when responding to incidents and the monitoring of raw ingredients. Advances in science have made it possible to reduce the me to result. This talk looks at different examples such as ELISA (proteins), PCR (DNA) and transcrip on mediated amplifica on (RNA). The talk concludes by looking at the challenges that these rapid methods face, such as cultural confirma on for molecular methods.
Frederic Mar nez, Bio‐Rad, France E‐mail: FREDERIC_MARTINEZ@bio‐rad.com Frederic Mar nez studied biochemistry, Agriculture Sciences and Engineering in France. He graduated his Masters degree at E.S.I.T.P.A in Rouen. He has almost 20 years of experience in Marke ng in food & animal feed produc on and diagnos c. He is Interna onal at Group Product Manager at Bio‐Rad Laboratories, in charge of the marke ng of food and water microbiology. He has been a member of AFNOR commi ee for 8 years and has followed AOAC‐RI and NordVal valida ons for 6 years.
Why and how – RAPID’Salmonella, an example from a test‐kit producer Bio‐Rad has developed alterna ve methods that reduce the me to results, save money, and are easy to use. 11 detec on and 4 enumera on methods have been validated according to the ISO 16140 protocol and 3 are pending. The valida on is an advantage for a laboratory using the method as it avoids the internal valida on to show the competent authority its’ reliability. Un l now, the third par es who validated the Bio‐Rad methods in Europe are AFNOR Valida on and NORDVAL. But 8 methods are also validated according to AOAC‐RI. For example, RAPID’Salmonella short protocol is an alterna ve method ISO 16140 validated for the detec on of Salmonella spp in 24h a er 18h enrichment in buffered peptone water + supplement. The results of the preliminary study for Salmonella detec on were good with a sensi vity of 90.6% equivalent to the standard ISO 6579 with a sensi vity of 91.6%. A high level of discrepant results is due to unpaired results and low level of contamina ons. An interna onal collabora ve study was successfully conducted with 15 laboratories. These results were obtained by ADRIA, an independent laboratory and sent to the AFNOR Valida on and NORDVAL commi ees. These two commi ees follow similar criteria to validate a method. Bio‐Rad however was s ll interested in submi ng the data to both because there are some differences: A be er recogni on in Northern Europe countries for NordVal and in Southern Europe countries for AFNOR Valida on. Specific requirements from each cer fica on bodies like 20% of naturally contaminated samples from AFNOR Valida on and specific matrices from NordVal.
26 Dr. Jeffrey Hoorfar, Technical University of Denmark E‐mail: [email protected] Jeffrey Hoorfar has a PhD in food science and is professor in food microbiology. Hoorfar is the Head of Molecular Diagnos cs at the Department of Microbiology and Risk Assessment.
In the past 20 years he has been working with the development of serological and microbiological de‐ tec on of zoono c pathogens in veterinary and food samples. He is the coordinator of BIOTRACER, a large EU Integrated Project under the 6th FPm and has been the Coordinator of Food‐PCR (5th FP) and Food‐PCR2 of MedVetNet (6th FP). He has extensive training in project management and has coordi‐ nated several large industrial and Nordic research projects, resul ng in several tests and more than 60 publica ons.
Current developments and future trends in rapid methods technology. MICROBIOLOGY
Jane e Handley, BioControl Systems Ltd , UK E‐mail: [email protected] Jane e Handley began her career as an MLSO in the Derby Royal Infirmary Public Health Laboratories and a er gaining her IMLS qualifica ons moved out to the
business world. Jane e has a very broad range of technical exper se covering food and clinical microbiology, molecular biology and protein chemistry from 25 years in technical sales and marke ng and during this me she had the opportunity to work among esteemed scien sts such as Sir Philip Cohen and Sir David Lane using biosensors in cell signaling and cancer research. She is an ac ve member of Campden and sits on the Microbiology and Quality Assurance panels for Biocontrol Systems. During the early 90’s she lived and worked in Germany, se ng up and running a charter airline for NATO employees and their families.
An easy, fast and accurate method for the detec on of the TOP 7 Shiga Toxigenic E.coli (STEC), including E.coli O157:H7. BioControl Systems has developed a new method to sa sfy industry’s need for an easy, fast and accurate method for the detec on of the TOP 7 Shiga Toxigenic E.coli (STEC), including E.coli O157:H7. The method consists of a simple and innova‐ ve IMS‐based sample prepara on procedure that helps narrow the field to the seven target O‐serogroups prior to molecu‐ lar analysis with the assurance GDS® Rotor Gene. With the Assurance GDS PickPen IMS procedure, magne c par cles coated with an bodies specific to the Top STEC O‐groups are combined with the enriched sample. If present, E. coli belonging to the target O‐groups bind to the an bodies a ached to the par cles. The PickPen device collects and removes the par cles with the bound E. coli, separa ng them from the non‐target O‐groups and helping to ensure detec on of the gene targets is lim‐
ited to the desired O‐groups of E. coli. Top STEC results are determined by the presence of the eae and stx1 and stx2 genes, while E.coli O157:H7 results are based on the same proven gene target found in the Assurance GDS E.coli O157:H7 kit. A study was conducted to validate the use of Assurance GDS MPX Top 7 STEC to detect low contamina on levels of the Top 7 serogroups of STEC (O26, O45, O103, O111, O121, O145, and O157) in 375 g beef trim samples. A total of 110 fresh 375g beef trim samples were inoculated with low levels of Shiga Toxigenic E. coli belonging to 1 of the Top 7 serogroups. Samples were enriched in 1,500 mL of pre‐warmed mEHEC® media at 42° C for 10 and 12 hours and tested with Assurance GDS MPX Top 7 STEC. All samples were culturally confirmed using immunomagne c separa on (IMS) and selec ve agar pla ng fol‐
lowed by PCR analysis of typical colonies for the presence of the eae and stx1 or stx2 genes as well as serogroup and bio‐ chemical iden fica on. The Assurance GDS method was demonstrated to be comparable to the reference culture method.
27
Julie Yang, 3M Company, St. Paul, MN, USA E‐mail: [email protected] Julie Yang received her Bachelor of Science and Master’s in Food Science from University of Minnesota, with a focus on molecular gene cs of Lactococcus bacteriophage resistance and Bifidobacterium produc on of an microbial pep de. Looking for new challenges, Julie joined the 3M Food Safety Global Technical Services team in 2007 where she provides tech‐ nical support and training for rapid pathogen tes ng to customers and colleagues world‐ wide. As the business pathogen subject expert ma er, she is leading customer‐inspired pro‐ jects to meet industry and regulatory needs. Julie is also a popular guest lecturer for the Food Safety and Microbiology laboratory course taught in the Food Science and Nutri on Department at University of Minnesota.
Performance of a New Molecular Pla orm for the Detec on of Salmonella and Escherichia coli O157 Current rapid pathogen methods are perceived to be complicated, lengthy or expensive. To address this need, a new molecular pla orm was designed. New Salmonella and E. coli O157detec on methods were evaluated for inclusivity and exclusivity. Frac onal recovery studies were performed in comparison to the ISO 6579 or ISO 16654 methods or to a commercial PCR method. Inclusivity and exclusivity rates of > 99% were determined. No significant differences were iden fied for the food matrices evaluated in comparison to ISO cultural or PCR methods. The new methods were deter‐ mined to be reliable and accurate and to offer advantages, including a quicker me to result compared to the cultural method and a smaller, more rugged instrument and less complex sample prepara on compared to the PCR method.
Alan Traylor, Business Manager‐Food Safety Products, USA E‐mail: [email protected]
Alan Traylor has over 30 years of experience in the development and marke ng of sensors, instruments and systems used in environmental, food and other analy cal applica ons. Mr. Traylor leads the marke ng and business development team for MOCON®’s new GreenLight® tes ng system for aerobic bacteria. Before MOCON, he was also involved in the successful deployment of sensor systems to defeat biological terrorism. Mr. Traylor holds a BS degree in Electrical and Electronic Engineering and a Masters in Business Administra on. MOCON® is a world leader in the measurement of gas and vapor permea on and packaging integrity for the food, medical and pharmaceu cal industries. The company is listed on the NASDAQ exchange under the cker symbol MOCO.
Oxygen‐Deple on Method for the Enumera on of Aerobic Bacteria‐ current status and further work Abstract: A novel approach to the enumera on of aerobic bacteria has been developed and marketed to the food safety community. The GreenLight® method is an equivalent to Total Viable Count (TVC). It has a ained AOAC‐RI as a PTM Validated Method and also has Microval cer fica on. A novel porphyrin sensor is able to detect very small changes in oxygen level in a food sample and relate it to bacterial load. Now, developments have been made to increase ease of use and throughput in order to produce 10 or more mes improvements in the me‐to‐result. This presenta on discusses the technical background of the system and examples of results ob‐ tained.
