coordinated research project (crp): d5.20 · analytical methods to strengthen national residue...
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Coordinated Research Project (CRP): D5.20.36
Development of radiometric and allied analytical methods to strengthen national
residue control programs for antibiotic and anthelmintic veterinary drug residues
REPORT OF THE 1ST RESEARCH
COORDINATION MEETING
Vienna, 19-23 October 2009
NOTE
The material in this document has been agreed by the participants and has not been edited by the IAEA. The views expressed remain the responsibility of the participants and do not necessarily reflect those of the government(s) of the designating Member State(s). In particular, neither the IAEA nor any other organization or body sponsoring this meeting can be held responsible for any material reproduced in the document.
Reproduced by the IAEA
Vienna, Austria, 2009
INTERNATIONAL ATOMIC ENERGY AGENCY
Report of the 1st Research Coordination Meeting (RCM) on the Development of
radiometric and allied analytical methods to strengthen national residue control programs for antibiotic and anthelmintic veterinary drug
residues
Vienna, Austria 19-23 October 2009
Working Material Produced by the IAEA
Vienna, Austria, 2009
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Introduction
The 1st Research Coordination Meeting (RCM) for the Coordinated Research Project (CRP) on the development of radiometric and allied analytical methods to strengthen national residue control programs for antibiotic and anthelmintic veterinary drug residues was held at the IAEA Headquarters, Vienna, 19 to 23 October 2009.
The Meeting was chaired by Philip Kijak (U.S. Food and Drug Administration, Center for Veterinary Medicine) and the scientific secretary was Rajendra Patel (IAEA). A list of participants (Annex A) and the meeting Agenda (Annex B) are attached.
The Meeting recalled that the main purpose of the CRP was to assist National Reference Laboratories of FAO and IAEA member states in meeting the need for effective and appropriate monitoring methods for residues of selected antibiotic and anthelmintic veterinary medicines through the development and application of screening methods that exploit the advantages (robustness, sensitivity, transferability) of radiotracer detection methods, in conjunction with confirmatory techniques using stable-isotope labelled molecules. The CRP also addresses issues pertaining to sources of natural antimicrobial compounds likely to impact the regulatory framework for veterinary drug residues and the development of new tools to understand and assess the environmental impact of the use of veterinary medicines.
Background
In many developing countries, rapid demographic changes and rising incomes have increased the demand for high value food commodities. Increasing international trade in these products has therefore led to dramatic growth in the livestock and aquaculture sectors. Changes in production practices and exacerbating factors such as climate change have resulted in a rise in disease outbreaks and an increased use of agrochemicals, including veterinary medicines, with the concurrent environmental contamination and development of microbial and parasitic resistance to these compounds. At the same time, awareness of food safety is rising and many importing countries have implemented food control regulations to guarantee the quality and safety of imported foods for their consumers.
Many developing countries have also taken steps to put in place control systems that encourage the responsible use of veterinary medicines to combat drug resistance and comply with international standards. However, they find it difficult to access the required know-how and skills, thus hindering their ability to access international markets for food products of animal origin. One significant constraint is the capacity of laboratory services to generate surveillance data using analytical methods validated to international standards.
The CRP was developed to address these issues after extensive consultation with stakeholders, including participants in previous CRPs, regulatory and scientific experts and authorities in member states, and consultants, to identify areas of concern to less developed countries. The report of the Consultants meeting is attached (Annex C).
Objectives of the Coordinated Research Project
Expected Research Outputs
1. Detailed knowledge on pharmacokinetics of veterinary drugs in aquaculture fish species not previously investigated.
2. Improved screening and confirmatory methods fit for use in National Residue Control Programs by utilizing isotopic and related technologies such as immunochemical, microbial, chromatographic and mass spectrometric techniques.
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3. Data on the sources and distribution of the naturally occurring antimicrobial compound, chloramphenicol, and an assessment of the impact of this information on the current regulatory framework for the control of chemical contaminants in food and animal feedstuff.
Expected Outcomes
1. Improved food/feed quality and safety in FAO/IAEA Member States and enhanced access for developing countries to major global food markets.
2. Availability of data to assist regulators in the development of new guidelines and regulations pertaining to food safety and environmental impact of veterinary drugs.
3. A unique and global network of scientific expertise addressing complex and important food safety challenges.
Objectives of the First Research Coordination Meeting
The objectives of this 1st RCM were to discuss the plans and proposed research of individual participants; to facilitate a broader understanding of the relationship each participant has to the overall objectives of the CRP; to promote interaction between the participants, and; to prepare recommendations and guidelines to facilitate project tasks and to agree on a common approach and way forward. The meeting recognized that research work plans, activities and the CRP framework may be refined if necessary in order to better meet the objectives of the CRP.
RCM Presentations
Andrew Cannavan, IAEA
Mr. Andrew Cannavan presented the work done during a previous CRP (D.3.20.22) on the development of strategies for the effective monitoring of veterinary drug residues in livestock and livestock products in developing countries (2002-2006). The main outcome of that project was the development of methods that have been applied in member states (e.g. Thailand, Sri Lanka, and Brazil). A main recommendation from the final meeting was that further work was needed to meet the increasing demand for robust, rapid and affordable analytical methods, especially in developing countries.
Philip Kijak, USA
Dr. Philip Kijak described the work done at the Division of Residue Chemistry, US Food and Drug Administration, to develop and validate methods for drug residues and other chemical contaminants in animal derived foods and animal feeds. He outlined the program to develop modern analytical methods to replace obsolete assays, the development of methods for the detection of antibiotic residues in distiller’s grains and research conducted to understand the metabolism of drugs in aquatic species.
Contract Holders Presentations
Each contract holder presented their recent and proposed future work under the project, with an emphasis on how their research will contribute to achieving the CRP objectives. The summaries of the presentations are attached (Annex D).
1. Guilherme Nogueira, Brazil: Development of multi-dot-ELISA for enrofloxacin and ciprofloxacin residues in
chicken tissue.
2. Alfredo Montes Nino (on behalf of Rodrigo Granja), Brazil:
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Development of RIA for florfenicol in fish tissue.
3. Grace Murilla, Kenya:
Preparation of antibodies in appropriate species for development of immunoassays
for antibiotics and anthelmintics.
4. Sasitorn Kanarat, Thailand:
Development of a microbiological screening method for the detection of residues of
antimicrobial substances.
5. Preeni Abeynayake, Sri Lanka: Development of HPTLC screening methods for anthelmintic and antibiotic
veterinary drug residues.
6. Guihua Liu, China:
Development of methods for aminoglycosides (LC-MS/MS and HPTLC).
7. Yang Shuming, China:
Development of multi-residue screening and confirmatory methods for
benzimidazoles and their metabolites.
8. Orlando Lucas, Peru: Development of multi-residue LC-MS/MS method for benzimidazoles and
avermectins.
9. Jin-Wook Kwon, Korea: Environmental impact studies on veterinary medicines: development of methods and
preparation of “incurred” soil, manure and water samples.
10. Aida Ben Mansour, Tunisia:
Pharmacokinetics of quinolones and tetracyclines in Tunisian farmed fish using
labelled drugs.
11. Tserendorj Enkhtuya, Mongolia:
Investigation of possible presence of chloramphenicol in pasture plants and in
animals fed with these plants.
Agreement Holders Presentations
These were made at a joint session with the Train-the-Trainers Workshop on Screening/Post-screening Techniques for Veterinary Drug Residues at the Agency’s Laboratories, Seibersdorf.
1. Chris Elliott, UK: Rapid screening techniques suitable for the CRP.
2. Heinrich Meyer, Germany:
Immunoassays: assay designs - amplification techniques, reagent development and
future potential.
3. Hubert De Brabander, Belgium:
Chromatographic and Mass Spectrometric Techniques.
4. Thomas Kuhn, Austria:
Method Validation.
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Conclusions of the First RCM
1. All projects have been rigorously scrutinized to ensure they meet high scientific standards and fulfil the objectives of the CRP.
2. The CRP addresses the following major elements for the safe and appropriate use of veterinary drugs to ensure food safety by:
a) utilizing isotopic techniques for pharmacokinetic studies, residue screening and confirmatory methods;
b) providing new tools to understand and assess the environmental impact of veterinary drugs;
c) assessing the impact of naturally occurring antimicrobial compounds on veterinary drug regulations.
3. There is good potential for high level dissemination of project findings through scientific publications and presentations to a wide range of stake holders (scientific and regulatory communities).
4. The use of radionuclides is vital to generate new knowledge, especially in the pharmacokinetics of veterinary drugs used in aquaculture, to underpin effective control programs.
5. The network has concluded that additional agreement holders are required to facilitate the research activities.
Recommendations1
1. The Agency should assist to make available the required radio-labelled drugs.
2. The regular evaluation of research progress and the renewed outline of future scientific work are of utmost importance. The Agency should put great emphasis on the regular organisation of RCMs.
3. Several projects need intense exchange of knowledge and experience. Where possible, the member states and the Agency, through its technical cooperation initiatives, should work together to facilitate bilateral scientific visits. These are of high strategic importance for the success of a number of the CRP projects.
4. Additional meetings should be organized concurrently with other scientific congresses.
5. The Agency should provide the necessary resources for additional agreement holders and technical cooperation.
___________________________________________________________________________
1 See Annex E for detailed recommendations and agreed work plans for the first phase of the project (May 2009 – December 2010)
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Agreed Action Plan and Logical Framework
Action Plan (Activities)
Activity 2009 2010 2011 2012 2013 2014
Advertise the CRP (December 2008).
Receipt of research contract and agreement proposals. Award contracts and sign agreements by end April 2009.
X
Organise 1st RCM (October 2009) to discuss overall CRP work plan, agree on research protocols, governance, quality assurance, record keeping and reporting.
X
Award Research Contracts X X X X X
Award Technical Contracts X X
Phase 1: 18 months work programme X X
Organise 2nd RCM in March/April 2011 to review the work conducted in Phase 1 based on progress reports and presentations. Develop the detailed work plan for Phase 2 and ensure that the CRP objectives are met.
X
Phase 2 : Work programme X X
Organise 3rd RCM (Sep/Oct 2012) to review work conducted in Phase 2 and agree final phase 3 work programme.
X
Phase 3: Work programme X X X
Final RCM: late 2013 / early 2014 to review Phase 3 work and prepare a TECDOC and /or research papers to an appropriate journal.
X X
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Logical Framework
Project Design Elements Verifiable
Indicators
Means of
Verification
Important
Assumptions
Overall
Objective:
Enhance food safety and
opportunities for
international trade in foods
of animal origin.
Rejections of
consignments
after
inspections.
Reports to
national
authorities.
Commitment by all
participating partners to
report on national data.
Specific
Objective:
Develop rapid / robust
screening / confirmatory
methods for antimicrobial
and anti-parasitic drug
residues, based on nuclear
and related techniques.
Monitoring
programs for
surveillance of
antimicrobial
and anti-
parasitic drug
residues.
Reports to
national
authorities.
Commitment by all
participating partners to
report on national data.
Outcomes: Methods for testing
antimicrobial and anti-
parasitic drug residues in
operational use in Member
States.
