Coordinated Research Project (CRP): D5.20 · on the development of radiometric and allied...

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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 2ND RESEARCH COORDINATION MEETING.

Kandy, Sri Lanka, 14-18 March 2011.

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, 2011

INTERNATIONAL ATOMIC ENERGY AGENCY

Report of the 2nd 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.

Kandy, Sri Lanka, 14-18 March 2011

Working Material Produced by the IAEA

Vienna, Austria, 2011

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Introduction The 2nd 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 University of Peradeniya, Sri Lanka, 14 to 18 March 2011. In spite of the very limited budget, the event was very well organised and the great efforts of the local counterpart, Prof Preeni Abayanayke and her team was greatly acknowledged and appreciated by all the participants.

The Meeting was chaired by Rodrigo Granja (Contract No. 15588, Microbioticos, Brazil) with Grace Murrila (Contract No. 15576, Kenya) as the Rapporteur and Rajendra Patel (IAEA) as Scientific Secretary. A list of participants (Annex I), the meeting Agenda (Annex II) and the Rapporteur’s notes (Annex III) are attached. Agreement holders Philip Kijak (U.S. Food and Drug Administration, Heinrich Meyer (Technical University, Munich, Germany) and Chris Elliott (Queens University Belfast, UK) were unable to attend the meeting but have continued to support the project by actively working with the contract holders. Ms Tserendorj Enkhtuya (Mongolia, Contract No. 15597) was also not able to attend.

The RCM was formally opened by the Vice Chancellor of the University of Peradeniya, Prof. S. B. S. Abayakoon, who in his opening speech highlighted the long standing association with NAFA/IAEA in the field of animal health and food safety and the success of this collaboration resulting in the setting up of a food contaminants laboratory in the Department of Veterinary Public Health and its ISO17025 accreditation by the Sri Lanka Accreditation Board (SLAB). The Accreditation Certificate was presented to Prof Preeni Abayanayke by a representative of SLAB

Objectives of the Second Research Coordination Meet ing. The meeting commenced with a presentation from the Scientific Secretary on the current activities of the Food and Environment Protection Section and the objectives of the CRP and the RCM. The meeting was reminded that the main aim of this 2nd RCM was to:

a) evaluate and discuss the progress made by each contract holder in relation to the work plans agreed at the 1st RCM (19 - 23 October 2009, Vienna);

b) prepare work plans for the next phase of the project. The meeting recognized that the activities and CRP framework may be refined if necessary in order to better meet the objectives of the CRP;

c) to facilitate a broader understanding of the relationship each participant has to the overall objectives of the CRP and to promote interaction between the participants;

d) prepare recommendations and guidelines to facilitate project tasks and for the participants to agree a common approach and way forward.

The Meeting was also reminded that the main objective 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 analytical methods utilizing radiotracer detection methods in conjunction with confirmatory techniques using stable-isotope labelled analogues. In this regard the importance of preparing Standard Operating Procedures with appropriate validation data was stressed.

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RCM Presentations. The Research Contract Holders’ presentations of their work focused on results and constraints and were followed by extensive discussion on overcoming the problems encountered. Working with the Agreement Holders and the Technical Contract Holder in small groups, each Research Contract Holder revised and finalised their individual work plans and formulated their planned activities and milestones for the next phase of the project (Annex IV).

All the Agreement Holders presented their current work on veterinary drug residues at a meeting organised by the Faculty of Veterinary Medicine and Animal Science as part of the events to commemorate World Veterinary Day 2011 (http://www.vet2011.org/). In addition to the RCM participants the meeting was attended by staff and students of the University of Peradeniya and representatives from the Industry. A presentation on analytical approaches to address food authenticity and traceability problems was made by the Scientific Secretary.

Conclusions. • The meeting recognized that this CRP has initiated the collaboration between

laboratories (network of laboratories) of different member states and that it provides information that can have a regulatory impact e.g. natural occurrence of substances like chloramphenicol and contamination of the environment by residues of pharmaceuticals will initiate additional risk assessment and evaluations.

• Each Research Contract Holder’s work was reviewed to ensure that high scientific standards and the objectives of the CRP were met and their work plans, activities and deliverables for the next phase of the project were finalised.

• To ensure that the primary objective of the CRP to provide National Reference Laboratories of FAO and IAEA member states with monitoring methods for residues of selected antibiotic and anthelmintic veterinary medicines is met, it was agreed that all methods developed and used by the contract holders will be presented as Standard Operating Procedures with appropriate validation data, using either Codex or EU guidelines. Agreement Holders will provide assistance where needed.

• The first phase of the project focussed on the development of multi-residue methods that are fit for purpose (easy to use and cost-effective). However the results clearly show this is not always possible and the meeting recognised that it may be more cost effective to have two separate methods, e.g. for benzimidazoles and ivermectins.