28 Cheryl Mooney, Microbiology Marke ng Manager – Food Safety Thermo Fisher Scien fic E‐mail: Cheryl.mooney@thermofisher.com
I have more than 20 years experience in the field of microbiology at Thermo Fisher Scien fic (previously Oxoid). Before joining the marke ng department in 2001, I worked ini ally in Oxoid’s research & development team and then later in UK sales. I have a BSc (Hons) degree in microbiology and a diploma in business management. As marke ng manager for food safety within the microbiology division, my key focus is to ensure that our products and services meet the evolving needs of our customers. I’m constantly looking for ways that enable the food safety microbiologist to get results faster and more efficiently, while keeping simplicity and value for money in mind. The Oxoid brand of the Thermo Fisher Scien fic Corpora on has been synonymous with microbiology for the last 50 years and served as a point of microbiological exper se. Our desire over the years has been for our products to be perceived as "developed by microbiologists for microbiologists". Now, we see laboratories searching for new ways to automate their work flows and are keen to adopt more advanced technologies. Thermo Scien fic, a premier brand of Thermo Fisher Scien fic, is associated with cu ng‐edge science
and innova on in many fields. We aim to build on our microbiology heritage using the MICROBIOLOGY Thermo Scien fic power of innova on to bring more to microbiology.
Thermo Scien fic Oxoid Listeria Precis™: a rapid, culture‐based method, validated to ISO 16140, for the detec on of Listeria monocytogenes and other Listeria species in food and environmental samples.
The Listeria Precis™ method comprises a single enrichment step followed by pla ng onto a single agar plate, with suspect colonies confirmed using a simple biochemical test. Results can be achieved in less than 48 hours; much faster than conven onal culture‐based methods, while being simple and cost effec ve to implement in almost any laboratory.
29
Sensory
Moderator: Dr. Grethe Hyldig, Na onal Food Ins tute, The Technical University of Denmark E‐mail: [email protected]
Grethe Hyldig, cand.lact., Ph.D., is Senior Researcher in Sensory Science and the leader of the sensory group in the Division of Industrial Food Research in DTU Food at the Technical University of Denmark(DTU). The sensory group has an extensive knowledge and experience within the area of sensory quality at the different steps in food chains especially of fish and fish products. The group works with consumer preferences, sensory quality and developing of sensory methods. An important part of the work is development of methods evalua ng ea ng quali‐ ty and shelf life of seafood, which makes it possible to predict quality and shelf life. Grethe is the core researcher in the development and valida on of the Quality Index Method (QIM) schemes interna onally. Her work includes communica on to the consumers and data mining of data from consumer studies. She gives lectures in academic courses at Copenhagen University and DTU. Training and supervision of BSc, MSc. and Ph.D. students is also included as well as, courses in basic sensory evalua on for the food industry. She gives na onal and interna onal QIM courses, both for implementa on and use of QIM and for developing new schemes at all academic levels. She is heading the organising commi ee of the Sixth European Conference on Sensory and Consumer Research in Copenhagen 2014.
30
Dr. Per Lea, Nofima, Norway
E‐mai: per.lea@nofima.no Sta s cian at Nofima ‐ Norwegian Ins tute of Food, Fisheries and Aquaculture Research for over 30 years and member of NMKL for over 20 of them. Main fields of
interest are design of experiments and General Linear Models (Analysis of variance and regression), both within sensory analysis as well as chemical/microbiological analyses. Co‐author of Analysis of Variance for Sensory Data (Wiley, 1997).
Sensory analysis: Measurement uncertainty and presentation of results When measurements are registered by human senses (taste/flavour, smell/odour, texture, tactile senstions, the human ear(!)) instead of chemical apparatus on a laboratory bench – what consequences does this have for the presentation and interpretation of the resulting data? The conclusions and advice from two NMKL Procedures on measurement uncertainty as well as ways of presenting the results from sensory analysis, respectively, are presented.
Chris an Dehlholm University of Copenhagen Faculty of Life Sciences E‐mail: [email protected] Chris an Dehlholm is an innova ve sensory scien st. He has been engaged within sensory science since 2002, holding posi ons both in the industry as well as at university. As project manager or consultant, he has been driving projects developing sensory quality control systems for various purposes. He has been working with sensory panels, products experts and consumers and performed projects as far as China. Through his educa onal background in food science and technology, both his bachelor and master thesis concerned sensory science. SENSORY Currently employed at University of Copenhagen in his final year of a PhD, he is working with rapid sensory evalua ons with industrial applicability. His research has addressed state‐of‐the‐art descrip ve methodologies, where he has proposed op mised ways of applying methods and interpre ng results.
Rapid sensory descrip ve methodologies – scope and applica ons
Rapid sensory tes ng methods are increasing in their popularity and applica ons in the industry and research. A number of new perceptual mapping methods, including the projec ve mapping variant Napping, Ultra Flash Profiling and the Flash Profile, have been proposed. This presenta on will focus on the differences and similari es between new rapid methodologies and their fields of applica on.
A convenient way to categorise methods is by varia ons in their sample approach. Methods can guide a more holis c or a more reduc onis c percep on, and where conven onal sensory profiles are more reduc onis c in a ribute selec on, newer rapid methodologies allow free choice vocabularies and broader individual inputs. Hence, other important differences between methods are the seman cs derived. Prac cal differences especially lie in the me spent on training and tes ng, but also on data treatment.
What method to apply in the sensory laboratory, truly depends on the ini al ques on. Does interest lie in the configura onal distances of the products or in the seman cs and is the study exploratory or meant to measure on specific concepts.
31
Dr. Grethe Hyldig DTU Food – Na onal Food Ins tute, Technical Univ. of Denmark E‐mail: [email protected] (Biography see page 30)
Quality index method – an objec ve rapid tool for determina on of sensory quality of fish.
Sensory evalua on is one of the most important method for assessing freshness and quality in the fish sector and in fish in‐ spec on services. Sensory methods performed in a proper way are a rapid and accurate tool providing unique informa on about food. European fisheries research ins tutes have developed such a tool, by which sensory assessment is performed in a systema c way with an objec ve quality assessment method called the Quality Index Method (QIM). It is foreseen that the QIM will be useful to give feedback to fishermen concerning the quality of their catch, which may in turn influence be er handling on board. The QIM is a promising method for quick and reliable assessment of the freshness of fish. It is expected to become the leading reference method for the assessment of fresh fish within the European community, as well as a part of labelling and iden fica on of the catch, par cularly in electronic auc oning of catch.
Dr. Ninino Federico, University of Udine, Italy E‐mail: [email protected]
Sensory characters of Cabernet Sauvignon dry red wine from Changli County (China)
The aromas of Cabernet Sauvignon red wines from eight vintages in Changli County (China) were evaluated by sensory analy‐ sis. A panel was trained to assess wine aroma by a ‘‘Le Nez du Vin” aroma it. Measurements of the olfactory threshold and aroma discrimina on ability of panelists were taken before and a er the training. Student t tests showed that training re‐ duced the olfactory threshold and improved the aroma discrimina on ability of the panelists. Sample wines were analyzed in duplicate by trained panelists over five sessions using a balanced, complete block design. Aroma descrip on of wine was ex‐ pressed by ‘‘modified frequency (MF)”. Principal component analysis (PCA) performed on ‘‘MF” data showed that Cabernet Sauvignon wines from Changli County were characterized by blackcur‐ rant, green pepper, smoke, redcurrant, cut hay, vanilla, bilberry, and cinnamon aromas.
32
Dr. Michele Suman, Barilla G.R. F.Ili.SpA E‐mail: [email protected] Michele Suman is Senior Scien st at Barilla SpA, headquartered in Parma, Italy. He also worked at the Na a Research Center (Ferrara, Italy) of Shell‐Montell Polyolefins, where he performed studies on catalysis for polyolefins produc on. Michele won the Na onal Prize for Young Researchers promoted by the Italian Chemistry Federa on (Federchimica) in 1998. In the same year he joined Barilla in the Packaging R&D Team and spent a long period studying food contact materials, developing ar ficial olfac ve systems and supramolecular sensors for packaging and food applica ons. In 2005 he received the PhD in Science and Technology of Innova ve Materials from University of Parma. Since the end of 2006 he is the head of Food Chemistry & Safety Research Department within the Food Research Labs of Barilla, working with public and private research centers on research projects in the field of food safety, sensing and mass spectrometry. Since 2007 he is Co‐Chairmen of Food Safety Pillar into Italian sec on of the European Technology Pla orm "Food For Life". He is member of working groups in the Italian Na onal Norma ve Organisa on (UNI) and in the European Commi ee for Standardiza on (CEN). He is also member of the Editorial Advisory Boards of Food Addi ves and Contaminants Journal, World Mycotoxin Journal and Interna onal Journal of Polymeric Materials. His scien fic produc on is documented by 40 presenta ons at na onal and interna onal conferences and 30 scien fic papers on interna onal journals.
Rapid and simultaneous analysis of xanthines and polyphenols as bi er taste markers in bakery products by FT‐NIR spectroscopy
The understanding of the molecular origin and the transduc on process of bi er taste on the human tongue represents a great challenge for scien sts because of the contemporaneous involvement of several receptors and many different target ‐compounds.
The food industry is con nuously looking for rapid methods useful to standardize different control parameters and has a direct interest into bi er‐tas ng substances, either for the iden fica on of nega ve off‐flavors or for the monitoring SENSORY of a desired organolep c quality. The exploita on of dedicated panel tests for sensory purposes is useful but it suffers of limita ons related to subjec vity, reproducibility and number of analysis per day. On the contrary, sophis cated analy cal approaches, such as LC‐MS, need trained personnel and are o en too expensive and/or me consuming.