Surveillance
reports
published.
Laboratory
reports
Continued commitment
by all partners to provide
laboratory data.
Outputs: Validated methods,
harmonized SOPs and
QA/QC protocols
recognized.
SOPs and
validation
dossier
produced.
Reports submitted
to the IAEA and
national
authorities.
Continued commitment
by all partners.
Activities: Consultant’s agreement on
specific technologies to be
developed.
Consultant’s
meeting.
Meeting report
and
recommendations.
Consultants identified,
available and meeting
held.
Research contract and
agreement holders
identified and contracts /
agreements signed.
First RCM. Meeting report. Continued commitment
by all parties.
Work programme to
develop, validate methods
for antibiotics and
anthelmintic drug residues.
1st, 2nd and 3rd
RCM.
Meeting reports. Continued commitment
by all parties.
Establish QA /QC protocols
for routine operation of
developed methods.
3rd RCM Meeting reports
and
recommendations.
Continued commitment
by all parties.
Prepare SOPs, scientific
papers and TECDOC.
Final RCM Meeting report,
TECDOC, SOPS
and papers
published.
Continued commitment
by all parties.
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Annex A: List of Participants
Mr Thomas Kuhn Österreichische Agentur für Gesundheit und Ernährungssicherheit GmbH (AGES) Spargelfeldstrasse 191
1220 WIEN, AUSTRIA Email: [email protected]
Prof Hubert De Brabander Université de Gent; Faculté de médecine vétérinaire
Salisburylaan 133 9820 MERELBEKE, BELGIUM
Tel: 0032 9 2647460
Fax: 0032 9 2647492 Email: [email protected]
Mr Alfredo Montes Nino (on behalf of Mr Rodrigo Henrique Granja)
Microbioticos Laboratorio
Avenida Santa Isabel 2120
Caixa Postal 6175
13084-471 CAMPINAS, S.P. BRAZIL
Tel: 0055 19 32525236
Fax: 0055 19 32899690 Email: [email protected]
Mr Guilherme de Paula Nogueira Universidade Estadual Paulista (UNESP); Curso de Medicina Veterinaria; Departamento de Apoio, Producao e Saude Animal Rua Clovis Pestana, 793, 16050-680 ARACATUBA, SP, BRAZIL Email: [email protected]
Ms Liu (Linda) Guihua Chemical Analysis and Physical Testing Center, Shenzhen CDC 21 Tianbei 1st Road, Luohu District
Shenzhen 518020 P.R. China
Tel/ Fax: 00852 25601549
Email: [email protected]
Mr Shuming Yang 12 Zhongguancun Nandajie,
Beijing, 100081 China
Tel: +86-10-82106561
Prof Heinrich Meyer Weihenstephaner Berg 5
85354 FREISING, GERMANY Tel: 0049 8161 713508
Fax: 0049 8161 714202
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Email: [email protected]
Ms Grace Mabel Adira Murilla KARI - Trypanosomiasis Research Centre
Off Naivasha Road
P.O. Box 362 00902 KIKUYU
KENYA
Tel: 00254 20 2700545 Fax: 00254 154 32397
Email: [email protected]
Ms Enkhtuya Tserendorj
Ministry of Food and Agriculture; State Central Veterinary Diagnostic Laboratory P.O. Box 53-03 ULAANBAATAR 210153 MONGOLIAcom
enxee627@yahoo
Mr Orlando Lucas Ministerio de Agricultura; Servicio Nacional de Sanidad Agraria (SENASA) Avenida La Molina 1915
LIMA 12
PERU Tel: +511 3133300 1621
Home: +511 3626494
Mr Jin Wook-Kwon National Veterinary Research and Quarantine Service (NVRQS)
480 Anyang-6dong, Manan-gu ANYANG, Kyonggi-Do 430-016
KOREA, REPUBLIC OF
Prof. Preeni Abeynayake University of Peradeniya Faculty of Veterinary Medicine and Animal Science Department of Veterinary Clinical Studies PERADENIYA 20400 Sri Lanka
Dr Sasitorn Kanarat Director of Veterinary Public Health Laboratory Bureau of Quality Control of Livestock Products, Department of Livestock Development, Tivanon Road., Bank-Kadi, Pathum-Thani 12000 Thailand Tel: 662 936 9215 Fax: 662 936 9215
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Ms Aida Ben Mansour Centre National des Sciences et
Technologies Nucléaires (CNSTN) Technopole Sidi Thabet CEDEX
B.P. 204
1080 SIDI THABET TUNISIA [email protected]
Prof Chris Elliott Institute of Agri-Food and Land
School of Biological Sciences, Room 1209A David Keir Building, Stranmillis Road
Belfast' Northern Ireland
BT9 5AG United Kingdom
Tel: +44 2890 976549 Fax: +44 2890 976513
Dr Philip Kijak Acting Director, Division of Residue Chemistry
Centre for Veterinary Medicine
8401 Muirkirk Road
Laurel MD 20708
United States of America Tel: +1 301-210-4589
Mr David Byron IAEA
Tel: +43-1-2600-21638
Mr Andrew Cannavan IAEA
Tel: +43-1-2600-28395 [email protected]
Mr Josef Brodesser
(for part of the meeting)
IAEA [email protected]
Tel:+43-1-2600-26058
Mr Carl Blackburn
(for part of the meeting)
IAEA
43-1-2600-21639
Mr Nima Mashayekhi-Tabrizi (administrative secretary)
IAEA
[email protected] 43-1-2600-26061
Mr Rajendra Patel
Scientific Secretary
IAEA
[email protected] 43-1-2600-21672
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Annex B: Agenda
Day 1: Monday, 19 October 2009
08:00 Registration (UN Pass Office, Gate 1, Vienna International Centre.
All
09:15 Welcome & Opening Address. David H. Byron, IAEA
09:45 Introductions and Presentation of Participants. All
09:55 Election of Chairperson and Adoption of Agenda All
10:00 CRP background: objectives and scope of the RCM in line with the conclusions and recommendations from consultants meeting.
Rajendra Patel, IAEA
10:30 Work done and outputs of a previous CRP (D.3.20.22) on The Development of Strategies for the Effective Monitoring of Veterinary Drug Residues in Livestock and Livestock Products in Developing Countries (2002-2006).
Andrew Cannavan, IAEA
11:00 Break
11:15 Analytical Method Development at FDA Center for Veterinary Medicine.
Philip Kijak, USA
12:00 Lunch / Administrative Arrangements
Session 1: Contract Holder Presentations (Proposed Research work plan, summary of work
done to date and future work plan meeting the objective of the CRP).
14:00 Development of multi-dot-ELISA for enrofloxacin and ciprofloxacin residues in chicken tissue.
Guilherme Nogueira
Brazil
14:30 Development of RIA for florfenicol in fish tissue. Alfredo Montes Nino (on behalf of Rodrigo Granja)
Brazil
15:00 Development of immunoassays for antibiotics and anthelmintics: preparation of antibodies in appropriate species.
Grace Murilla
Kenya
15:30 Break
15:45 Development of a microbiological screening method for the detection of the multi-residues of antimicrobial substances.
Sasitorn Kanarat
Thailand
16:15 Development of HPTLC screening methods for anthelmintic and antibiotic veterinary drug residues.
Preeni Abeynayake
Sri Lanka
16:45 Development of methods for aminoglycosides (LC-MS/MS and HPTLC).
Guihua Liu
China
17:15 End of Day 1
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Day 2: Tuesday, 20 October 2009
08:45 Summary of Day 1 Rajendra Patel, IAEA
09:00 Development of multi-residue screening and confirmatory methods for benzimidazoles and their metabolites.
Yang Shuming
China
09:30 Development of multi-residue LC-MS/MS method for benzimidazoles and avermectins.
Orlando Lucas
Peru
10:00 Environmental impact studies on veterinary medicines: development of methods and preparation of “incurred” soil, manure and water samples.
Jin-Wook Kwon
Korea
10:30 Break
10:45 Pharmacokinetics of quinolones and tetracyclines in Tunisian farmed fish using labelled drugs.
Aida Ben Mansour
Tunisia.
Session 2: Presentations from Agreement Holders (a joint session with Train-the-Trainers
Workshop on Screening/Post-screening Techniques for Veterinary Drug Residues at the
Agency’s Laboratories, Seibersdorf.
11:45 Bus to Seibersdorf
13:00 Lunch at Seibersdorf
14:00 Rapid screening techniques suitable for the CRP. Chris Elliott
United Kingdom
14:40 Immunoassays: assay designs - amplification techniques, reagent development and future potential
Heinrich Meyer
Germany
15:20 Break
15:30 Chromatographic and Mass Spectrometric Techniques
Hubert De Brabander
Belgium
16:10 Methods Validation Thomas Kuhn
Austria
16:50 Discussion of work plans: Formation of work groups (led by Agreement Holders) to put together detailed work plans during Session 3.
All
17:15 End of Day 2, Return to Vienna.
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Day 3: Wednesday, 21 October 2009
08:45 Summary of Day 2 Rajendra Patel, IAEA
09.:00 Investigation of possible presence of chloramphenicol in pasture plants and in animals fed with these plants.
Tserendorj Enkhtuya
Mongolia
Session 3: Preparation of Work Plans, Recommendations and Meeting report.
09:45 Quality Assurance and Control guidelines for all CRP participants.
Thomas Kuhn, Austria and Rajendra Patel, IAEA
10:00 Formation of working groups
Group 1: Screening Assays (immuno and microbiological
assays)
Group 2:Chromatographic Techniques (HPLC / TLC/ Mass
spectrometry)
Ecotoxicology: (Environmental contamination and natural
sources)
Chairperson and Agreement Holders
10:15 Brainstorming Session 1: to identify tasks for the 2nd year of the project.
The tasks may include comparative assessment; benchmarking;
exchange of data; cooperation in method development, etc.
The outputs of this session are expected to be written suggestions for certain tasks to be performed by participants of each group – actually, a plan for coordinated research for the
next phase of the project
All
11:00 Break
11:15 Brainstorming Session 1 (continued) All
12:30 Lunch Break
14:00 Drafting Session 1. The objective of this session is to prepare presentations (one for each group) of the findings of Brainstorming Session 1.
Agreement Holders and participants of groups
15:30 Break
16:00 Presentation of Group 1, Q&A Chairperson of Group 1
16:30 Presentation of Group 2, Q&A Chairperson of Group 2
17:00 Discussion on outcomes of Brainstorming Session 1. All
17:30 End of Day 3
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Day 4: Thursday, 22 October 2009
08:45 Summary of Day 3 Rajendra Patel, IAEA
09:00 Drafting Session 2. The objective of this session is to prepare a consistent write-up of the findings of Brainstorming Session 1 (i.e. work plans for the next phase of the project).
All
10:15 Break
10:30 Review the written reports of the working groups. All
11:00 Brainstorming Session 2. The objective of this session is to define the number, the titles, the scope and the structure of Reports to be produced within this CRP, by each Contract Holder and cumulatively.
• The suggestions are expected to be based on the
outputs of Brainstorming Sessions 1.