• It was also agreed that rather than continuing with further method development work, Contract Holders working on benzimidazoles would work towards setting up a ‘harmonized’ method for benzimidazoles in specific matrices (muscle, liver and milk), based on acetonitrile extraction, solid phase clean-up and LC-UV/MS detection. The method would be collaboratively tested by exchanging samples.

• The work on preparation of reagents (antibodies and enzyme-conjugates) for development of immunoassays using radiolabelled substances has encountered some difficulties. It was agreed that the Technical Contract Holder will provide participants with appropriate protocols and reagents.

• It was recognised that the time required obtaining appropriate import licences for reagents and biological samples should not be underestimated. Failure to comply with import regulations could mean the refusal of a carrier to transport the material or cause

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delays in customs clearance. It was agreed that Contract Holders will examine their national regulations concerning export/import of biological and chemical materials and standards and put in place all the necessary clearances before shipment.

Recommendations. 1. In line with the work plans agreed at the second RCM, specific reagents (e.g. antibodies

and radiolabelled materials) should be transferred to relevant members. 2. Methods used and developed by the participants should be validated according to one

specific fit for purpose validation procedure (CODEX/EU) and should be described in a standard operating procedure and distributed between all CRP contract and agreement holders.

3. Collaborative studies for methods developed in the CRP should be organised by relevant members:

a. Benzimidazoles in meat (China) b. Aminoglycosides in meat (China) c. Florfenicol in fish (tilapia and catfish (Brazil))

4. To facilitate appropriate risk assessments and development of regulatory policy, the data regarding the contamination of the environment with residues of pharmaceuticals including veterinary medicines and the naturally occurring pharmacologically active substances (e.g. chloramphenicol) should be collated and disseminated to a wider group of interested stakeholders.

5. All participants should endeavour to communicate on a regular basis with appropriate CRP contract and agreement holders and with the IAEA. The platform dedicated for the CRP at the University of Gent (Zephyr) should be used to disseminate and exchange information.

6. Finally the meeting recommended that additional IAEA support should be sought to address emerging issues on food and environmental contamination by veterinary drug residues, e.g. generation of pharmacokinetic data in fish species where this is not available, and environmental impact of increasing aquaculture.

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Agreed Work Plan and Logical Framework

Work Plan (Activities)

Activity 2009 2010 2011 2012 2013 2014

CRP advertised (December 2008) and research contract and agreements awarded.

Contracts and agreements signed by end May 2009.

X

1st RCM, Vienna (19 to 23 October 2009) agreed CRP strategy and research work plans.

X

Awarded Research Contracts X X X X X

Awarded Technical Contracts X X

Phase 1: 18 months’ work programme completed. X X

2nd RCM (14 to 18 March 2011) held in Kandy, Sri Lanka to review the work conducted in Phase 1 based on progress reports and presentations. Detailed work plans for Phase 2 agreed to ensure that the CRP objectives are met.

X

Phase 2 : Work programme X X

Organise 3rd RCM (Oct 2012) to review work conducted in Phase 2 and agree final phase 3 work programme.

(Proposed Venue: Nairobi, Kenya. This is subject to Host country and IAEA approval).

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 I: RCM Attendees

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 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]

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]

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 Institute of Quality Standards and Testing Technology for Agro-Products (IQSTAP) Chinese Academy of Agricultural Sciences (CAAS) 12 Zhongguancun Nandajie Beijing, 100081, China Tel: +86-10-82106561 Email: [email protected]

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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 Alida (Linda) A. M. Stolker RIKILT, Wageningen UR Akkermaalsbos 2 P.O.Box 230 6700 AE Wageningen, The Netherlands Tel: +31-317-480390

Email: [email protected]

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

[email protected]

Mr Jin Wook-Kwon National Veterinary Research and Quarantine Service (NVRQS) 480 Anyang-6dong, Manan-gu ANYANG, Kyonggi-Do 430-016 KOREA, REPUBLIC OF [email protected]

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]

Ms Preeni Abeynayake &

Mr Madura Munasinghe Ms Sanda Kottawatta

Krishanthi Premarathne

University of Peradeniya Faculty of Veterinary Medicine and Animal Science Department of Veterinary Clinical Studies Peradeyia 20400 Sri Lanka Tel: +94 81 2388205 / +94 81 2395700 [email protected] [email protected]

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Mr Terry Fodey Institute of Agri-Food and Land Use Queens University Belfast David Keir Building, Stransmillis Road Belfast, Northern Ireland BT9 5AG Tel: +44 (0) 289097 6535 Fax: +44 (0) 289097 6513 [email protected]

Mr Rajendra Patel Food and Environmental Protection Section Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture Department of Nuclear Sciences and Applications International Atomic Energy Agency – IAEA Wagramer Strasse 5 1400 Vienna, Austria Tel: +43 1 2600 21672 [email protected]

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Annex II: Agenda 14-18 March 2011

University of Peradeniya, Kandy, Sri Lanka.