The target of the present research work is therefore the development of a rapid technique based on a Fourier
Transform‐ Near InfraRed Spectroscopy (FT‐NIR) pla orm poten ally able to detect taste molecular markers in bakery commodi es, mainly xanthines (caffeine, theobromine, theophylline) and polyphenols (catechins, epicathechins) which are considered responsible for the bi er‐taste of coffee\cocoa\chocolate based products. In par cular, eleven types of commercial biscuits made with different amounts of various ingredients like cocoa beans, chocolate, nuts, cream, etc., were here considered. Relevant concentra ons of xanthines and polyphenols were, firstly, checked using a confirmatory LC‐ESI\MS‐MS procedure and then used for the calibra on of the FT‐NIR spectrophotometer by using par al least squares (PLS) regression. Values of the standard errors of predic on (lower than 10% either for polyphenols or xanthines) were comparable to the values of the standard errors of cross‐valida on. Coefficients of determina on indicated a good predic ve power in the calibra on model (R2 xanthines = 0.97, R2 polyphenols = 0.96) and a similar sa sfying discrimina ng power among different contents in the valida on models (R2 xanthines = 0.96, R2 polyphenols = 0.96). A tes ng phase of the generated model was executed by a comparison of further LC‐MS and sensory panel data with FTNIR responses recorded on unknown biscuits: concentra on differences between found and predicted levels were generally below 5 % and the best predictability was achievable in chocolate‐based biscuits. This methodology is able to work directly on solid products, has the poten al to be expanded on other categories of gusta ve molecular markers (like sugars in the case of sweet taste percep on) and can be conceived as applicable for a rou ne control of a standardized bi er taste quality in a real industrial produc on field.
33
Dr. Lene Meinert, DMRI, Danish Technological Ins tute, Denmark Email: [email protected]
Dr. Lene Meinert is a consultant, since 2008, at the Danish Meat Research Ins tute, department of raw meat quality. Lene has a master and Ph.D. from the University of Copenhagen within the field of food science with focus on sensory science, chemistry and microbiology. Today, she is involved in sever‐ al R&D projects including development of rapid sensory methods for indus‐ trial applica on, mathema cal model for shelf‐life predic on of fresh pork, bioac ve components in pork and bi‐products and finally boar taint detec‐ on by human nose method. Lene has wide experience in the coordina on
Holis c approach for consumer surveys Holis c by DMRI is an easy and rapid sensory method based on holis c everyday words like “tradi onal”, “familiar”, “exci ng” etc. By using these words in the sensory assessment, a link is created between the sensory department and the department for marke ng and/or sales. This link is unique and improves the understanding for sensory assessments within the company. Friland A/S, a producer of organic meat, used successfully Holis c by DMRI in a consumer survey concerning the appearance of marinated pork chops in a retail situa on. Friland A/S wanted to know which associa ons consumers of the future (18‐25 years) and consumers of today (35‐45 years) had when looking at retail packed pork chops marinated with seven different marinates varying in appearance. The first step was to select the words, and for this a CATA (Check all that apply) was used. A small group of consumers (7 persons in each of the 2 segments) were asked to mark all the words from a list of approx. 30 words that they could associate with the appearance of the marinates. The list both consisted of more posi ve words e.g. delicious and more nega ve words e.g. boring. The number of marks was subsequently summed, and the “top ten” words were selected for step 2. This second step is the “heart” of the method. Here 35‐40 consumers in each seg‐ ment graduated the ten words on a 15 cm unstructured line scale with the end points “not at all” to “very much”. So, look‐ ing at the seven marinates one by one, the consumers had to evaluate “how tradi onal” and “how surprising” etc., each marinate looked. Even though there was ten words for each of the seven marinates, a total of 70 words, the survey took only ten minutes to complete, and the consumers found the method very easy to perform. The survey generated a very clear answer for Friland A/S, as the consumers grouped the seven marinates in dis nct groups. The marinates with a bright colour and visual herbs and spices were described as “delicious”, “appe sing” “summer‐like” and “exci ng”. On the other hand, the more common marinates (e.g. BBQ Texas marinate) with bright colour but no herbs and spices were described as “boring”, “familiar”, “easy” and “tradi onal”. In this way Friland A/S got a clear indica on of which marinates to launch for the next barbeque season.
34 Dr. Thomas Lindblad, Royal Ins tute of Technology, Sweden E‐mail: lindblad@par cle.kth.se
Thomas Lindblad was born in 1945, got his Ph.D. in physics at the University of Stockholm in 1972 and became associate professor i 1974. Although originally the research was in nuclear and par cle physics, his main interest has been in measuring techniques and informa on processing. This was also the name of his department at the Manne Siegbahn Ins tute (MSI). Later on he became a professor at the Royal Ins tute of Technology (KTH) in Stockholm were he carried on the research. But at KTH he was also teaching environmental physics and became the Director of Undergraduate Studies. The electronic nose was developed during the me at MSI and KTH, won several prizes and was used in environmental sciences. Later on the company NoseLabs AB was formed and professor Lindblad is one of the co‐founders.
State of the art of the ar ficial nose; What we are up against, can do and expect The olfactory system of mammals is quite fantas c, but also has some limita ons. Thus, for example, bomb dogs cannot dis nguish between different explosives and also cannot work for hours and hours. Although an electronic nose today is not en rely as sensi ve as a dog’s nose, it can separate between different explosives and it never gets red. The electronic bomb nose once developed for the police, however, turned out to have a rather broad range of use. The fields included environmental sciences and diagnos cs of cancer. The electronic nose was therefore modified to meet specific requirements and is now applied to fields like quality control in produc on lines of organic material. The electronic nose is based on mul ‐ sensor technology using standard semiconductor sensors and an elaborate feature extrac on and iden fica on process. The background, development and state of the art of this electronic nose and some comparisons to other similar products will be discussed during this presenta on.
Dr. Urd Bente Andersen, Chair of the NMKL Norwegian Na onal Commi ee E‐mai: [email protected]
Quality control test for drinking water (NMKL 183) and use SENSORY of PanelCheck. NMKL Method No 183 is a descrip ve qualita ve and quan ta ve method for quality tes ng of drinking water, which is well suited for opera ons control and
quality control of large series of drinking water. It is tested collabora vely and was published in 2005. The method follows principles of sensorial quality control tests that have been developed for other foods such as dairy products. The samples are evaluated by comparing them to reference water that is described in a specifica on. Two to five assessors trained in typical tastes and odours of drinking water, give scores by using a scale from 0 to 4 where point 0 means no devia on and point 4 means strong devia on from reference water. If a devia on is detected, the assessors indicate this by using terms from a nomenclature of odours/tastes that might occur in drinking water. A short presenta on of the method and the results of the collabora vely study will be given. The nomenclature describes odours/tastes like earthy/mouldy, fishy, cucumber, snow, petrol, plas c or medicine. These fragrances can be created by using chemicals, and the method describes how to make actual chemical delu ons. Most of the chemicals are toxic and concerns have been raised about the possible risk posed by svallowing the tas ng sample. Therefore toxicolists were asked to do a safety assessment of the actual chemicals, and their conclusions will be presented. To get reliable results it is important to do a follow‐up of the assessors. The method describes how to use control cards to check repeatability of assessors and the panel. Another possibility is to use the sta s cs so ware PanelCheck (Nofima, Ås, Norway) for determining panel reliability, and an example of using PanelCheck for analyzing drinking water data will be demonstrated.
35 Poster Abstracts, Tuesday 9 May P1 Op miza on and Valida on of a commercial ELISA kit for the analysis of T‐2 and HT‐2 toxins in cereal flours. Lucie RACAULT, Nestec Ltd, Nestlé Research Center, Switzerland E‐mail: [email protected]
Rapid methods like ELISAs represent nowadays a rac ve tools to lighten sample prepara on while increasing the sample throughput. These methods are highly beneficial at factory level allowing more frequent safety controls as well as quicker decisions for raw materials acceptance.A compe ve ELISA (NeogenVeratox® for T‐2/HT‐2 toxin) was op mized and validated to quan fy T‐2 + HT‐2 in cereals flours (wheat, oat, rye, barley, and corn) and corn‐by product (corn gluten). Its sensi vity al‐ lows achieving an LOD of 10 µg/kg and a LOQ of 20 µg/kg for T‐2+HT‐2 toxins with results being obtained within 30 minutes. The ELISA kit is highly specific to T‐2 and HT‐2 and did not demonstrate cross‐reac ons with the others mycotoxins from trichothecene family. Results demonstrated that this analy cal method is reliable and very valuable for monitoring occurrence of T‐2 and HT‐2 in cereals flours.
P2 Fast Mul Element Screening with ICP‐MS Barbro Kollander and Joakim Engman, Na onal Food Agency,Box 622, 751 26 UPPSALA E‐mail: [email protected]
A fast mul element screening method has been adapted for the semi quan fica on of 70 elements in food. The screening method gives an es mate of the chemical composi on of the sample which could be used in applica ons such as informa on of nutri on value or contamina on. The method could also be used as a first step in the analysis of an unknown sample. The analy cal instrument ICP‐MS (induc vely coupled plasma mass spectrometry) is able to detect elements with masses between 6 and 240 in the sample, and the described method is a combina on of the semi quan ta ve so ware of the instrument, and the rou ne method used in our laboratory for the quan fica on of elements in food. Different types of reference materials have been evaluated with sa sfactory results, mostly within 80 to 120 % of the cer fied value, which is far be er than ex‐ pected for a semi quan ta ve method.