All
12:30 Lunch
14:00 Drafting Session 3. The objective of this session is to prepare a consistent write-up of the proposals of Brainstorming Session 2
All
15:15 Break
15:30 Continue Drafting Session 3. All
16:30 Review the outputs of Drafting Session 3. All
18:00 End of Day 4
Day 5: Friday, 23 October 2009
08:45 Summary of Day 4 Rajendra Patel, IAEA
09:00 Discussion on the conclusions and recommendations of the meeting.( the draft will be prepared by the scientific secretary and distributed prior to this session)
All
10:30 Break
10:45 Finalizing conclusions and recommendations All
12:15 Lunch
13:30 Adoption of the Final Report of the 1st RCM. All
15:45 Any Other Business All
16:00 Closing Remarks and End of Meeting.
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Annex C
Consultants Meeting for the Coordinated Research Project on the Development of
Multiresidue Methods for Antibiotic and Anthelmentic Veterinary Drug Residues
16 to 19 September 2008, Vienna, Austria
Meeting Report
1. Purpose of the meeting
The purpose of the meeting was to advise the Food and Environmental Protection Subprogramme (FEP) of the Joint FAO/IAEA Programme for Nuclear Techniques in Food and Agriculture (NAFA) on the proposed Coordinated Research Project (CRP), Development
of multiresidue methods for antibiotic and anthelmentic veterinary drug residues to combat
drug resistance and promote food safety in developing countries.
2. Background
In many developing countries, rapid demographic changes and rising incomes have increased the demand for high value food commodities. Increasing international trade in these products has therefore led to dramatic growth in the livestock and aquaculture sectors. Changes in production practices and exacerbating factors such as climate change have resulted in a rise in disease outbreaks and increased use of agrochemicals including veterinary medicines, with the concurrent development of microbial and parasitic resistance to these compounds. At the same time, awareness of food safety is rising and many importing countries have implemented food control regulations to guarantee the quality and safety of imported foods for their consumers. Many developing countries have also taken steps to put in place control systems that encourage responsible use of veterinary medicines to combat drug resistance and comply with international standards. However, they find it difficult to access the required know-how and skills, thus hindering their ability to access international markets for food products of animal origin. One significant constraint is the capacity of laboratory services to generate surveillance data using analytical methods validated to nationally and internationally agreed standards.
Approaches for the detection of veterinary drug residues include microbiological, immunochemical, chromatographic and spectrometric techniques. While these may fulfil suitability criteria, such as method sensitivity, they require multiple time-consuming steps for extraction, sample clean-up or pre-concentration prior to measurement. These measurement techniques also often lack the robustness necessary for their successful application in developing countries. These issues can be addressed by developing methods utilising nuclear and related technologies for the detection of veterinary drug residues. These methods could also meet the need for robust analytical methods required by other international bodies such as the Joint FAO/WHO Codex Alimentarius Commission during the standards setting process.
3. Objectives
The objectives of the meeting were to:
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i. critically review the draft Contract Research Project Proposal. ii. identify robust nuclear and related technologies suitable for the screening and
confirmatory analysis of residues of veterinary medicines. iii. address issues relating to drug resistance by identifying anti-parasitic drugs of
importance in developing countries and to select suitable antimicrobials for methods development from the list provided by the Joint FAO/WHO/OIE Expert Meeting on Critically Important Antimicrobials.
iv. address the systems needed to assure the quality of research processes used in this CRP and provide guidelines to implement them.
v. assist with identifying suitable agreement and contract holders with suitable laboratories and resources.
4. Presentations and Discussions
The Provisional Agenda for the meeting was adopted as proposed (Annex 1). The meeting was opened by Mr. David Byron, Head, Food and Environmental Protection Section. He provided background information on the Joint FAO/IAEA Division and the IAEA Contract Research Programme, including the role of agreement and contract holders in the process.
Mr Andrew Cannavan presented the work done during a previous CRP (D.3.20.22) on The Development of Strategies for the Effective Monitoring of Veterinary Drug Residues in
Livestock and Livestock Products in Developing Countries (2002-2006). The main outcome of that project was the development of methods that have been applied in member states (e.g. Thailand, Sri Lanka, and Brazil). The main recommendation from the final meeting of the CRP was that further work is needed to meet the increasing demand for robust, rapid and affordable analytical methods, especially in developing countries.
The current situation in different regions (Southern Africa, Thailand and India) was discussed following presentations from the consultant experts, Prof Mathew Nindi, Dr Sasitorn Kanarat and Dr Vishweshwaraiah Prakash. Key points were:
i. Recognition that veterinary drug residues are a problem in commodities produced in many developing countries.
ii. The development of drug resistance is a problem in terms of reduced effectiveness of the drugs both in animal husbandry and for human health.
iii. There are problems with availability of analytical standards and reagents and often difficulty in obtaining those that are commercially available.
iv. Most control programmes are focussed on export markets and the need for affordable technologies to ensure domestic consumer food safety was highlighted.
Presentations on the technologies available for residues detection were made by Prof. Chris Elliott and Prof. Hubert De Brabander. Dr. Thomas Kuhn presented the regulatory framework in the EU for methods validation and performance criteria. Prof. De Brabander also presented the IT solutions used to teach and disseminate information at the Faculty of Veterinary Medicine, Ghent University. Detailed discussions concluded that:
i. For the benefit of the countries with limited resources, the CRP should include development of screening methods that do not require purchase of sophisticated equipment. Transfer of these methods to these countries would empower them to start establishing the national preliminary monitoring programs for veterinary drug residues.
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ii. The concept utilized in a previous CRP to develop a screening method using radioactive labelled streptavidin in a radio-immunoassay format should be further developed to provide screening assays for antibiotics and anti-parasitic drugs.
iii. Issues relating to extraction of residues from complex food matrices, sample clean-up and pre-concentration should be addressed by the CRP.
iv. Taking into account new developments in the field of high performance thin layer chromatography (HPTLC), the use of this technique for rapid detection of drug residues should be investigated as part of the CRP.
v. The use of stable isotope labelled internal standards is vital for successful development of robust analytical methods.
vi. The methods developed during the CRP should be validated using the relevant guidelines and detailed SOPs written using guidelines to be agreed upon at the first RCM.
vii. A protected website for interaction between IAEA, contract and agreement holders should be established.
viii. Appropriate experts should be invited to the RCMs to present up to date developments in the residues field to all participants. A component of training was felt to be important to the success of the project and this could best be achieved in tandem with RCMs.
ix. Up to 10 research contracts should be awarded to Member States submitting appropriate research proposals. Each Principal Scientific Investigator will have to submit his/her project highlighting current equipment and experience levels in their laboratory together with an indication of availability of adequate funds for relevant research activities. Research Agreement holders (3-4) will be invited to support the CRP with their expertise.
x. Technical contacts will be awarded to selected institutions to provide radioactive and stable isotope labelled reagents and internal standards for the CRP.
xi. The Agrochemicals Unit of the FAO/IAEA Agriculture and Biotechnology Laboratory at Seibersdorf will provide scientific and technical support to the CRP.
5. Conclusions and recommendations
Based on the FEP proposal and the activities needed to meet the objectives of the CRP, the consultants concluded that
i. In the context of rapid demographic and climate changes, international trade, human health, the development of drug resistance, food security and food safety, veterinary drug residues in food is a growing concern for developing countries. The meeting strongly supports the CRP submission (Annex 2) for approval and implementation, which should lead to an establishment of a harmonised network of expertise able to share knowledge and transfer technology to strengthen the national residue control programs of Member States so as to comply with harmonized international standards.
ii. In order to promote effective control policies to prevent/minimize drug resistance, the CRP should focus on anti-parasitic drugs widely used in developing countries and significant antibiotics highlighted by the Joint FAO/WHO/OIE Expert Meeting on Critically Important Antimicrobials.
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iii. The CRP should aim to develop for operational use assays using nuclear and related techniques that are rapid, robust, affordable and sustainable. The approach developed in the previous CRP using radioactive labelled streptavidin in an assay to detect prohibited veterinary medicines should be further extended to cover veterinary antibiotic and anti-parasitic compounds. The use of radioactive and stable isotope labelled compounds and internal standards for method optimisation and validation studies to establish quality control and method performance criteria should be facilitated by the Agrochemicals Unit of the IAEA Laboratories at Seibersdorf.
iv. The CRP should aim to generate detailed laboratory protocols, including validation data. These should be widely disseminated for use by Member states and standards setting bodies. Additionally, the meeting noted that these protocols would play an important role in training programmes, including those initiated by the IAEA Technical Cooperation Programme.
v. In order to ensure that the protocols are robust, repeatable, transferable and suitable for publication, the CRP agreement and research contract holders should adhere to a defined quality system. To facilitate this, a guideline based on the Joint Code of Research developed by the UK Biotechnology and Biological Sciences Research Council, the Department for Environment, Food and Rural Affairs, the Food Standards Agency and the Natural Environment Research Council will be prepared by the FEP section prior to the first RCM.
vi. The Consultants agreed on the need to focus on analytes of significance in international trade of high value commodities and to accept appropriate proposals from contract and agreement holders based on these criteria.
6. Potential CRP contract and agreement holders.
The importance of selecting technically competent laboratories and contract holders from developing countries was stressed and in this regard, the consultants proposed potential CRP participants and laboratories (Annex 3).
7. List of Participants.
Prof. Dr. Hubert De Brabander, Faculty of Veterinary Medicine, Ghent University, Belgium
Prof Chris Elliott, Queens University, Belfast, UK
Prof Mathew Muzi Nindi, University of South Africa
Dr Sasitorn Kanarat, Thailand
Dr Vishweshwaraiah Prakash, CFTRI, Mysore, India
Dr Thomas Kuhn, Austrian Agency for Health and Food Safety, Vienna, Austria
Mr David Byron (NAFA)
Mr Rajendra Patel (NAFA) (Scientific Secretary)
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Mr Gary Luckman (NAFA)
Mr Josef Brodesser (NAFA)
Mr Andrew Cannavan (NAAL)
Ms Marivil Islam (NAAL)
Mr James Sasanya (NAAL)
Ms. Britt Maestroni (NAAL)
Mr. Nasir Rathor (NAAL)
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Annex D
ABSTRACTS OF PRESENTATIONS
THAILAND. Development of screening method for the detection of the presence of Multi-residues of
Antimicrobial substances.