Scientific secretary: Rajendra Patel (FEP/NAFA) [email protected] Provisional Agenda

Day 1: Monday, 14 March 2011

08:00 Registration. All

09:15 Welcome & Opening Address and presentation of Certificate from the Sri Lanka Accreditation Board.

Prof Preeni Abeynayake Prof S B S Abayakoon, Rajendra Patel

09:45 Introductions and Presentation of Participants. All

09:55 Election of Chairperson and Rapporteur. All

10:00 Adoption of Agenda. All

10:05 CRP objectives and scope of the RCM. Rajendra Patel, IAEA

10:45 Break

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).

11:00 Development of multi-dot-ELISA for enrofloxacin and ciprofloxacin residues in chicken tissue.

Guilherme Nogueira, Brazil.

11:45 Development of RIA for florfenicol in fish tissue. Rodrigo Granja, Brazil.

12:30 Lunch / Administrative Arrangements

14:00 Development of immunoassays for antibiotics and anthelmintics: preparation of antibodies in appropriate species.

Grace Murilla, Kenya.

14:45 Development of a microbiological screening method for the detection of the multi-residues of antimicrobial substances.

Sasitorn Kanarat, Thailand

15:30 Break

16:00 Development of HPTLC screening methods for anthelmintic and antibiotic veterinary drug residues.

Krishanthi Permarathne, Sri Lanka

16:30 End of Day 1

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Day 2: Tuesday, 15 March 2011

08:45 Summary of Day 1 Rajendra Patel, IAEA

09:00 Development of methods for aminoglycosides (LC-MS/MS and HPTLC).

Guihua Liu, China

09:45 Development of multi-residue screening and confirmatory methods for benzimidazoles and their metabolites.

Yang Shuming, China.

10:30 Break

11:00 Development of multi-residue LC-MS/MS method for benzimidazoles and avermectins.

Orlando Lucas, Peru.

11:45 Environmental impact studies on veterinary medicines: development of methods and preparation of “incurred” soil, manure and water samples.

Jin-Wook Kwon, Korea

12:30 Lunch

14:00 Pharmacokinetics of quinolones and tetracyclines in Tunisian farmed fish using labelled drugs.

Aida Ben Mansour, Tunisia.

14:45 Investigation of possible presence of chloramphenicol in pasture plants and in animals fed with these plants.

Tserendorj Enkhtuya, Mongolia (presented by Linda Stolker).

15:30 Break All

15:45 Evaluation of the work done during the first phase

16:30 End of day 2

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Day 3: Wednesday, 16 March 2011


Session 3: Preparation of Work Plans, Recommendations and Meeting report.

09:00 Quality Assurance and Control guidelines for all CRP participants.

Thomas Kuhn, Austria and Rajendra Patel, IAEA

9:30 Formation of working groups

Group 1: Screening Assays (immuno and microbiological assays)

Group 2:Chromatographic Techniques (HPLC / TLC/ Mass spectrometry)

Chairperson and Agreement Holders

9:45 Working 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 Working Session 1 (continued) All

12:30 Lunch Break

13:30 to 18:00

Field trip to animal production units.

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Day 4: Thursday, 17 March 2011


08:45 Drafting Session 1. The objective of this session is to prepare presentations (one for each group) of the findings of Working Session 1.

Agreement Holders and participants of groups

10:30 Break

11:00 Presentation of Group 1, Q&A Chairperson of Group 1

11:30 Presentation of Group 2, Q&A Chairperson of Group 2

12:00 Discussion on outcomes of Working Session 1. All

13:00 Lunch

Session 2: Presentations from Agreement Holders (a joint session with the Department of Veterinary Public Health).

14:00

Presentations from Agreement Holders and the Scientific Secretary on their current work on Veterinary drug residues and other emerging issues on food safety, quality, authenticity and traceability at a meeting organised by the Faculty of Veterinary Medicine and Animal Sciences as part of the events to commemorate World Veterinary Day 2011.

Hubert De Brabander, Thomas Kuhn, Terry Fodey, Linda Stolker, Leen van Ginkel and Raj Patel.

16:50 Round Table discussions on issues which rose during the presentations.

All

17:15 End of Day 4.