P3 Isotope ra o analysis as tool to determine the origin of food products Søren Dalby, Bruker Daltronics, Denmark E‐mail: [email protected]
Scien fic disciplines like Food Chemistry, Geochemistry, Paleontology are not only interested in the total concentra on of an element but also in the determina on of isotopic ra os. The isotope ra os can help to determine the age or the origin of sam‐ ples. The verifica on of the origin of food products like honey, olive oil, wines is important as original, high quality products are some mes blended with cheaper ones. Different elements can be used to determine the origin of natural food products with the help of isotopic ra os. Quadrupole ICP‐MS as an analy cal mul ‐element technique can help to characterize the composi on of samples and isotopic ra os of elements. To achieve accurate results, the challenge for ICP‐QMS is to obtain a required precision below 0.1%. Differ‐ ent approaches to improve the precision of the isotope ra o analysis with ICP‐QMS are discussed.
36 P4 Evalua ng Porous Materials for Directly Sampling Pes cides from Produce Surfaces Us‐ ing Direct Analysis InReal Time (DART) High Resolu on Mass Spectrometry Elizabeth Crawford*, Brian Musselma IonSense, Inc. Saugus, MA, USA *Corresponding author‐E‐mail: [email protected],
Rapid screening of pes cides present on the surface of fruits and vegetables has been facilitated by using direct analysis in real me (DART) open air high resolu on accurate massmass spectrometry. These experiments focus on the use of various materials to collect pes cides from large objects, including plants and produce commodi es by using a swabbing sampling approach and then direct analysis of the swab material. Evalua on of the efficiency of various polymeric foam and co on swabs for capture of analytes will be examined. Suitability of different materials as both sampling and desorp on ioniza‐ on support will be reported. These experiments build on the original pes cide screening experiments where polyethylene foam was used as both the collec on and desorp on substrate for screening small fruits and nuts for pes cides using “Transmission‐mode”DART‐MSanalysis.
P 5 Quan ta ve Analysis of Carbendazim and other Pes cides in Fruit Juices by Direct Analy‐ sis in Real Time (DART®) Mass Spectrometry Elizabeth Crawford*, Brian Musselman IonSense, Inc. Saugus, MA, USA *Corresponding author ‐ E‐mail: [email protected]
Rou ne pes cide and fungicide use in the United States, as well as abroad warrants the need for analy cal techniques that can rapidly screen and quan fy residues in order to efficiently screen products against maximum residue limits (MRLs) before reaching the consumer market. Of par cular interest in the United States, carbendazim, which is not allowed at any levels on citrus fruits in the US was recently found in imported orange juice from Brazil where the use of that fungicide is legal. Ambient ioniza on offers the ability to screen fruit juice samples directly in seconds and with automated sample introduc on quan ta‐ ve measurements can be assessed using direct analysis in real me (DART®) mass spectrometry. Limits of detec on using the DART ioniza on quan ta ve method coupled with Q Exac ve and API 4000 QTRAP mass spec‐ trometers were below 10 ppb for a 10 pes cide mixture detected in a variety of fruit juices, as well as for carbendazim directly analyzed from fruit juices. A number of the orange juices from the EU (5 juices), India (4 juices) and USA (3 juices) were screened for carbendazim and three juices all purchased in the EU tested posi ve for the fungicide at levels ranging from < 5 ppb up to 21 ppb.
P 6 Rapid development of mul ‐residue GC‐QQQ methods using a new 1000+ component MRM database Juan‐Luis Aybar1, Chris Sandy 2, Chin‐Kai Meng3 1Agilent Technologies Spain S.L., Carretera N ‐ VI, Km. 18,2, Las Rozas, Madrid 28230, Spain 2 Agilent Technologies Ltd., 610 Wharfedale Road, Winnersh,Berkshire, RG41 5TP, UK 3 Agilent Technologies Inc., 2850 Centerville Rd, Wilmington, DE 19808‐1610, USA John Lee [email protected]
Pes cide residue analysis is a challenging task poten ally requiring the search for hundreds of target compounds in a wide variety of complex matrices. GC/MS Tandem Quadrupole (GC/QQQ) instruments provide excellent sensi vity and selec vity in order to analyze trace organic contaminants in complex sample matrices. The availability of pre‐configured and pre‐tested GC/ QQQ methods can simplify and speed‐up method development and having access mul ple op mized MS/MS transi ons for
37 data acquisi on is important in order to avoid poten al matrix interferences.
The new Pesticides and Environmental Pollutants MRM Database from Agilent has an average of 8 optimized MS/MS transitions for more than 1000 organic contaminants. Macro tools incorporated in the database enable the development of a customized multi-residue MRM method in minutes. Additionally, the database provides 3 pre-configured GC Methods with locked retention times for all target analytes. These methods incorporate capillary flow technology and back flush to provide additional chro- matographic method robustness.
P7 Development and valida on of an UHPLC‐MS/MS based method for mul ‐mycotoxin analysis in cereals and nuts Elisabeth Varga a, Thomas Glauner b, John Lee b, Franz Berthiller a, Rudolf Krska a, Rainer Schuhmacher a, Michael Sulyok a a Center for Analy cal Chemistry, Department for Agrobiotechnology (IFA‐Tulln), University of Natural Resources and Applied Life Sciences, Vienna, Konrad Lorenz Str. 20, 3430 Tulln, Austria b Agilent Technologies Sales & Services GmbH & Co. KG, Chemical Analysis Group, Hewle ‐Packard‐Str. 8, 76337 Wald‐ bronn, Germany John Lee [email protected]
A new mul ‐mycotoxin UHPLC‐MS/MS screening method was developed and validated for raw cereals, like wheat, oat, and maize, as well as nuts like almonds, hazelnuts, peanuts, and pistachios. Samples were extracted with an acidified acetonitrile/water mixture on a rotary shaker. 5 µL of the 1:1 diluted raw extract were then directly injected.
Apparent recoveries and matrix effects were evaluated by spiking blank samples of model matrices before extrac on on one level in triplicate, and a er extrac on on mul ple levels.
The use of UHPLC improved the chromatographic resolu on for the target analytes and poten ally reduced matrix effects. The Dynamic MRM acquisi on mode allowed for maximized dwell mes and easy method setup and implementa on. One posi ve and one nega ve run covered in total 242 mycotoxins . LODs were determined and were in the low µg kg‐1 range with RSD’s below 15 % for most analyte‐matrix combina ons.
P8 A PHOSPHATASE INIHIBITION ASSAY ‐OKATEST‐ FOR QUANTITATIVE DETERMINATION OF OA‐TOXINS GROUP IN MOLLUSCUS BIVALVES: COLLABORATIVE STUDY Domínguez E.,Smienk H., Calvo L., Razquin P., Mata L. ZEU‐INMUNOTEC, C/Bari 25 Dpdo. 50197. Zaragoza, SPAIN. [email protected]
The toxicity of OA‐toxins group (Okadaic acid, DTX‐1, DTX‐2 and DTX‐3) is directly related to their inhibitory ac vity against protein phosphatase (PP) enzymes. OkaTest is a colorimetric phosphatase inhibi on assay for determina on of OA‐toxins in shelfish.
An interna onal collabora ve study was carried out to evaluate OkaTest method performance. A total of 8 materials, includ‐ ing mussels, scallops, clams and cockles were analysed by 16 laboratories.
The es mated reproducibility standard devia on (SR) was from 10.7 to 23.2 µg/Kg, with reproducibility rela ve standard devia‐ on (RSDR) values between 7.6 % and 13.2 %. The HORRAT values, obtained were between 0.4 and 0.6. The results obtained in this valida on study indicate that the colorimetric phosphatase inhibi on assay, OkaTest, is suitable for determina on of the OA‐toxins group. OkaTest complies with the current legisla on requirements (EC 15/2011) and can be used for monitoring this group of toxins.
38 P9 Colorimetric method for nitrites trace analysis in milk products Marine Nicolas (a), Janique Richoz‐Payot (a) and Eric Poitevin (a) Quality & Safety Department, Nestlé Research Center, Lausanne, Switzerland [email protected]
Nitrites and nitrates are found in a variety of food as naturally occurring ions, which are part of the nitrogen cycle, with drink‐ ing water and vegetables being substan al sources of nitrite and nitrate intake. Nitrite can also be formed by nitrate reduc on during food processing leading to significant contamina on in finished product. Looking at nitrite implica on in either blue baby syndrome by reac on with hemoglobin, or as carcinogeni N‐nitroso com‐ pounds by reac on with secondary or ter ary amines present in the body, nitrite intake is of health concern. A rapid colorimetric method has been developed for milk products, using commercial kits a er sample prepara on aiming at removing matrix effects. The method shows suitable performance, in terms of linearity of response, LoD and LoQ, selec vity, accuracy and measurement uncertainty.
P10 Rapid analysis of DON (deoksynivalenol) using dips ck –kit Chris n Plassen*, Per‐Erik Clasen, Aksel Bernho Na onal Veterinary Ins tute, Oslo, Norway E‐mail: chris n.plassen@ve nst.no
The aim of the project was to test dips ck kit to analyze samples of barley and oats for the grain producers. Chromatographic methods are me‐consuming and therefore expensive analysis, and the grain mill industry wanted to have an opportunity to differen ate the grain quality from the different deliveries. We decided to use dips ck kit from two different manufacturers. Both methods are immunological tests for determina on of DON in cereals. Before analyzing the samples we performed a standard valida on at different concentra on. We analyzed 60 samples of barley and oat using both kits. Finally we compared the dips ck‐results with results from GC‐MS analysis of the same samples. The conclusion was that one of the test‐kit had much more accordance to the GC‐MS results than the other, and the grain de‐ liveries now use this kit for control.