Sasitorn Kanarat Department of Livestock Development, Ministry of Agriculture and Cooperatives, Thailand. The study has been conducted to find out the popular antimicrobial substances commonly used in the past and present in Thailand by questionnaires; the bacterial strains that are susceptible to the antimicrobials obtained from this study; the characteristics of the selected bacterial strains and investigate the suitable dyes to be used as the indicator of bacterial growth. From the study of 3,764 poultry farms, we found out that the top 10 antimicrobial substances most commonly used in Thailand at present are Doxycycline, Amoxicillin, Tylosin, Tilmicosin, Lincomycin, Trimethoprim, Colistin, Neomycin, Sulfonamide group and Erythromycin with the percentages of 79.36, 79.06, 77.42, 50.85, 50.48, 28.51, 26.73, 26.65, 26.57 and 2.02 respectively. The susceptible bacterial strains selected for this study are Geobacillus stearothermohilus ATCC 10151, Bacillus cereus ATCC 11779, Bacillus megaterium ATCC 9885, Bacillus subtilis BGA strain, Bacillus pumilus CN607 , Kocuria rhizophila ATCC 9341, E.coli ATCC 11303, and Yersinia ruckeri NCIM 13282. Three dye indicators including 0.2% bromocresol purple, 0.2 % bromothymol blue and 0.2% phenol red were studied by adding into the medium used for growing the abovementioned bacterial strains. The results showed that bromothymol blue is a suitable dye giving clear differentiation between growth and non-growth of all the bacterial strains under study. To evaluate the sensitivity of the bacterial strains, susceptibility test according to NCCLS has been conducted against commonly used antimicrobial substances; i.e., Erythromycin, Norfloxacin, Oxytetracyclin, Trimetoprim, Amoxycillin, Enrofloxacin, Sulfadiazine, Neomycin, Tilmicosin, Doxycycline, Cefoperazone, Ampicillin, Ciprofloxacin, Chlortetracycline, etc. The results have not been completed yet. Proposed work conducted in Oct-Dec 2009 and Jan-Oct 2010 -To complete the susceptibility study against antimicrobial substances. -To optimize the medium which supports growth of each bacterial strain. -To study on optimal temperature and incubation period for each bacterial strain to make the test rapid and accurate. - To study on the most appropriate concentration of each bacterial strain to be used in the screening method to obtain the method able to detect the residues of antimicrobial substances at the level as low as possible, preferably below MRLs for drugs with MRLs. Contribution to CRP Objectives: It is expected that at the end of this study, the screening method for the detection of antimicrobial residues which is inexpensive, reliable, practicable and affordable to developing countries will allow those countries to be able to start establishing a simple residue monitoring system.
CHINA Development of methods for aminoglycosides (LC-MS/MS and HPTLC).
Guihua LIU, Zhou ZHU, Chemical Analysis and Physical Testing Center, Shenzhen Center for Disease Control and Prevention, China
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Aminoglycosides (AGs) are commonly used antibiotics, which are in the list provided by the Joint FAO/WHO/OIE Expert Meeting on Critically Important Antimicrobials. It is necessary to have suitable methods in place to screen and confirm the presence of these drug residues in animal derived foods. Until now, a confirmatory and quantitative method based on solvent-based standard mixtures by LC–ESI-MS/MS has been developed for simultaneous determination of AGs antibiotics. The instrumental of an Agilent 1100 HPLC system was employed, which coupled with an API 3000 triple quadrupole tandem mass spectrometer. For chromatographic separation, an Atlantis® C18 column (150mm×2.1mm, particle size 5 µm) was chosen and heptafluorobutyric acid was added into in the mobile phase as an ion-pair agent. The separation of AGs was achieved within a 40 min elution program. However, it was observed that the precisions for streptomycin and dihydrostreptomycin were poor, so the optimisation of the mass spectrometric conditions will be further undertaken in the future. For sample-preparation, since AGs are water soluble, highly polar, acid and base resistant compounds which are not extensively bound to proteins, the AGs were released and extracted from different matrices with aqueous trichloroacetic acid solution (5%, w/v). Due to different pKa values of the compounds, seven AGs were quantitatively retained on Oasis HLB cartridges at pH< 1 and then six AGs were retained at pH 8.5 as reported by Zhu et al. Thus, the combination of two HLB SPE cartridges with different pH values was involved to simultaneously purify AGs. The AGs mixed standard was fortified at 51~160 µg kg-1 in Pork, mutton, beef, milk and honey samples. It was obtained that the good peak shapes of AGs in all the spiked samples of five tested matrices, and the retention time of each analyte varied less than ±1.8% compared with standard solution. Future Work Plan: 1. To introduce a suitable injection internal standard and further optimize MS parameters. As the stable isotope labelled reagent of AGs could not be purchased at present, it is necessary to find an approach to improve the precision and recoveries. Matrix-match standards will be used for quantification. 2. To validate the quantitative LC-MS/MS based confirmatory method according to the 2002/657/EC guideline, and to write a detailed SOP of this method. 3. To utilize HPTLC technique for rapid detection of AGs drug residues. HPTLC conditions will be developed and optimized with respond to separation of all the AGs. Developing solvent and coloration solution will be employed and be fully investigated. 4. Sample preparation should be simple and rapid for HPTLC. Trichloroacetic acid will be considered to be used to remove protein. A suitable buffer solution should be chosen to extract the AGs, and the extract should be concentrated if needed. The methods need to be assessed. The positive sample results will be confirmed by the confirmatory LC–MS/MS method. Contribution to CRP Objectives: It is expected that a LC-MS/MS confirmatory method and a HPTLC screening method for aminoglycosides will be developed to strengthen the capacity for monitoring drug residues in animal derived foods.
BRAZIL (Microbioticos) Development and Validation of an Immunoassay Kit for the Screening of Florfenicol in Fish
Tissue Samples
Rodrigo H. M. M. Granja, Microbioticos Labs.- Brazil
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The project includes the development of a Radioimmunoassay kit for the determination of Florfenicol in Fish tissue Samples. In a recent report produced by the FAO/WHO/OIE, florfenicol was pointed as a critically important antimicrobial to be monitored. The AOAC int. also pointed out interest on florfenicol molecule as it was considered by the organization’s seafood community as an important molecule to have fast methods available. During this first 6 months it was performed the following activities in attention to the project:
a) Technical meetings and contract with the florfenicol antibody manufacture; b) Sampling procedures considering the country production sites and dimensions; c) Development of a chromatography method for the equivalence studies with the
immunoassay method to be developed. The next steps of the project are:
a) Contact with tritium labelled compound suppliers to obtain the tritium labelled compound
b) Development and Validation of the RIA method for florfenicol residues in fish tissue samples
c) Validation of the HPLC method florfenicol residues in fish tissue samples d) Development and validation of MS confirmatory method for florfenicol residues in
fish tissue samples Future work
a) Promote the equivalence of the three methods (interlaboratoy rounds) b) Analyze real samples c) Report the results obtained (including publications).
BRAZIL Development, standardization and application of multiparametric Dot-ELISA assay to detect
enrofloxacin and ciprofloxacin in chicken.
Guilherme de Paula Nogueira; Sarita Priscila Gobbo, Sao Paulo State University- UNESP, Department of Animal Health and Production, Endocrinology Laboratory We intend to develop a analytic method for two antibiotic residue detection in poultry meat that can be performed outside a laboratory: a colorimetric multiparametric dot-ELISA. Work done to date: First step after contract agreement (May 12th) was to purchase the reagents, usually it takes 45-60 days after order to be at the Lab, fortunately some Enrofloxacin and mcKLH was borrowed from a friends Lab, the rabbits came from the university rabbit warren. At 01/07 animals started to be immunized, 3 rabbits (New Zealand) with 45 days of age, weighing 1 kg were immunized with 0.1 mg of the conjugate with Enrofloxacin on 01/7; 16/7; 31/07; 14/08; 28/08 and 11/09; blood form ear collected before immunization, animals were sacrificed 24/09 (with 3.2 kg) with Ketamin/Xilazin, maximum amount of blood collected by ear vein and heart puncture. For Ciprofloxacin other 3 rabbits weighting 2.0 kg were used, received 0.1 mg of conjugate with mcKLH every 15 days, started on 13/08; 28/08; 11/08; 25/09 and 9/10, blood was collected from ear before every immunization. Both antibiotics were also conjugated with BSA for plate coating. For ELISA plate was incubated with 2.0 µg/ml antibiotic-BSA, 3h at 37oC plus 13h at 4oC with, casein (2.5%) was added and after 2h incubation and washed 100µL of rabbit plasma was added in different dilutions, after 1h at 37oC anti rabbit IgG+Po (1/5000) was added for 1h at 37oC. The TMB (100 µL) was added for 30s, reaction stopped with H2SO4 (2M), plate read at 450 nm. From the indirect ELISA results it was possible to verify that all rabbits were producing
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antibodies against Enrofloxacin and the rabbits were sacrificed. Ciprofloxacin is still being injected in the other 3 rabbits. Proposed for future work for the next 3 years: Next step is the antibody purification, the ELISA development and standardization including in vivo experiment. Then the Multi-Dot ELISA using a plastic frame need to be developed together with a simple method for residue extraction from poultry meat, to be done by farmers outside the laboratory. Another evaluation and standardization and a in vivo test to validate the final product. Link with the CRP objectives: When this simple analysis method will be working, with little steps, fast, affordable it can increase the surveillance for the exportation and domestic food quality. It can work without the need of sophisticated equipment, radioactive material the only limitation will be the residue extraction from tissue. UNITED KINGDOM
Rapid screening techniques for drug residues
Professor Chris Elliott, Institute of Agri-Food and Land Use, Belfast. Screening techniques for drug residues are required to determine if there is a strong possibility that samples contain residues of target analytes at or above critical concentrations. These methods should be rapid, easy to use, low cost, have low false positive rates and generate no false negatives. A wide variety of analytical methods can be used for screening purposes. These range from simple microbiological inhibition assays, tests based on physiochemical properties such as HPLC/TLC, procedures high reply on specific molecular interactions such as receptor and antibody based tests to more sophisticated hyphenated procedures such as GC and LC coupled to mass spectrometry. A review of the important features of each procedure in terms of delivering all the essential criteria outlined above will be given.
GERMANY Immunoassays: assay designs - amplification techniques - reagent development - future
potential
Heinrich H.D. Meyer, Physiology Weihenstephan, Technical University Munich, Weihenstephaner Berg 3, 85354 Freising, Germany Hormones, veterinary drug residues, endocrine disruptors etc often need to be detected in the nano- or picogram range. The direct determination using physico-chemical methods is possible nowadays, but needs expensive equipment like GC-MS or LC-MS-MS. Imunoassay techniques offer an alternative as absolute sensitivities down to the attomol range can be achieved. Immuno assays are always based on the indirect estimation of the analyte of interest. Larger molecules like oligo peptides of proteins are big enough to provide at least two epitopes for antibody bindings. The quantification of the analyte is commonly based on quantification of one ore more bound antibodies. Depending on the technology of labelling, rather different sensitivities can be achieved (table 1). All technics are still in use – however, the advantages and disadvantages are quite different. Smaller molecules (less than 1000 dalton) represent only one antibody binding epitope, and sandwich technics are not possible. Competitive assay designs need to be applied, and almost similar sensitivities can be achieved. Usually, the specific antibody is the limiting reagent in competitive assays.
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Table 1: Label detection and approximate absolute sensitivity photometry micromoles fluorescence nanomoles radionuclide 3H femtomoles radionuclide 125I attomoles enzyme amplification + photometry femtomoles enzyme amplification + fluorescence attomoles time resolved fluorescence attomoles laser fluorescence attomoles surface plasmon resonance attomoles
Anyhow, sensitivity and specificity of the assay are mostly depending on the antibody. Hence, it is necessary to develop optimal antibodies with excellent affinities. The quality is influenced by coupling design, immunisation protocol and maybe further antibody clean up. Future optimisation of immuno assays will mainly depend on the availability of optimal antibodies, but also on new amplification technologies, kinetics of immuno reactions, selectivities, cost-efficiency etc.
UNITED STATES OF AMERICA
Analytical Method Development at FDA Center for Veterinary Medicine
Philip J. Kijak, US Food and Drug Administration. At the Division of Residue Chemistry, the mission is developing and validating methods for drug residues in animal derived foods, and drugs and contaminants in animal feeds. One of the major research efforts currently underway is a program to develop modern analytical methods to replace obsolete assays. Another program focuses on the development of methods for the detection of antibiotic residues in distiller’s grains. In addition to method development, research is conducted to understand the metabolism of drugs. Currently, studies are being conducted to better understand the metabolism of several drugs in aquatic species to determine metabolism. The research will enable appropriate marker compounds to be used in analytical methods for monitoring and compliance programs. An overview these current research studies will be presented. TUNISIA. Development and Implementation of Physico-Chemical Techniques for the Detection of
Authorized Antibiotics Using Radioactive Tracers for the Effective Monitoring of Veterinary
Drugs Residues in the Aquaculture Production.
BEN MANSOUR Ayda, National Center for Nuclear Sciences &Technologies, Tunisia. Fish farming is a promoting sector in Tunisia. Traditionally the main fish species commercialized are the common sea bass (Dicentrarchus labrax) and the gilthead sea bream (Sparus aurata). One of the most important problems in fish husbandry is effective control of infectious diseases. In that respect one of several strategies is the use of antibacterial agents. Due to their low minimum inhibitory concentration (MIC) value for most susceptible fish pathogens and effective systemic distribution, quinolones and tetracyclines are the main group of antibacterial agents prescribed by the Tunisian veterinarians to treat bacterial diseases in the farmed sea bass and gilthead sea bream. In our studies, we will focus in our first step on the absorption, distribution and elimination of an active antibiotic to be selected at the first RCM in the body tissues and organs of the farmed sea bass or gilthead sea bream. All the studies will be carried out using a whole body autoradiography and Liquid Scintillation
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counting. The active antibiotic will be administered to the sea bass or gilthead sea bream orally at a precise dose. The absorption and distribution rate of the medicine to the major tissues and organs of the treated animals will be reported. After the distribution phase, groups of fish will be collected and comparative studies will be carried out to distinguish the different levels of the active antibiotic between the different tissues and organs at several post administration sampling points. The second main step of this project is to implement a RIA screening method for an antibiotic (to be selected at the 1st RCM) using radioactive labelled streptavidin in a competitive immunoassay format. (The technology for this will be transferred at the 1st RCM). The several therapeutic regimes are designed to maximise efficiency and minimise the risk of the development of resistant pathogens. In that respect, the study of the pharmacokinetic properties of the active drug to be selected, in combination with the RIA screening test through this CRP, can be an important analytical tool for the determination of the optimal dosage regimes and thus the promotion of their correct use in the selected species. In addition to that, microbiological tests (Premitest & Delvotest) are used as screening methods for the detection of authorized antibiotics in national foodstuffs. According to their low sensitivity for some compounds such as quinolones, sub-contracting is the strategy chosen by the Tunisian Government to ensure surveillance and control of the aquaculture products for international trade. In order to improve the national monitoring programme, the implementations of new affordable methods complying with the international standards in national laboratories are needed. The analytical methods to be implemented and validated through this CRP can be further used in routine analysis. Thus, more data will be available for the improvement of the awareness for reasonable use of veterinary drugs. CHINA
Research on screening and confirmatory methods for detecting multi-benzimidazoles,
probenzimidazoles and their metabolites in animal products.
Yang Shuming, Institute of Quality Standard and Testing Technology for Agri-Products (IQSTAP), Beijing. Up to dated Sept. 30, 2009, the 13 of standard agents which include CMB, 5-hydroxythiabendazole, thiabendazole, albendazole 2-aminosulfone, albendazole sulfoxide, oxibendazole, oxfendazole, albendazole sulfone, albendazole, fenbendazole sulfone, fenbendazole, Febantel and netobimin have been brought. Both CMB-BSA and CMB-KLH were conjugated by the acid anhydride method. The UV spectrogram and time of flight mass spectrogram of the conjugated CMB show that CMB is combined to carrier protein. The calculated link ratios are 1:9 and 1:25 for CMB-BSA and CMB-KLH respectively. The mixtures were dialyzed to remove free small molecular substances. The further purification of the CMB-BSA and CMB-KLH will be done by molecular sieve column in this month. The immunization plan of the two antigens will be carried out in end of this year ahead of the original plan. The parameters of HPLC and mass have been optimized for qualitative and quantitative analysis. A c18 column is used for separation of the 12 drugs with acetonitrile/acetic acid as mobile phase by gradient elution. Qualitative and quantitative Ion pairs for each of 12 drugs were determined. Relation curve of solutions from 0 to 80ppb of 12 drugs is good enough for next researching, correlation coefficient is more than 0.998. Extract and cleanup of matrix and recovery of spiked samples will be done in the following two months. Mr. Liu Hongbing, my Msc. student, has joined the mission, he will graduate in summer of 2011 with a thesis titled as screening and confirmatory methods for detecting multi-
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benzimidazoles, probenzimidazoles and their metabolites in animal products. We will join proficiency testing to validate the established screening and confirmatory methods, when they are been completed. KOREA
The Development of Microbiological Multi-Residue and Allied LC-MS-MS Methods to
Monitor the Veterinary Drug Residues in the Environment.
Jin-Wook Kwon, Shin-Hee Kim, Dong-Hwan Oh, Hye- Jeong Chung National Veterinary Research & Quarantine Service (NVRQS), Republic of Korea Veterinary drugs are widely used in large quantities for the treatment of disease or to promote animal growth. The Korean animal farming industries have a very intensive rearing system, but farmer's still self-prescribe and treat their animals with drugs without a veterinarian's direction. The use of veterinary medicine as feed additives is also prevalent. Resistance to antibiotics has increased every year, furthermore environmental contamination through the excretion of animal faeces and urine, and the subsequent dispersion of contaminated manure onto land is an issue of great concern in Korea. In addition, recent and increasingly frequent occurrences of avian influenza and brucellosis outbreaks have required greater quantities of veterinary pharmaceuticals for application and treatment. Nowadays, Veterinary drugs are recognized as not only medicines but also as ‘Newly Emerging Contaminants’ in the environment. The purpose of this project is to develop and determine if the microbiological multi-residue screening/confirmatory methods used with animal products, can be used for environment samples with the modern LC-MS-MS techniques. Target veterinary pharmaceuticals have been selected by prioritizing with the following parameters; annual sales amount, toxicity, incidents, and some situations of interest in Korea. These pharmaceuticals are chlorotetracycline, oxytetracycline, penicillin, amoxicillin, cephalexin, tylosin, erythromycin, streptomycin, neomycin, sulfamethazine, sulfamethoxazole, sulfathiazole, enrofloxacin, and fluorophenicol. Soil, manure, and ground/stream water were selected as major target samples to be analysed in this project. Microbiological multi-residue screening methods, including EC four-plate test (EEC FPT) and microbial receptor-assay multi-method, have been widely used to screen the residue of veterinary drugs in meat and other animal products in Korea. EEC FPT, and its modification as screening methods, was a general purpose measurement. Microbial receptor-assay multi-methods, Charm-Test II was also a subsequent confirmatory test. Effective extraction and clean-up steps for environmental matrices need to be set for the applicability of these two phase method and the LC-MS-MS quantitative method. Method validation is in accordance with the minimum requirements for IUPAC and other relevant guidelines, in addition to the requirements of multi-residue screening methods: easy-to-use, low set-up cost, high through-put, reduced time and low running cost for result, sensitivity, repeatability, and reproducibility. To develop the effective sample preparation for multi-residue screening and confirmatory methods in various environmental samples, as blanks, soil, manure compost, and water samples, which are non-contaminated by veterinary drugs, with typical Korean soil properties, typical Korean solid and liquid manure compost properties, groundwater as livestock drinking water, were collected in several animal drug free-regions in Korea since SPF animal farms also use restricted drugs for breeding. To apply in-use methods, EC four-plate test and its modification and microbial receptor-assay multi-method, Charm-Test II, were tested respective soil, manure compost, and water with
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fortifications. In microbial bioassay and Charm-Test II, more than 80 % false positive results, only with direct buffers or solvents extract application without any purification steps of soil and manure composts, were shown, but water samples were directly applicable only with filtration. Detection limits of chlortetracycline, oxytetracycline, penicillin, neomycin, erythromycin from water were 0.02, 0.02, 0.02, 0.5, and 0.1 µg/g in microbial bioassay with B. megarerium (ATCC 9885), B. subtilis (ATCC 6633), B. stearothermophilus (ATCC 10149), B. cereus (ATCC 11778), and E. coli (ATCC 11303), respectively. As a confirmatory method, Charm-Test II, matrices matched control points of liquid samples for chlortetracycline, oxytetracycline, sulfonamide and erythromycin were set, and only erythromycin matrices matched control point of manure sample’s was reproducible. To develop the effective extraction and clean-up method, which is applicable to screening and LC-MS-MS at the same time, 3 sulfonamides with stable isotope labelled internal standards and 2 tetracyclines were primary tested according to various reported methods. Considering fit for purpose, EDTA-McIlvain buffer (pH=6) with MeOH (10:90, v/v) was selected as an extraction solvent and shown more than 70% of recoveries. On column clean-up, various reported methods were also applied, including MCX and/or HLB, but SPE C18 cartridge was cost effective with equivalent efficiency and easy to use for simultaneous purification of sulfonamides and tetracyclines in all the different matrices. Application to microbial screening and Charm-Test II with purified samples is now on going. Most of the LC-MS-MS analytical conditions for selected drugs were set with specified m/z for MRM, besides some epimers of 2 kinds of tetracycline are also applying to microbial screening, Charm-Test II and LC-MS-MS. Since, microbiological multi-residue methods and microbial receptor-assay multi-methods have long been widely used to screen and subsequently confirm the presence of veterinary drug residue in meat or animal products in Korea. And most of the laboratories are performing veterinary drug residue analysis without expensive and sophisticated LC-MS-MS. Furthermore, regulations on veterinary drug residues are more getting stringent in food and extend to fertilizer, drinking water, and soil in Korea. Through this proposed and future CRP, following outputs are expected - Defining the applicability of muti-residue screening and confirmatory methods for different types of environmental sample processing, - Validated sample processing and general-purpose residue analytical methods, - Reference material preparation of the representative environment samples for veterinary drug survey/monitoring purposes as blanks or references, - Publications of the results in a special issue of a scientific journal or e-journal/internet, and to change the governmental policy and related research work in Member States, and - Proposals for the inclusion of the results into the recommended procedures and methods of the National Survey/Monitoring Programme. MONGOLIA. Investigation of possible presence of Chloramphenicol in pasture plants and animals fed with
these plants.