P 11 Fast element screening and origin analysis of edible oils by TXRF spectroscopy Armin Gross, Hagen Stosnach Bruker Nano GmbH, Schwarzschildstrasse 12, 12489 Berlin, Germany armin.Gross@bruker‐nano.de
In order to ensure that dietary intake is providing adequate levels of essen al elements, these trace elements must be deter‐ mined accurately in food stuff. In addi on, certain forms of elements can be toxic, which requires con nous monitoring during food processing. The most common analy cal techniques for the analysis of trace elements in edible oils or other food material are ICP‐AES and ICP‐MS. But as these analy cal techniques demand a laborious and me‐consuming sample prepara on, their suitability for a fast screening of large sample batches is limited. In this paper the opportuni es and limita ons of total reflec on X‐ray fluorescence (TXRF) analysis for the quan fica on of nutri on‐relevant and toxic trace elements in edible oils are summarised. In the first part different prepara on methods were developed and validated by using reference standard samples. In the second part different edible oils from several regions in Europe were analyzed for trace metal content a er microwave ashing and compared by mul variate analysis. We conclude that TXRF in combina on with microwave ashing is a rapid and cost‐effec ve method for origin analysis and quality control of edible oils.
39 P 12 Development and valida on of an UHPLC‐MS/MS based method for mul ‐mycotoxin analysis in cereals and nuts Elisabeth Vargaa, Thomas Glaunerb, John Lee b, Franz Berthillera, Rudolf Krskaa, Rainer Schuhmachera, Michael Sulyoka a Center for Analy cal Chemistry, Department for Agrobiotechnology (IFA‐Tulln), University of Natural Resources and Applied Life Sciences, Vienna, Konrad Lorenz Str. 20, 3430 Tulln, Austria b Agilent Technologies Sales & Services GmbH & Co. KG, Chemical Analysis Group, Hewle ‐Packard‐Str. 8, 76337 Wald‐ bronn, Germany Juan Aybar juan‐[email protected]
A new mul ‐mycotoxin UHPLC‐MS/MS screening method was developed and validated for raw cereals, like wheat, oat, and maize, as well as nuts like almonds, hazelnuts, peanuts, and pistachios. Samples were extracted with an acidified acetonitrile/water mixture on a rotary shaker. 5 µL of the 1:1 diluted raw extract were then directly injected. Apparent recoveries and matrix effects were evaluated by spiking blank samples of model matrices before extrac on on one level in triplicate, and a er extrac on on mul ple levels. The use of UHPLC improved the chromatographic resolu on for the target analytes and poten ally reduced matrix effects. The Dynamic MRM acquisi on mode allowed for maximized dwell mes and easy method setup and implementa on. One posi ve and one nega ve run covered in total 242 mycotoxins. LODs were determined and were in the low µg kg‐1 range with RSD’s below 15 % for most analyte‐matrix combina ons.
P13 Increasing selec vity in LC/MS/MS analysis using techniques such as MRM3 (MS/MS/MS), differen al ion mobility and high resolu on LC/MS/MS Dr Stephen Lock, ABSCIEX [email protected]
The demand for speed and cost reduc on in food analysis has meant that sample prepara on is o en simplified. Techniques such as liquid /liquid extrac ons or QuEChERS are commonly employed in Food Tes ng but the resul ng extracts are more complex leading to matrix interferences which can in turn lead to increased number of false posi ves in food tes ng and issues with quan ta on. There is therefore a need to increase the selec vity of detec on in LC/MS/MS, to get around the issues of matrix interferences but s ll maintain speed of analysis and the sensi vity needed to reach the regulatory limits. This talk will discuss several different ways to overcome these issues using various types of mass spectrometry techniques. Techniques including MRM3 and ion mobility separa on using the new differen al ion mobility interface will be discussed and examples where these techniques have benefits will be shown. In addi on the principles of high resolu on LC/MS/MS will be described together with examples of where this technique has been applied to food tes ng.
P14 The detec on of polyphenolics in food and drinks by LC‐MS Dr Stephen Lock , ABSCIEX [email protected]
Catechins are polyphenolic an oxidant plant metabolites found in a variety of foods including fruits, wine, beer, chocolate, and most abundantly in tea. By reac ng with free radical forming compounds before they can cause cell damage, an oxidants protect the body against oxida ve stress and as such these compounds are now been considered for their health benefits and can be present in some dietary supplements. This study shows the use of LC‐MS for determina on of the 8 known catechins in samples such as tea extracts and wines. The method demonstrates the efficient separa on and detec on and characteris c of these compounds by LC‐MS analysis and uses advances in HPLC column phases and high pressure separa ons to improve sen‐ si vity and speed up analyses.
40 The samples inves gated in this study included tea and wine samples. Tea samples were dissolved in 70% methanol at 70ºC, filtered and diluted before injec on. Wines were just filtered before injec on. Injec ons were separated by reverse phase chromatography using a small par cle size reverse phase HPLC column in order to speed up analyses. Detec on and quan fica on was achieved by Mul ple Reac on Monitoring (MRM) method and electrospray ioniza on. The LC‐MS method developed in this study has been shown to be appropriate for the simultaneous quan fica on of catechins and gallic acid in complex matrixes. Commercially available polyphenolics were obtained, standards prepared and samples analyzed by reverse phase chromatography using a new method incorpora ng a small par cle size HPLC column and a more rapid HPLC separa on as well as the scheduled MRM™ algorithm. The use of the small par cle column was shown to speed up the separa on and increase sensi vity over a tradi onal longer. The sensi vity varied with polyphenolic with limits of detec on always in the low parts per billion range. When this method was applied to the analysis of wine and tea samples it was shown to be robust and sensi ve producing linear responses, r value > 0.98 over the range tested.
P15 Mul ‐residue on‐line sample prepara on LC‐MS/MS method for the determina on an bio cs in animal ssue Katerina Bousova a and Klaus Mi endorf b a Thermo Fisher Scien fic, Food Safety Response Center, Im Steingrund 4‐6, 63303 Dreieich, Germany; katerina.bousova@thermofisher.com b Thermo Fisher Scien fic, Food Safety Response Center, Im Steingrund 4‐6, 63303 Dreieich, Germany; klaus.mi endorf@thermofisher.com
A fast and reliable mul ‐residue method is reported for the iden fica on and quan fica on of thirty‐five different an bio cs from seven different classes (aminoglycosides, sulfonamides, macrolides, quinolones, tetracyclines, lincosamides and trimethoprim) in animal ssue. Automated on‐line sample clean‐up was applied using turbulent flow chromatography (TLX system), directly coupled to a mass spectrometer (MS/MS) for sensi ve and specific detec on. The method involved a simple extrac on using mixture of acetonitrile and trichlorace c acid, followed by centrifuga on and filtra on. A er this preliminary step, the extract was injected into the TLX‐ESI‐MS/MS using op mized turbulent flow and HPLC condi ons. Single‐laboratory valida on of the method was carried out according to the Direc ve 2002/657/EC, clearly demonstra ng the suitability of this method for quan ta ve determina on of this wide range of an bio cs in animal ssue. A small survey, which covered samples of muscles and organs of all food producing species demonstrated the robustness of this method and its suitability for enforcement purposes.
P16 Rapid development of mul ‐residue GC‐QQQ methods using a new 1000+ component MRM database Juan‐Luis Aybar1, Chris Sandy 2, Chin‐Kai Meng3 1 Agilent Technologies Spain S.L., Carretera N ‐ VI, Km. 18,2, Las Rozas, Madrid 28230, Spain 2 Agilent Technologies Ltd., 610 Wharfedale Road, Winnersh, Berkshire, RG41 5TP, UK 3 Agilent Technologies Inc., 2850 Centerville Rd, Wilmington, DE 19808‐1610, USA juan‐[email protected]
Pes cide residue analysis is a challenging task poten ally requiring the search for hundreds of target compounds in a wide variety of complex matrices. GC/MS Tandem Quadrupole (GC/QQQ) instruments provide excellent sensi vity and selec vity in order to analyze trace organic contaminants in complex sample matrices. The availability of pre‐configured and pre‐tested GC/ QQQ methods can simplify and speed‐up method development and having access mul ple op mized MS/MS transi ons for data acquisi on is important in order to avoid poten al matrix interferences.
41 The new Pesticides and Environmental Pollutants MRM Database from Agilent has an average of 8 optimized MS/MS transitions for more than 1000 organic contaminants. Macro tools incorporated in the database enable the development of a customized multi-residue MRM method in minutes. Additionally, the database provides 3 pre-configured GC Methods with locked retention times for all target analytes. These methods incorporate capillary flow technology and back flush to provide addi onal chromatographic method robustness.