Ts.Enkhtuya, Mongolia Chloramphenicol (CAP) is a bacteriostatic antibiotic used for treating both humans and animals against Gram positive and negative bacteria. Although this antibiotic has a broad spectrum of antibacterial activity, the use of chloramphenicol is totally banned within the European Union, and many other countries since 1994, because of its toxicity, Therefore EU The only exportable animal product from Mongolia into EU countries is currently the now small intestines of sheep and goats. However, since 2003 the antibiotic residues have been detected several times in sheep small intestines that were exported by a numbers of producers in Mongolia. Tests by the State Central Veterinary laboratory, Mongolia and the Hamburg
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Institute for Sanitation and Environment, Germany have revealed the residue of chloramphenicol was higher than MRL. Chloramphenicol is not so broadly used for animals in our country used, mostly only in spring season, if necessary. The animals, from whom small intestines were exported to the EU were slaughtered only during the autumn season and no CAP treatments were done in summer and autumn. On the basis of multilateral conclusions, we propounded the hypothesis, that animals might eat some bitter tasting pasture plants, which contain materials which can be confused with chloramphenicol in terms of their natures. To examine our hypothesis or to detect chloramphenicol in several bitter tasting plants grown in the pasture lands of Mongolia, first of all we measured CAP in such plants as Artemisia sieversiana, Artemisia sp, Artemisia frigida, Thalictrum simplex, Tanacetum vulgare L, Ledum palustre L, Rheum undulatum L, Thymus goblicus, and Thermopsis daurica, most of which have bitter taste and some animal specimen by ELISA test (Table 1 and 2). Table 1. Results of ELISA test of measuring chloramphenicol in plants
Chloramphenicol residue (µ/kg) Name of plants
2006-11 2007-3 2007-5 2007-8 2008-9
1 A.anethifolia Web ex Stechm
0,015 0.08
2 Thalictrum minusL 0,016 3 Thalictrum petaloideum 0,78 4 Tanacetum vulgare L 0,86 5 Padix rheum undulatum 0,018 6 Thymus dahuricus serg 0,016 7 A.Frigida white 0,013 8 Thymus dahuricus serg 0,017 9 A.Intricata Franch 0,72 10 Ledum palustre L 0,012 11 Iris dichotoma 0,013 0.000 12 Artemisia Sieversiana Willd 1,3 13 A.SericeaWeb.ExStechm 0,013 14 Artemisia frigida 0,87 0.257 0.1672 15 A.Frigida 0,013 16 Artemisia macrocephal 0,013 17 Artemisia drancunculusl 0,014 18 Artemisia Sieversiana 1,56 0.9 1.46 3.073 19 Thalictrum simplex 1.02 0.73 0.360 0,091 20 Thermopsis daurica 1.13 0.299
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Table 2. Chloramphenicol residue in organs of animals fed by some bitter tasting plants
Chloramphenicol residue (µ/kg)
plants Animal type
specimen 2006 10-4
2007 3-14
20075-10
20078-28
2007 8-29
2007 10-25
2007 11-05
liver 0,53
0,044
0.37
kidney 0,15
0,043
2.5
muscle 0,65
0,014
3.55
Small intestine
0,94 0,02 3.68
Sheep
spleen - - 3.62 liver 0.91 kidney 1,97 muscle 3.34 Small intestine
3.13
spleen 1,28
1
A.frigida
Rabbit
heart 3,48 liver 0,72 0,63 0.11 0.04 0.81 kidney
0,61 0,054
0.32 0.24 2.63
muscle 1,88 1,05
0.56
1.21 0.29 3.4
sheep
Small intestine
1,95 1,12 0.56 0.24 3.46
liver 2,69 lidney 1,96 muscle 3,62 Small intestine
1,69
2
A.macrocephala
rabbit
liver 0.11 0.03 kidney 0.32 0.12 muscle 1.21 0.07
3
Artemisia sp
sheep
Small intestine
0.56 0.20
liver 2.11 kidney 2.52 muscle - Small intestine
3.31
4
Thalictrum simplex
sheep
spleen 3.29 Liver 0.62 kidney 0.55
Thermopsis dahurica
sheep
muscle 3.57
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Small intetsine
3.30 5
spleen 2.54 Liver 1,72 1,72 0.24 0.10 2.15 kidney 1,77 0,37 1.72 0.96 2.01 muscle 1,63 1,59 1.4 3.95 1.59 3.46 Small intetsine
1,95 1,47 2.5 4.8 2.85
6
CAP
sheep
spleen 3.19 Liver 1,17 kidney 2,09 muscle 3,59 Small intetsine
2,98
7
CAP
Rabbit
heart 3,38 Liver 0.03
5
kidney 0.080
muscle 0.186
Small intetsine
0.141
8
Green hay
sheep
spleen - From the above table it is demonstrated that bitter plants, including A.macrocephala, Artemisia sp and Thalictrum simplex contain higher amounts of CAP, and the specimen from the animals, which ate these plants also contain the antibiotic. Having seen that the results of testing should be confirmed by the testing in laboratory with higher technical capacity, we joined related staff at RIKILT-Institute of Food Safety, Wageningen, the Netherlands and measured the residue in 15 plant samples by using ELISA kit, made by RIKILT liquid chromatography in combination with triple –quadrupole mass-spectrometric, and HPLC-Time of Flight-Mass Spectrometric techniques. Results of this testing fully confirmed that the plants contain CAP. Conclusion; This study revealed the detection of CAP residue in the small intestines of pasture Mongolian sheep can be associated with their feeding, which includes grazing of some bitter plants on the pasture. KENYA
Development and validation of sensitive and specific methods for the detection and
monitoring of selected anti-helmintic drug residues and their metabolites in livestock
products destined for human consumption.
Grace Murilla and Raymond Mdachi Kenya Agricultural Research Institute – Trypanosomiasis Research Centre, Kenya. Some of the major consequences of extensive veterinary drug use in food producing animals are presence of drug residues in products and development of drug resistance. In Africa, millions of dollars are used to procure trypanocides, antibiotics and anti-helmintics to treat
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various animal diseases. To-date KARI – Trypanosomiasis Research Centre has been involved in the development and validation of methods for the detection and monitoring veterinary drug residues in animal products, resulting in the publication of methods for the trypanocidal drugs diminazene, isometamidium and ethidium and also for chloraphenicol, a banned drug in food producing animals. These screening methods have been adopted for routine testing of meat and meat products under contracted services with the Kenya Meat Commission and the Veterinary Department of the Ministry of Livestock, Kenya. The proposed project aims to develop ELISA-based screening methods and confirmatory tests for albendazole and levamisole, the two commonly used anti-helmintics. So far, two animal species have been selected, screened for their suitability, and are ready for use in the production of antibodies. Some of the reagents for the preparation of immunogen have also been procured. It is expected that the primary immunization of the animals will commence by first week of November 2009. With increased awareness on public health issues amongst consumers, the development, validation and adoption of these methods for routine use will not only enhance consumer confidence and protection but also enhance trade in quality animal products. Field sampling procedures, transportation, storage, processing and analysis will be developed. Routine screening will ensure that residues in edible products do not exceed the Maximum Residue Levels (MRLs) as set by the FAO/WHO Expert Committee on Food Additives. It is also hoped that data and information generated will contribute to policy dialogue and inform policy change regarding veterinary drug residues, public health and trade issues in Kenya and the eastern Africa region. SRI LANKA
Current Status of Antimicrobial Residue Monitoring Project in Sri Lanka.
P Abeynayake, K Premarathne & DMS Munasinghe Faculty of Veterinary Medicine & Animal Sciences, University of Peradeniya The residue monitoring project commence in Sri Lanka in the year 2002 with the establishment of the simple bioassay technique for antimicrobial residues in broiler meat. Since then the project expanded steadily by acquiring analytical capability for many food commodities including chicken, eggs, milk, fish, shrimp, pork bees honey and animal feed. The analytical service being provided to producers, processors, exporters, regulatory authorities. Quarantine division and Sri Lanka Standard Institute. The two regulatory authorities are the Department of Animal Production & Health and the Ministry of Fisheries & Aquatic Resources, who are responsible for implementing Animal Disease Act and Fisheries & Aquatic Resources Act respectively. Currently only the screening techniques have been established. The Six Plate Method (SPM) is now routinely used for screening of almost all commodities for Group B antimicrobial agents. The ELISA kits are being used for screening shrimp for chloramphenicol having CCα of 0.04, CCβ of 0.3 and at 102% recovery rate. The HPLC-UV technique is being used to screen shrimp for four nitrofuran metabolites with the liquid- liquid extraction at the wave length of 275 nm. Futher the HPLC-UV method is now being validated to detect nitrofurans in animal feed after liquid-liquid extraction at the wave length of 375 nm. The HPTLC method is now applied to screen shrimp for sulphonamide residues at the wave length of 375 nm. The future research activities are aimed to establish confirmatory analytical techniques. These include establishment of HPLC-UV method for Group B antimicrobials. This laboratory set-up at the Faculty of Veterinary medicine & Animal Sciences provides the antimicrobial analytical service as the National Independent Laboratory in Sri Lanka which has contributed to the national economy of the country by enhancing food quality assurance
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capacity, enhancing exports, providing employability at all levels. Further the laboratory service being provided as cost recovery basis and had become a business unit hence ensured financial sustainability. This project had already generated several Post Graduate degrees hence trained personal are available to disseminate the technology. PERU Develop and Validation of a Multiresidue Method for Simultaneous Determination of
Benzimidazoles and Avermectins Anthelmintic Residues in Bovine Muscle and Milk by Liquid
Chromatography Coupled to Tandem Mass Spectrometer (LC-MS/MS).
Orlando Lucas and Roxana Ventocilla, National Agricultural Health Service-SENASA. Peru. Benzimidazoles and avermectins are the most frequently anthelmintics used in animal husbandry for protecting or treating the animals mainly against gastrointestinal nematodes, lungworms and liver fluke. However, if the recommended withdrawal times are not respected or if dosages are higher than that recommended, the residues levels in food of animal origin, such as bovine milk, muscle and other edible tissue, can be higher than maximum residue limit (MRL). Anthelmintic residues in food cause some toxic effect in consumers, as teratogenic and embryotoxic effect. Moreover, some benzimidazoles metabolites are more toxic than the parent drug. Codex Alimentarius Committee and the national or regional food safety authorities have been set MRLs values in food in order to protect the consumer’s health. Codex and EU MRLs for benzimidazoles and avermectins in bovine muscle and milk range from 10 to 225 ug/kg. EU legislation not authorizes use of Triclabendazole, Mebendazole, Doramectin and Abamectin in animals which milk is produced for human consumption. Marker residues of benzimidazoles include mainly benzimidazole metabolites and specific compound for avermectin, yielding a total of 21 target compounds that should be analyzed. Benzimidazol and avermectin anthelmintics residues have been mostly determinate by liquid chromatography with UV of fluorescence detectors. However, these methods require extensive cleanup, detect a limited number of compound and often require derivatization of analytes. LCMSMS technique is more sensitive and specific than HPLC with UV or Fluorescence detector. Moreover, mass spectrometry detection doesn’t depend of chromatographic separation for detection of compound, and then it can be used for simultaneous quantitative determination of several compounds (multi-residue method) with the same chromatographic system and same extraction process. The high selectivity of tandem mass spectrometry permit to use a relatively simple and fast sample extraction process that permit its application as a routinely method in regulatory laboratories. Ruyck et al. has been published an analytical method validation for residues of Benzimidazoles in milk, which include extraction with ethyl acetate followed by liquid chromatographic separation and detection with tandem mass spectrometer with electrospray ionization; the main advantage of this method is the relatively simple and fast sample extraction process that permit its application as a routinely method in regulatory laboratories. Nevertheless, this method was validated only for bovine milk samples. Then, due to extended consumption of bovine muscle by local consumer and for international trade, would be important expand and validate the above-mentioned fast and simple analytical method for simultaneous determination of benzimidazoles and avermectins residues in bovine muscle and milk. The aim of this research is to develop and validate an analytical method for simultaneous determination of Benzimidazol and Avermectin anthelmintics residues in bovine muscle and milk by liquid chromatography-tandem mass spectrometer (LC-MS/MS), in order to provide to official national laboratories a fast and relatively simple validated analytical method for their monitoring program.
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Advances of this project include the development of the liquid chromatographic-tandem mass spectrometric analytical method for the simultaneous detection and quantification of Albendazole (ABZ), Thiabendazole (TZB) and Tricabendazole (TCB) in milk. Milk samples were extracted with ethyl acetate. The liquid chromatographic separation was performed in a reverse phase C18 column with gradient elution. The analytes were detected with an LCMSMS instrument in ESI positive ionization mode and multiple reaction monitoring. Preliminary results obtained showed good results for selectivity, linearity and repeatability. Recoveries were lower than 70%. The method showed very high sensitivity with limit of detections (LOD) lower than 1 ug/kg. However, these results are only preliminary, so is necessary to will perform the following future work for complete this research: To complete the development of the analytical method: Expand the development of the analytical method, including 21 target analytes of benzimidazoles and avermectins for milk and muscles tissue. Optimize the chromatographic and spectrometric parameters for the 21 target analytes. Investigate different strategies for improve extraction performance (Use of Internal Standard, including Isotopic labelled standards, test SPE technique and compare with other method of extraction (QuEChERS). To validate the analytical method Elaboration of the validation protocol using Codex and EU (2002/657/EC) guidelines. Determination of validation parameters, including Trueness (Recovery), Repeatability, Within-laboratory Reproducibility, Linearity, Specificity, Limit of detection (LoD), decision limit and detection capability. Redaction of Standard Operative Procedure (SOP) of the validated analytical method. BELGIUM.
Chromatographic and mass spectrometric techniques.
Lynn Vanhaecke, Hubert F. De Brabander Ghent University, Faculty of Veterinary Medicine, Department of Veterinary Public Health and Food Safety, Lab Chemical Analysis, Salisburylaan 133, B-9820 Merelbeke, Belgium. The use of chromatographic and mass spectrometric methods in residue analysis should be seen in the light of the 96/23/EC. For A (banned) substances -at first instance- the presence (or not) of a substance must be demonstrated. For B substances (registered veterinary drugs) the method used should be able to detect if the concentration of a substance is above the MRL (or not). The introduction of RPAs (Reference Points for Action) with the 479/2009/EC increases the need for quantification also for A substances. Within a period of 40 years there was a tremendous change in performance of apparatus. In the field of chromatography –in recent years- analytical progress has especially been made in the field of liquid chromatography. The introduction of UPLC (Ultra Pressure LC) and U-HPLC (Ultra-High Performance LC) can improve and speed up analysis. This is very important for the time management of the sophisticated (and expensive) instruments coupled to this separation technique. In the field of mass spectrometry magnetic sector instruments are mostly replaced by smaller, cheaper and faster instruments: quadrupoles and/or ion traps. At first these were coupled almost exclusively to gas chromatographs. Later on (ca 1995) the hyphenation between LC and MS became available for routine residue laboratories. LC-MS introduction was also enhanced by the possibility of MS-MS (qQq) or even MSn (Ion Trap), reducing the background noise. In the latest years, high mass accuracy measurements by TOF (Time of Flight) or Ion cyclotron (Orbi Trap) benchtop instruments opened new horizons. Series of publications demonstrate that the accurate determination of a large number (100, even 300) of
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substances in one single run is no longer a fantasy. The classical strategy, screening with a cheap method and confirmation of the (fewer) suspects may change into screening and confirmation with Fast LC (FLC) hyphenation with high mass accuracy apparatus. However, laboratories and inspection services need to balance local situations, human resources and time management versus financial consequences.
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Annex E: Work plans for 1st Phase May 2009 to December 2010.
BRAZIL (Contract no. 15588).
Project Title Development and Validation of an Immunoassay Kit for the Screening of Florfenicol in Fish Tissue Samples
LEAD Partner Rodrigo Granja (Alfredo Montes Nino), Microbioticos, Campinas, SP, Brazil
Start Month May 2009
End Month December 2010
Interaction with other partners
Chris Elliott, Heinrich Myer and Guilherme de Paula Nogueira
Technical support required
Some advice from antibodies for immunological screening methods expert would be welcomed.
OBJECTIVE(S). 1. To develop, standardize and validate a radioimmunoassay kit for the determination of florfenicol residues in fish tissues;
2. To develop, standardize and validate an analytical method, using the product outcome from topic A);
3. To conduct an equivalence study of the validated method (topic B)) with other technology methods (such as HPLC and MS);
4. To apply the validated method to samples collected and further to the Official Monitoring Program in Brazil;
5. To participate on interlaboratory rounds conducted by Microbioticos and International Bodies.
DESCRIPTION OF WORK
Task 1 Meetings with antibodies manufacturers, contract
Task 2 Sampling project considering production sites
Task 3 Development of HPLC method for the determination of florfenicol residues in fish tissue samples
DELIVERABLES
Number Description Month
1 Radioimmunoassay validated HPLC method for the determination of florfenicol residues in fish tissue samples
Oct/2010
2 HPLC and MS methods for the determination of florfenicol residues in fish tissue samples and equivalence
Sept/2010
3 Scientific publications (submission) Dec/2010
EXPECTED RESULTS:
Date Description
December 2010 A reliable method for the rapid screening of florfenicol in fish tissue samples and equivalence with confirmatory techniques.
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BRAZIL (Contract no. 15596).
Project Title Development o f a Multi-Dot ELISA for enrofloxacin and ciprofloxacin residue in chicken tissues
LEAD Partner Guilherme de Paula Nogueira
Start Month May, 2009
End Month December 2010
Interaction with other partners
Chris Elliott, Heinrich Meyer, Alfredo Nino
Technical support required
Antibody purification, assay validation
OBJECTIVE(S). 1. Rapid and simple screening device for antibiotic residue in poultry meat
2. Production of antibody and purification and characterization
3. Development and validation of an immuno-assay (ELISA, RIA)
DESCRIPTION OF WORK
Task 1 Conjugation with protein, Immunization
Task 2 Antibody characterization: cross reaction, specificity
Task 3 Test other samples, other materials, in vivo study
DELIVERABLES
Number Description Month
1 Antibody May to December 2009
2 Assay January to May 2010
3 In vivo study June-December 2010
EXPECTED RESULTS
Date Description
December 2010 Validated assay for enrofloxacin and ciprofloxacin with necessary sensitivity
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CHINA (Contract no. 15598).
Project Title Development of methods for aminoglycosides (LC-ESI-MS/MS and HPTLC)
LEAD Partner Guihua LIU
Start Month May, 2009
End Month Dec. 2010
Interaction with other partners
Preeni Abeynayake(Sri Lanka), Philip Kijak, Hubert De Brabander
Technical support required
Method validation
OBJECTIVE(S). 1. To develop a LC-MS/MS confirmatory method for the determination of Aminoglycosides(AGs) drug residues.
2. To utilize HPTLC technique as an additional screening method of AGs drug residues.
DESCRIPTION OF WORK
Task 1 Literature search of possible methodologies.
Task 2 HPLC conditions will be developed and optimized.
Task 3 Optimisation of the mass spectrometric conditions.
Task 4 The extraction and clean-up will be developed to ensure that required recoveries are achieved.
Task 5 HPTLC conditions will be developed and optimized with respond to separation of all the AGs.
Task 6 Sample preparation should be simple and rapid for HPTLC
DELIVERABLES
Number Description Month
1 Procedure for sample preparation December 2010.
2 Methodology for AGs on HPTLC and LC-MS/MS December 2010
3
EXPECTED RESULTS
Date Description
December 2010 Method ready for validation and accreditation.
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CHINA (Contract no. 15672).
Project Title Screening and Confirmatory Methods for Detecting Multi-
benzimidazoles, Probenzimidazoles and Their Metabolites in Animal Products
LEAD Partner Yang Shuming, Institute of Quality Standards & Testing
Technology for Agro-Products �
Start Month Oct. 2009 (5th month)
End Month Dec. 2010 (19th month)
Interaction with other partners
Grace Murilla, Preeni Abeynayake, Olando Lucas.
Technical support required
Organizing validation and promoting confirmatory SOP as ISO standard
OBJECTIVE(S). To solve the shortcomings of the existed screening and confirmatory methods for BZs detection in animal products, a ELIA or RIA will be developed for screening BZs in animal foodstuff, moreover, a LC-ESI-MS/MS method will be developed for simultaneously detecting BZs, pro-benzimidazoles and their metabolites in animal foodstuff.
DESCRIPTION OF WORK
Task 1 Immunization: mouse will be inoculated with routine procedure for mono-clone antibody.
Task 2 Identifying individual mice
Task 3 Testing bleeds to find the mouse with high titer of antibody
Task 4 Completing cell fusion
Task 5 Testing clones by specification and sensitivity
Task 6 Choose of the wanted clone
Task 7 Production and purification of monoclonal antibodies
Task 8 Obtaining HRP conjugated hapten or radio-labelled hapten
Task 9 Procedures of extraction and cleanup: for different matrix including meat, liver, milk, egg, fish; fresh and cocked samples.
Task 10 Determination of LOD and LOQ by the spiked samples.
Task 11 Determination of accuracy testing by a set of concentrations spiked samples.
Task 12 Anti-:influence testing.
DELIVERABLES
Number Description Month
1 Monoclonal antibody 13th month
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2 HRP conjugated CMB 15th month
3 Sample preparation procedure for different matrix 14th month
4 The first version SOP of the confirmatory method 19th month
EXPECTED RESULTS
Date Description
December 2010 1. Ab: Monoclonal antibody, hopefully, the IC50 is about 5ug/kg.