P17 Fast GC‐FID method for the determina on of iridoids in Plantago species Silvia Sponza, Alexandra Dockal, Sabrina Wlaschitz, Chlodwig Franz, Remigius Chizzola Ins tute for Botany and Pharmacognosy, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria [email protected]
Plantago apecies are perennial herbs of the Plantaginaceae family and they are widely distributed in Europe and America (1). The leaves of some species, like for example Plantago lanceolata, and their polar extracts are used in folk and phytotherapy medicine for a wide range of diseases that include problems related with diges ve and respiratory organs, skin diseases and pain relief (2). These species are also im‐ portant for the animal feeding point of view as their medicinal effect can improve the physiological condi ons of the animals (3). Literature data reported that the iridoid aucubin and catalpol can be used as analy cal markers to determine the quality of extracts from different sources (4‐5). The iridoids aucubin and catalpol are commonly analyzed in plant extracts by HPLC. Therefore our objec ve was to develop an alterna ve gas chromatographic method for their separa on and quan fica on. The analyses were carried out using a GC‐FID (6890N Network GC system Agilent Technologies, Palo Alto, CA, USA) equipped with a DB‐5 narrow column (10m x 0,1mm id.; 0,17μm film thickness; Agilent). The method parameters are: split 1:100, injec on volume 0,2μL, inlet temperature 280°C, oven temperature was increasing from 200 to 280°C at 15°C/min and held for 5min at 280°C. Before analysis, the plant extracts were silylated. GC‐MS and comparison with reference compounds were used for the compound iden fica on. For the quan fica‐ on, phenyl‐D‐glucoside was used as internal standard. The fast GC‐FID method with a run of 10min gives a good separa on of aucubin and catalpol, as well as a quan fica on of them.
Galvez, M.; Mar n‐Cordero, C.; Houghton, P.J.; Jesus Ayuso, M. An oxidant Ac vity of Methanol Extracts Obtained from Plantago Spe‐ cies. J. Agric. Food Chem. 2005, 53, 1927‐1933 Samuelsen, A.B. The tradi onal uses, chemical cons tuents and biological ac vi es of Plantago major. A review. J. Ethnopharmacol. 2000, 71, 1‐21. Rumball, N.; Keogh, R.G.; Kane, G. E.; Miller, J.E.; Claydon, R. B.; „Grasslanad Lancelot“ Plantain (Plantago lanceolata L.). N. Z. J. Agric. Res. 1997, 40, 373‐377. Rischer, M.; Adamczyk, M.; Ratz, H.; Hose, S.; Marchesan, M.; Paper, D.H.; Franz, G.,; Wolf‐Heuss, E.; Engel, J. Quan ta ve Determina‐ on of the Iridoid Glycosides Aucubin and Catalpol in Plantago lanceolata L. Extracts by HPTLC and HPLC. J. Planar Chrom. 1998, 11, 374‐378. Ronsted, N.; Franzyk, H.; Molgaard, P.; Jaroszewski, J.W.; Jensen, S.R. Chemotaxonomy and evolu on of Plantago L. Plant Syst. Evol. 2003, 242, 63‐82.
P18 Migra on of cadmium and lead concentra ons in ceramic materials used for food: Four year monitoring programme (2008‐2011) with ICP/MS Beril Atamer, Mehtap K. Evcimen, Pelin Ulca, A&T FOOD LABS. [email protected]
Ceramic cookware and ar cles used for serving food and drink are frequently coloured with pigments which can give rise to migra on of metals. Regulatory limits range from 1.5 to 4.0 mg/L for lead and 0.1 to 0.3 mg/L for cadmium depending on the size and geometry of the ceramic ar cles (in some cases controlled as mg/dm2). In this study, tes ng was conducted with an acidic food s mulant (4% ace c acid), following defined procedures for repeat‐use ar cles. A total of 330 samples comprising plates, mugs, cups, kitchenware, bowls and trays were obtained over 4 years from 2008‐2011. A er following prescribed tests for intended condi ons of use (temperature & me) analysis was conducted by ICP‐MS to determine levels of lead and cadmi‐ um in acidic extracts. Over the 4 year period a total of 3 samples (1%) were not compliant with the regula ons giving lead concentra ons exceeding the maximum of 0.8 mg/dm2.
42 P19 Detec on of pork adultera on of raw and cooked meat products using PCR Authors: Handan Balta, Ilknur Cagin, Pelin Ulca A&T FOOD LABS. [email protected]
There is a need for consumer protec on to monitor meat and meat products for deliberate or inadvertent cross‐ contamina on with pork. The economic adultera on of meat products is difficult to visually detect in the raw state and impos‐ sible a er cooking. However, the polymerase chain reac on (PCR) offers a highly sensi ve and very specific technique based on detec on of porcine DNA. In this poster we report the performance characteris cs of SureFood® ANIMAL ID Pork Sens PLUS kits for the detec on of pork adultera on of raw and cooked Turkish meat products. The performance of kits was as‐ sessed using fresh meat samples of pork, beef, chicken, turkey and comminuted meat products (meatball, doner, cured spiced beef and sucuk). The meat products were analysed in the raw state and a er cooking for 20 min at 200oC. For raw and cooked meats it was demonstrated that the kit could reliably detect the addi on of pork at a level of 0.1%.
P20 Inclusivity and exclusivity performance of a Scorpion® probe‐based real‐ me PCR assay for Salmonella. Authors: Jacqueline M. Harris, Andrew D. Farnum, Morgan Wallace, Daniel Demarco, Stephen Varkey, Thomas Moeller Presenter Name & Title: Thomas Moeller, DuPont Qulicon [email protected]
The purpose of this study was to evaluate the inclusivity and exclusivity capabili es of a Scorpion® probe‐based real‐ me PCR assay for genus Salmonella detec on using the same primer sequence as the commercial BAX® System assay for Salmonella. Four hundred and nine different strains across the six main subgenera of Salmonella were tested for inclusivity. The exclusivity panel included 44 different non‐Salmonella species. For inclusivity and exclusivity tes ng, each pure culture was inoculated into prepared BHI media and incubated overnight at 37°C. Inclusivity cultures were diluted in culture media to a cell density of 105cfu/mL and exclusivity cultures were diluted to approximately 108cfu/mL. The commercial BAX® System assay for Salmonel‐ la was tested for comparison. The real‐ me assay, as well as, the BAX® System assay for Salmonella returned posi ve results for the 409 inclusivity strains and nega ve results for the 44 non‐Salmonella strains. This study demonstrated that the real‐ me PCR assay for Salmonella is rapid and sensi ve.
P21 Comparison of two official methods for the enumera on of Escherichia coli in Manila clams (Tapes philippinarum) Authors: Silva Rubini1, Sonia Casaro2, Laura Bianchi1, Rossano Melloni1, Guido Govoni3, Giorgio Galle 1 Silva Rubini [email protected]
The classifica on criteria of the areas where shellfish are harvested, are laid down by the UE Regula ons 853/2004, 854/2004, 2073/2005. According to European Legisla on, the enumera on of Escherichia coli in bivalve molluscs must be performed with the ISO method 16449‐3:2005 (MPN). The aim of this work is to compare MPN and pour plate on TBX tests (both ISO methods) to ascertain whether the la er could subs tute the former. Because of the very short shelf‐life of bivalve molluscs the analysis to determine their fitness to human consump on should be as fast as possible. The MPN method requires two media and a two‐day analysis, whereas the TBX method requires one medium and one‐day analysis only. We tested 56 samples of Manila clams with both methods. The results were compared with McNemar’s test and by calcula ng rela ve Sensi vity and Specificity. The two methods do not show any significant differences.
43 P22 Single laboratory valida on of a microbiological method for screening of inhibitory residues in sheep, goat and cow milk Sanz D., Domínguez E., Razquin P. & Mata L. Elena Domínguez [email protected] ZEU‐INMUNOTEC, C/Bari 25 Dpdo. 50197. Zaragoza, SPAIN.
ECLIPSE 3G is a qualita ve microbiological test designed for detec on of veterinarian an bio c residues in raw, heated and powder milk. The test has been validated following the ISO 1369:2003 (E) regula on and the Guidelines for the valida on of screening methods for residues of veterinary medicines. Ruggedness, detec on capability and applicability to milk of different species were determined for this test. The LODs calculated for 18 an bio cs were below or equal to the MRL. (Penicillin G 2‐3ppb, tetracycline 100ppb, sulfathiazole 50ppb, tylosin 40ppb, neomycin 1500ppb, lincomycin 150ppb). ECLIPSE 3G can be used as screening method for cow, sheep and goat milk, as it is able to detect a wide range of an microbials at MRL EU levels.
P23 A phosphatase inhibi on assay –OkaTest ‐ for quan ta ve determina on of OA‐Toxins group in molluscus bivalves: collabora ve study Domínguez E., Smienk H., Calvo L., Razquin P., Mata L. Elena Domínguez [email protected] ZEU‐INMUNOTEC, C/Bari 25 Dpdo. 50197. Zaragoza, SPAIN.
The toxicity of OA‐toxins group (Okadaic acid, DTX‐1, DTX‐2 and DTX‐3) is directly related to their inhibitory ac vity against protein phosphatase (PP) enzymes. OkaTest is a colorimetric phosphatase inhibi on assay for determina on of OA‐toxins in shelfish. An interna onal collabora ve study was carried out to evaluate OkaTest method performance. A total of 8 materials, including mussels, scallops, clams and cockles were analysed by 16 laboratories.
The es mated reproducibility standard devia on (SR) was from 10.7 to 23.2 µg/Kg, with reproducibility rela ve standard devia on (RSDR) values between 7.6 % and 13.2 %. The HORRAT values, obtained were between 0.4 and 0.6. The results obtained in this valida on study indicate that the colorimetric phosphatase inhibi on assay, OkaTest, is suitable for determina on of the OA‐toxins group. OkaTest complies with the current legisla on requirements (EC 15/2011) and can be used for monitoring this group of toxins.
P24 Microbial screening methods for an bio c residues M. G. Pikkemaat, J.W.A. Elferink* , H. J. van Egmond RIKILT ‐ Ins tute of Food Safety, Wageningen University and Research centre P.O. Box 230, 6700 AE Wageningen. Alexander Elferink: [email protected]
Monitoring of food products from animal origin for the presence of an microbial residues is preferably done using microbial screening methods, because of their high throughput poten al and cost effec veness. RIKILT has developed a series of improved tests, dedicated to an bio c residue screening in typical animal ssue matrices at the EU Maximum Residue Limits. These NAT and SCAN tests are mul plate bacterial growth inhibi on assays. A sample is applied on five individual test plates, each comprising a balanced combina on of test‐organism, growth medium and synergis c compounds, yielding them preferably sensi ve to a specific group of an bio cs. A er overnight incuba on the test plate showing the largest inhibi on zone reveals the group specific iden ty of the residue, which significantly reduces confirmatory efforts and costs.
44 The methods have been validated according to EU Commission Decision 2002/657/EC and are currently used in Na onal Monitoring programs. Addi onally they are used in private monitoring programs, and disseminated to a number of European and non‐European countries. RIKILT offers support on implementa on and quality assurance of this new genera on of microbial inhibi on tests.
P25 Valida on of Charm® Enrofloxacin Test for Raw and Pasteurized Milk M.Sc., Clinical Miicrobiologist KatariinaPekkanen, Finnish Food Safety Authority Evira katariina.pekkanen@evira.fi
Charm® Enrofloxacin Test (Charm Sciences Inc., U.S.A.) is a rapid one step assay for the detec on of enrofloxacin residues in raw and pasteurized milk, egg and ssue. in this valida on work the assay was validated for tes ng milk samples. Charm® EnrofloxacinTest was validated to supplement our current method, Delvotest® SP‐NT (DSM, Netherlands) that doesn’t detect enrofloxacin on a sufficient (ie. EU‐MRL) level. The valida on study was done according to Comission Decision 2002/657/2002 using spiked samples of different concentra ons of enrofloxacin to find the detec on capacity (CCβ –value) and 20 parallel samples to confirm the limit of detec on (LOD). The test specificity was assayed spiking milk samples with other an bio cs or by several interfering agents. The assay was also tested for robustness by using old/spoiled milk or incuba ng the test strips for a longer or shorter me than recommended. The test was found well suited to its purpose.
P26 Performance of a New Molecular Pla orm for the Detec on of Salmonella and Escherichia coli O157 Yang, Julie [email protected] 3M Company, St. Paul, MN, United States.
Current rapid pathogen methods are perceived to be complicated, lengthy or expensive. To address this need, a new molecular pla orm was designed. New Salmonella and E. coli O157detec on methods were evaluated for inclusivity and exclusivity. Frac onal recovery studies were performed in comparison to the ISO 6579 or ISO 16654 methods or to a commercial PCR method. Inclusivity and exclusivity rates of > 99% were determined. No significant differences were iden fied for the food matrices evaluated in comparison to ISO cultural or PCR methods. The new methods were determined to be reliable and accurate and to offer advantages, including a quicker me to result compared to the cultural method and a smaller, more rugged instrument and less complex sample prepara on compared to the PCR method.
P27 Real‐ me PCR Detec on of Pathogenic E. coli Strains Florian Waldherr, Dana Nelson, Almut Richter and Ma hias Kuhn CONGEN Biotechnologie GmbH, Berlin, Germany; [email protected] Ronald Niemeijer, R‐Biopharm AG, Darmstadt, Germany r.niemeijer@r‐biopharm.de
Pathogenic Escherichia coli worldwide repeatedly cause foodborn outbreaks with high mortality rates. Latest case was the outbreak of serotype O104:H4 in Germany during the summer months of 2011. Near future legal regula ons makes an approach by monitoring for defined serotypes and serogroups. E.g. the USA were ini ally focusing on serotype O157:H7 and recently extended the spectrum to the “Big 6” including serogroups O26, O103, O45, O111, O121 and O145. Serotyping is based on surface an gen structures which are not directly correlated to the pathogenic poten al. Pathogenicity in E. coli is based on virulence factors like genes for toxin produc on (stx1/stx2) and others. Real‐ me PCR is the most powerful tool for food safety tes ng. In the context of pathogenic E. coli serogroups can be specifically detected by qPCR. Addi onally a more risk based approach based on the detec on of pathogenicity factors like stx can also be performed.
45 P28 Determina on of Vet Drugs in Meat and Fish with Quechers Extrac on and LCMSMS detec on Paolo Ma eini, Francesco Rosi, Alessandro Minia , Tommaso Cafissi, Pierre Metra Silliker Italia S.p.A. [email protected]
1 Introduc on Following the QuEChERS philosophy, Silliker Italia S.p.a. has validated a set of simple, rapid and reliable methods to determine residue of vet drugs in meat and fish. These methods consists in simultaneous extrac on and LCMSMS detec on of 63 Veterinary Drugs belonging to 8 different families (Sulphamidics, Macrolides, Beta La amics, Quinolones, Coccidiostats, Ionofores An helmin cs, An mico cs, Tranquillizers).
2 Experimentals The methods consist in a simple extrac on with direct detec on in LCMSMS using MRM and ESI(+) or ESI(‐). The validated matrixes are fish and meat ssues, but the study is con nuing and the method will be extended to other commodi es (e.g. dairy). For each matrix, two concentra on levels have been inves gated, one of the levels being the LOQ.
3 Conclusions A er valida on studies, the trueness, linearity and precision of the methods have been fully assessed. Sensi vity and consequently LOQs (1 µg/Kg to 10 µg/Kg) fits the requirements of Reg. 37/2010/EU. Expanded Uncertainty has been es mated with metrological approach, and it ranges from 32% to 55%.
P29 Development and valida on of an UHPLC‐MS/MS based method for mul ‐mycotoxin analysis in cereals and nuts
Elisabeth Varga a, Thomas Glauner b, John Lee b, Franz Berthiller a, Rudolf Krska a, Rainer Schuhmacher a, Michael Sulyok a a Center for Analy cal Chemistry, Department for Agrobiotechnology (IFA‐Tulln), University of Natural Resources and Applied Life Sciences, Vienna, Konrad Lorenz Str. 20, 3430 Tulln, Austria b Agilent Technologies Sales & Services GmbH & Co. KG, Chemical Analysis Group, Hewle ‐Packard‐Str. 8, 76337 Waldbronn, Germany
A new mul ‐mycotoxin UHPLC‐MS/MS screening method was developed and validated for raw cereals, like wheat, oat, and maize, as well as nuts like almonds, hazelnuts, peanuts, and pistachios.
Samples were extracted with an acidified acetonitrile/water mixture on a rotary shaker. 5 µL of the 1:1 diluted raw extract were then directly injected.
Apparent recoveries and matrix effects were evaluated by spiking blank samples of model matrices before extrac on on one level in triplicate, and a er extrac on on mul ple levels.
The use of UHPLC improved the chromatographic resolu on for the target analytes and poten ally reduced matrix effects. The Dynamic MRM acquisi on mode allowed for maximized dwell mes and easy method setup and implementa on. One posi ve and one nega ve run covered in total 242 mycotoxins . LODs were determined and were in the low µg kg‐1 range with RSD’s below 15 % for most analyte‐matrix combina ons.