2. Extract and cleanup: Sample preparation procedures using stable isotope internal standard.
3. SOP: first version.
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KENYA (Contract no. 15576)
Project Title Development of immunoassays for selected antibiotics and antihelmintics for the detection and monitoring of the drugs in livestock and livestock products
LEAD Partner Grace Murilla, Kenya Agricultural Research Institute
Start Month May 2009
End Month December, 2010
Interaction with other partners
China (albendazole) and Belfast (tetracylines)
Technical support required
multi-residue antibody production and characterization; training in LC-MS
OBJECTIVE(S). 1. To develop immunoassays for a range of anti-helmintics and antibiotics
2. To produce novel conjugates for tetracyclines
DESCRIPTION OF WORK
Task 1 To prepare/acquire conjugates of interest
Task 2 Select and screen experimental animals
Task 3 To undertake immunizations in a range of animal species
Task 4 To monitor antibody development through test bleeds
Task 5 To characterize antibodies
Task 6 Sample preparation: Undertake literature review on target analytes, including PK data. Information synthesized and documented for use to develop future strategies
Task 7 To develop methods and protocols
Task 8 To undertake in-house validation of developed assays
DELIVERABLES
Number Description Month
1 A range of Conjugates Month 8
2 Characterized antibodies by month Month 8
3 Prototype immunoassays Month 11.
4 A summary of sample preparation techniques Month 12
EXPECTED RESULTS
Date Description
December 2010 Validated, sensitive, specific, affordable, easy to use assays for detection and monitoring of selected antibodies and antihelmintics
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KOREA (Contract no. 15578)
Project Title Development of Microbiological Multi-Residue and Allied LC-MS-MS Methods to Monitor the Veterinary Drug Residues in the Environment.
LEAD Partner Jin-Wook Kwon, National Veterinary, Research & Quarantine Service (NVRQS),
Republic of Korea
Start Month May, 2009
End Month May, 2010
Interaction with other partners
York Univ., FERA (CSL) in UK, RIKILT Wageningen in Netherlands, NAAS & NIER in Korea
Technical support required
If available
OBJECTIVE(S). To develop and determine if the microbiological multi-residue screening/confirmatory methods used with animal products, can be used for environment samples with the modern LC-MS-MS techniques
DESCRIPTION OF WORK
Task 1 To Define the Applicability to In-use Bioassay Methods
Task 2 Development of LC-MS-MS Technique
Task 3 Application of LC-MS-MS Extraction & Purification Samples to Microbial-Bioassays
DELIVERABLES
Number Description Month
1 Publication/Presentation of the results and relevant topic in a special
issue of a scientific journal or scientific society (including technical report)
December 2010
2 SOP and Method Validation Guideline for this purpose
December 2010
3 Holding a workshop/seminar of relevant research group
July 2010.
EXPECTED RESULTS
Date Description
December 2010 1. Publications of the results in a special issue of a scientific journal or e-journal/internet
2. Proposals for the inclusion of the results into the recommended procedures and methods of the National Survey/Monitoring Programme
3. Publication of SOP and Method Validation Guideline for this purpose
4. Technical coordination among the authorized organizations (Nat’l Institute of Environment Research, Nat’l Academy of Agricultural Science in Korea)
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Mongolia (Contract no. 15597)
Project Title Confirmatory Analysis on Chloramphenicol Presence in Certain Number of Pasture Grass in Mongolia
LEAD Partner State Central Veterinary Laboratory of Mongolia
Start Month May 2009
End Month December, 2010
Interaction with other partners
Technical University of Munich RIKILT Wageningen Queens University Belfast FAO/IAEA laboratory Vienna
National Veterinary Research and Quarantine service Anyan
Technical support required
analytical support
-plant physiological
-q-PCR technique
OBJECTIVE(S). Find the answer to the question:
How is it possible that CAP occurs in plant material?
-is it produced by the plant?
-is it absorbed from the soil?
If CAP is absorbed from the soil then: what is the source which produces CAP in soil?
DESCRIPTION OF WORK
Task 1 Investigate if it is possible that the plant produces CAP
Growing plants (without soil) in clean water; analysis of the plant for containing CAP
Task 2 Investigate if the plant can absorb CAP from soil
Growing plants (without soil) in water containing (labelled)CAP: analysis of the plant for containing CAP
Task 3 Prove that CAP is produced in the soil (find the source)
Find out by using for example q-PCR techniques that the soil contains S. Venuzuela
DELIVERABLES
Number Description Month
1 The results of the performed analysis will be published (occurrence of CAP in plant material)
December 2009
2 Confirm that the plant absorbs CAP (=in combination with deliverable no. 1 the results will be presented during ‘Gent’ symposium June 2010)
Month 8
3 Detect the source which produces CAP in the soil December 2010
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EXPECTED RESULTS
Date Description
December 2010 Results of experiments regarding isotope labelled CAP;
if it is possible a plant can adsorb CAP from soil
Training for using q-PCR in Mongolia/technical assistance from TUM To set up q-PCR in Monglia ; find the source in the soil which produces the
CAP
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PERU (Contract no. 15606).
Project Title Development and Validation of a Multiresidue Method for Simultaneous Determination of Benzimidazol and Avermectin Anthelmintics Residues in Bovine Milk by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).
LEAD Partner Orlando Lucas, SENASA-Peru.
Start Month November 2009
End Month December 2010
Interaction with other partners
Yang Shuming (China), Hubert De Brabander and Philip Kijak
Technical support required
EU legislation for validation of analytical method.
Chromatographic tandem-mass analytical method
OBJECTIVE(S). 1. To complete the development of the analytical method for ten prioritized representative target analytes of benzimidazoles and avermectins for milk.
2. To validate the tandem-mass spectrometric method as a screening method.
DESCRIPTION OF WORK
Task 1 To optimize the chromatographic and tandem-mass spectrometric parameters for the ten prioritized representative analytes (seven benzimidazoles and three avermectins)
Task 2 To optimize extraction performance, using internal standard and solid phase extraction.
Task 3 To carry out validation experiments of the chromatography-tandem-mass spectrometric as a screening method.
DELIVERABLES
Number Description Month
1 Protocol of the extraction procedure March 2010
2 Protocol of the validation of tandem-mass screening analytical Method.
July 2010
3 Validated method for screening of benzimidazole, avermectins and anthelmintics by chromatography-tandem mass spectrometry (LC-MSMS).
December 2010
EXPECTED RESULTS
Date Description
December 2010 Validated LCMSMS screening method.
To provide to official national laboratories a fast and relatively simple validated chromatographic-tandem-mass spectrometric screening method for the simultaneous determination of benzimidazoles and avermectins anthelmintics in milk for their monitoring program.
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SRI LANKA (Contract no. 15586).
Project Title Development of HPTLC screening methods for antibiotic Veterinary Drug residues
LEAD Partner Preeni Abeynayake
Faculty of Veterinary Medicine, University of Peradeniya
Start Month May 2009
End Month December 2010
Interaction with other partners
Guihua Liu (China), Yang Shuming (China), Orlando Lucos (Peru), Thomas Kuhn (Austria), Prof Hubert De Brabander (Belgium)
Technical support required
Access to scientific literature & procurement of standards
OBJECTIVE(S). To expand the laboratory capability for secondary screening and confirmatory testing for antibiotic residues using HPTLC and HPLC respectively
DESCRIPTION OF WORK
Task 1 Literature review on HPTLC & sample preparation for the purpose
Task 2 Ensure smooth supply of chemicals
Task 3 Setting –up the assay procedure of sulphonamides & tetracyclines
Task 4 Preparing the SOPs
Task 5 Validation of techniques
DELIVERABLES
Number Description Month
1 Availability of the assay technique for sulphonamides & teteracyclines
May 2010
2 Availability of capable staff August 2010
3 Availability of SOPs December 2010
EXPECTED RESULTS
Date Description
December 2010 Being able to carryout routine analysis of samples for the Industry & Regulatory Authorities
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THAILAND (Contract no. 15604)
Project Title Development of microbiological screening method for the detection of multi-residues of antimicrobial substances
LEAD Partner Sasitorn Kanarat
Start Month May 2009
End Month Dec 2010
Interaction with other partners
QUB, TUM, Korea National Vet. Research & quarantine service.
Technical support required
OBJECTIVE(S). To develop a microbiological screening method for the detection of antimicrobial residues which is inexpensive, reliable, practicable and affordable to developing countries in order to enable these countries to start establishing a simple residue monitoring plan.
DESCRIPTION OF WORK
Task 1 To find out the most commonly used antimicrobials in Thailand in order to prioritize the antimicrobials study.
Task 2 Undertake literature review on pharmacokinetics of the most important antimicrobials used in Thailand.
Task 3 Undertake MIC study to select the most appropriate bacterial strains.
Task 4 To optimize the medium which support growth of each bacterial strain.
Task 5 To study on the dye indicators in order to select the dye that gives clear differentiation between growth and non-growth of all bacterial strains for including into the medium used.
Task 6 To study on optimal temperature and incubation period for each bacterial strain to make the test rapid and accurate.
Task 7 To study on the most appropriate concentration of each bacterial strain to be used in the screening method to obtain the method able to detect the residues of antimicrobials at the level as low as possible, preferably below MRLs for authorized substances.
Task 8 Production of prototype assay in laboratory environment.
Task 9 Selection of matrices and development of sample preparation protocol.
DELIVERABLES
Number Description Month
1 A list of antimicrobials commonly used in Thailand December 2010
2 A summary of literature review December 2010
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3 MICs of antimicrobials under study. December 2010
4 Appropriate, temperature and incubation period to be used
December 2010
5 Appropriate dye and concentration of each bacterial strain to be used
December 2010
6 Report on the prototype assay December 2010
EXPECTED RESULTS
Date Description
December 2010 To have a rapid test capable of detecting the most important antimicrobials
commonly use in Thai poultry industry
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TUNISIA (Contract no. 15625).
Project Title Development and Implementation of Radio-Chemical Techniques for the Detection of Authorized Antibiotics Using Radioactive Tracers for the Effective Monitoring of Veterinary Drugs Residues in the Aquaculture Production.
LEAD Partner Ms Aida Ben Mansour, CNSTN, Tunisia
Start Month May 2009
End Month December 2010
Interaction with other partners
1. Rodrigo Granja and Alfredo Montes Nino, Microbioticos Laboratorio Avenida Santa Isabel Campinas, Brazil,
2. Philip Kijak, Division of Residue Chemistry, FDA, USA. 3. Raj Patel, International Agency for Atomic Energy, NAFA,
Vienna. 4. Heinrich Myer, Weihenstephaner Berg 5 Freising, Germany.
5. Chris Elliott, Queen’s University of Belfast, Institute of Agri-Food and Land Use, United Kingdom.
Technical support required
1. Protocols for pharmacokinetics studies using chromatographic techniques
2. Protocol & antibody for RIA Method
OBJECTIVE(S). 1. Pharmacokinetics studies on sea bass of an active drug.
2. Implementation and validation of RIA Screening method.
DESCRIPTION OF WORK
Task 1 Search for an available 14C Labelled drug
Task 2 Perform Pharmacokinetics studies
Task 3 Develop an RIA method for the antibiotic to be selected
DELIVERABLES
Number Description Month
1 Report to the agency containing the information of the available 14C labelled compound to be used.
December 2009
2 Report to the agency containing the results of the pharmacokinetics studies
December 2010
EXPECTED RESULTS
Date Description
December 2010 Tissue distribution & Elimination rate of the selected active select drug in sea bass