46 Participants
First name Surname Company Country Kjell Rehn 3M Sweden Anja Sanberg 3M Denmark Julie Yang 3M USA Thomas Andersen 3M Norway Anne‐Lise Grøholt 3M Norway Simen Rutlin‐Sæter 3M Norway Beril Atamer A&T FOOD LABS. Turkey Mehtap K. Evcimen A&T FOOD LABS. Turkey Pelin Ulca A&T FOOD LABS. Turkey Stephen Lock AB SCIEX UK Kasper Oland AB SCIEX Germany Karleen le Louedec ADM Cocoa BV Netherlands Valen ne Digonnet AFNOR CERTIFICATION France Stéphanie Sammartano AFNOR CERTIFICATION France Juan Aybar Agilent Technologies UK John Lee Agilent Technologies UK Gorm Karstens Agilent Technologies Denmark Gunnar Warnke Agilent Technologies Denmark Sune Eriksson AOAC Europe Sweden Pierre Metra AOAC Europe France Klaus Reif AOAC Europe Germany Eric Vernon AOAC Europe France Roger Wood UK Krystyna McIver AOAC INTERNATIONAL USA Annie Kaalby Arla Foods Denmark Jan‐Olof Jörnryd Arlafoods Sweden Michele Suman Barilla G.R. F.lli SpA, Italy Frederic Mar nez Bio‐Rad France Jane e Handley Biocontrol Systems England Erling Markussen Biolab A/S Denmark Jawaid Baig BIOTECON Diagnos cs GmbH Germany Benjamin Junge BIOTECON Diagnos cs GmbH Germany Gordon van't Slot Bruker Dalthronics Netherlands Søren Dalby Bruker Daltronics Denmark Per Nilsson Bruker Daltronics Denmark Armin Gross Bruker Nano GmbH Germany Flemming Hansen Danish Technological Ins tute Denmark Niels L. Nielsen Danish Veterinary and Food Administra on Denmark Karine Bertrand DANONE RESEARCH France
47 First name Surname Company Country Lene Meinert DMRI ‐ Danish Meat Research Ins tute Denmark Susanne Mansdal DMRI Teknologisk Ins tut Denmark Camilla Bejerholm DMRI, Teknologisk Ins tut Denmark Grete Hyldig DTU Food, Na onal Food Ins tute, Technical Univ. Denmark Thomas Møller DuPont Qualicon Denmark Nicole Hsiao Ecolean Development AB Sweden Roman Lilleorg Estonian Veterinary and Food Laboratory Estonia Charlo a Engdahl Axelsson Eurofins Sweden Fredrik Westerberg Eurofins Food & Agro Tes ng AB Sweden Kari Johanne Einvik Eurofins Food & Agro Tes ng Norway AS Norway Heidi Camilla Sagen Eurofins Food & Agro Tes ng Norway AS Norway Nina Skammelsrud Eurofins Food & Agro Tes ng Norway AS Norway Ragnhild Skyrud Eurofins Food & Agro Tes ng Norway AS Norway Birthe Madvig Soerensen Eurofins Steins Laboratorium A/S Denmark Anne‐Helen Larsen Felleskjøpet Rogaland Agder Norway Maria Speichert Felleskjøpet Rogaland Agder Norway Anu Kallinen Finnish Customs Laboratory Finland Elina Vatunen Finnish Customs Laboratory Finland Minna Anthoni Finnish Food Safety Authority Evira Finland Satu Hakola Finnish Food Safety Authority Evira Finland Katariina Pekkanen Finnish Food Safety Authority Evira Finland Annika Pihlajasaari Finnish Food Safety Authority Evira Finland Jan‐Erik Carlsson Food Diagnos cs AB Sweden Maiken Lynge Eriksen Food Diagnos cs ApS Denmark Katrine Nørrelund Food Diagnos cs ApS Denmark Hanna Tidblom Food Diagnos cs ApS Denmark Arne Højgaard Jensen Fødevarestyrelsen Denmark Jy e Warming Fødevarestyrelsen Denmark Osman Inay Goverment Food Control Lab Turkey Zhiyong Li Guangdong Entry‐Exit Inspec on & Quaran ne Bureau China Katriina Luoma HK Ruokatalo Oy Finland Jürgen Möller Independent consultant Sweden Silvia Sponza University of Veterinary Medicine Vienna Austria Elizabeth Crawford IonSense, Inc. / MS Consult Denmark USA Tore Vulpius IonSense, Inc. / MS Consult Denmark USA Is tuto Zooprofila coSperimentale della Lombardia e Silva Rubini Italy dell'Emilia Romagna Feng Xue Jiangsu Entry‐Exit Inspec on & Quaran ne Bureau China Thomas Lindblad KTH ‐ Physics Department and NoseLabs AB Sweden Ann Kris n H. Gule Kystlab as Norway Anne Kris n Gussiås Kystlab as Norway Juhana Riskala Labema Oy Finland Iiris Ylöstalo Labema Oy Finland Dietrich Maede Landesamt für Verbraucherschutz Sachsen‐Anhalt Germany Franklín Georgsson Ma s ohf Iceland Anna Pala Vignisdó r Ma s ohf Iceland
48
First name Surname Company Country Rodrigo Granja Microbio cos Labs. Brasil Alan Traylor Mocon Inc. (Biolab) Denmark Barbro Kollander Na onal Food Administra on Sweden Ylva Sjögren Na onal Food Administra on Sweden Monica Ferm Na onal Food Agency Sweden Mia Hallgren Na onal Food Agency Sweden Sara Åkerström Na onal Veterinary Ins tute Sweden Marc Barret Neogen Europe Ltd Scotland, UK Pauline McCrystal Neogen Europe Ltd Scotland, UK Jennifer Rice Neogen Europe Ltd Scotland, UK Lucie Racault Nestec Ltd, Nestlé Research Center Switzerland Thomas Bessaire Nestec S.A. Switzerland Marine Nicolas Nestec S.A. Switzerland Chris an Bruno Nexus A/S Denmark Urd Bente Andersen NMKL Norway Ulla Edberg NMKL Sweden Harriet Wallin NMKL Finland Nina Bakkelund NMKL/NordVal Norway Hilde Skår Norli NMKL/NordVal Norway Mats Carlehøg Nofima AS Norway Per Lea Nofima AS Norway Sven Qvist NordVal Denmark Mika Tuomola NordVal Finland Kjell Hauge Norwegian Food Safety Authority/NordVal Norway Arne Holst‐Jensen Norwegian Veterinary Ins tute Norway Chris n Plassen Norwegian Veterinary Ins tute Norway Inngunn Anita Samdal Norwegian Veterinary Ins tute Norway Michael Wainø Novozymes Denmark Helvi Mustonen Orion Diagnos ca Oy Finland Tiina Tomperi Orion Diagnos ca Oy Finland Kris na Eriksen Svendsen Oxoid & Remel, Thermo Scien fic Denmark Anders Thomsson Oxoid & Remel, Thermo Scien fic Sweden Philippe Leroux PhL Consultant France Cecilia Wallen n Lindberg Probi AB Sweden Torben Skou Professionshøjskolen Metropol, Denmark Chris ne Gutschelhofer R‐Biopharm AG, Germany Ronald Niemeijer R‐Biopharm AG, Germany Adrianne Klijn RDLS Nestle Research Centre Switzerland Alexander Elferink RIKILT‐Ins tute of Food Safety, Wageningen UR Netherlands Nathalie Smits RIKILT‐Ins tute of Food Safety, Wageningen UR Netherlands Elisabeth Hammer ROMER LABS Division Holding GmbH Austria Alois Schiessl ROMER LABS Division Holding GmbH Austria Ervin Tanyi ROMER LABS Division Holding GmbH Austria Auréline Tilly Royal Canin France Mélanie Tréhiou Royal Canin France
49
First name Surname Company Country Josefin Sterky ScanBi Diagnos cs Sweden Gregory Bone Sensient Colors LLC USA Connie Johansen SFK Food Denmark Russ Flowers Silliker USA Olena Korytniuk SLL SOCTRADE Ukraine Me e Bendixen Statens Serum Ins tut, SSI Diagnos ca Denmark Árný Árnadó r Syni Laboratory service Iceland Harpa Hlynsdó r Syni Laboratory service Iceland Lars Kristoffersson Tetra Pak Packaging Solu ons AB Sweden Mikael Turunen The Absolut Company Sweden Ola Jörgensen The Absolut Company ‐ Pernod Ricard Sweden Willy Bjørklund Thermo Fisher Scien fic Denmark Katerina Bousova Thermo Fisher Scien fic Germany Michal Godula Thermo Fisher Scien fic The Czech Republic Inger Ødegård TINE SA Norway Abraham Aharon TNUVA ‐ TENE NOGA DAIRY Israel Tonje A ret Trondheim Kommune Analysesenteret Norway Camilla Moen Trondheim Kommune Analysesenteret Norway Chris an Dehlholm University of Copenhagen Denmark Alessandro Bedini University of Parma Italy University of Udine department of food science and Ninino Federico Italy technology Alexandra von Trotha University of Wuppertal Germany Sonja Latvakoski Valio Ltd, R&D Finland Anne‐Maria Riihimäki Valio Ltd, R&D Finland Elena Domínguez Zeu Inmunotec S.L. Spain Melanie Pa erson Life technologies UK Mikael Pedersen DTU Na onal Food Ins tute Denmark Cheryl Mooney Thermo Fisher Scien fic UK
50
Available NMKL Procedures
No 1, 2. Ed. 2005 Calibration and performance checking of laboratory balances
No 2, 1995 Performance check and in-house calibration of thermometers
No 3, 1996 Control charts and control materials in internal quality control in food chemical laboratories
No 4, 3. Ed. 2009 Validation of chemical analytical methods
No 5, 2. Ed. 2003 Estimation and expression of measurement uncertainty in chemical analysis
No 6, 1998 Yleiset ohjeet aistinvaraisten laboratorioiden laadunvarmistukseen (avail. Danish/Finnish)
No 7, 1998 Checking of UV/VIS spectrophotometers
No 8, 4. Ed. 2008 Measurement of uncertainty in quantitative microbiological examination of foods
No 9, 2. Ed. 2007 Evaluation of method bias using certified reference materials.
No 10, 2001 Control of microbiological media
No 11, 2.Ed. 2010 Procedure for sensory analysis of drinking water
No 12, 2002 Guide on sampling for analysis of foods
No 13, 2003 Volumetric control
No 14, 2004 SENSVAL: Guidelines for internal control in sensory analysis laboratories
No 15, 2004 Temperature control in microbiological laboratories
No 16, 2005 Sensory quality control
No 17, 2006 Guidelines for requirement specifications for food analyses
No 18, 2006 The use of reference materials, reference strains and control charts in a food microbiological laboratory
No 19, 2007 Guideline for sensorial analysis of food containers/packages
No 20, 2007 Evaluation of results from qualitative methods
No 21, 2008 Guide for sensory analysis of fish and shellfish
No 22, 2008 Considerations regarding evaluation of immunochemical test kits for food analysis
No 23, 2008 Guide on quality assurance in microbiological laboratories (replacing NMKL Report No. 5)
No 24, 2010 Guidelines for quality assurance for food chemical laboratories
No 25, 2012 Recovery information in analytical measurement
51 SPONSORS
AOAC Europe and NMKL /NordVal thank the support of these sponsors
52