Day 5: Friday, 18 March 2011

08:45 Summary of Day 4 All

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)

10:30 Break

10:45 Finalizing conclusions and recommendations All

12:15 Lunch

13:30 Adoption of the Final Report of the 2nd RCM. All

15:45 Any Other Business All

16:00 Closing Remarks and End of Meeting. All

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Annex III: Rapporteur’s notes. Report of Proceedings, Second RCM of CRP D5.20.36 on Analytical Methods for

Veterinary Drugs

The Meeting was held at the University of Peradeniya, Faculty of Veterinary Medicine and Animal Science, Department of Veterinary Clinical Studies, Kandy, Sri Lanka

Following a very successful and colourful official opening of the meeting, all the contract holders presented the research work covering the period Aug 2009 to December 2010, whose focus was on development, validation and application of enzyme and radio-immunoassays, TLC and LC-MS/MS in the analysis of various veterinary drugs. Below is a brief summary of each presentation and the issues that were raised during the discussions:

Development of multi-dot ELISA for enrofloxacin and ciprofloxacin residues in chicken tissue (Guilherme Nogueira, Brazil))

• The objective was to produce polyclonal antibodies, optimize and develop methods for the detection and monitoring of the residues in chicken.

• Several attempts have been made which have not been very successful due to the following reasons:

a) Low antibody titres of the polyclonals b) Persistent low titres of the purified antibody c) Cross-reactivity of the reagents with unidentified non-target analytes d) Change in protocols, adapting those from other laboratories, for antibody

production and also breed of the rabbit did not improve the ODs • In vivo experiments using chickens to which various doses of the drug had been

administered did not show any conversion and there were no differences between the low and high doses

• It was observed that the experiments design may not have been appropriate, including the unhygienic housing of the chicken

• Issues on animal welfare were not observed • The following were suggested:

a) There is need to review the current immunization protocols b) Adjuvant should be used all the time from primary to booster immunizations c) There is need also to select appropriate protein carrier, e.g. KLH, since all

drug molecules do not exactly work the same way d) All experiments that utilize animals have to be cleared by the appropriate

institutional ethics committees

Development of RIA for florfenicol in fish tissue (Rodrigo Granja, Brazil)

• Focus only on parent compound • It was reported that the immunogen was prepared by conjugating the drug to

KLH as the protein carrier • Low titres were obtained following the primary immunization; this was expected.

However, following second immunization, high antibody tires were obtained; the optimal dilution of 1:10,000 was used

• Method still under optimization • It was observed that regulatory agencies tend to use LCMS as confirmatory test,

however there is need for assessment bioequivalence with other methods • Using various methods, samples from different parts of Brazil were analyzed and

levels found not to be violative

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• It was recommended that: a) Calculations of CCα and CCβ need to be reviewed b) These values need to be calculated for each species, comparing the

fresh water and salt water fish c) Methods for determining MRLs should also be species specific e.g.

the salmon • Issues regarding importation of reagents and the time taken were discussed and it

was agreed that they should be addressed as availability of appropriate reagents can significantly delay the implementation of work plans.

Development of immunoassays for selected antibiotics and anthelmintics for the detection and monitoring of the drugs in livestock and livestock products (Grace Murilla)

• It was reported that antibodies have been developed in the rabbit against two classes of compounds (the tetracyclines and sulphonamides)

• An immunogen consisting of a cocktail of tetracycline, oxytetracycline and chlortetracycline was conjugated to Keyhole limpet heamocynin

• Initial bleeds after the primary immunizations gave low titres, these have however improved with later booster immunizations

• Two rabbits immunized with immunogen prepared using sulpahthiazine-KLH are being monitored through test bleeds. Results appear promising

• Various drug conjugates are under preparation in an effort to optimize both antigen and antibody dilutions

• It was recommended that: a) Use of eggs in the production of antibodies be explored b) Confirmatory tests be considered c) There are methods for detection of tetracycline in honey; these need also to be

explored including fluorescence methods (chicken bones) d) Efforts be made to register the two PhD students; the country Liaison office

on IAEA matters be approached

Development of microbial screening method for the detection of multi-drug residues of antimicrobial substances (Sasitorn Kanarat)

• A comprehensive report was give showing that a lot of work has been accomplished in the validation of various methodologies

• Issues were raised regarding acquisition of bacteria for use in the assays. • Ways to be explored in which Sasitorn’s laboratory would supply the other laboratories

with the various bacteria needed for different assays • It was recommended that:

a) Sample extraction be extended to different matrices e.g. honey, drinking water for food producing animals since nitrofurans have previously been found in drinking water. This is important especially in poultry farming

b) Also to be considered are environmental matrices; to work collaboratively with contract holder from Korea

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Development of HPTLC screening methods for anthelmintic and antbiotic veterinary drug residues (Preeni Abeynayake, Sri Lanka)

• It was observed that the veterinary drugs market in Sri Lanka is very well regulated and that there are no uncontrolled imports

• It was recognized that TLC is a good and powerful technique in which fluorescence can be enhanced through the use of appropriate sprays. This will enhance detection (lower detection limits)

• Other approaches for enhancing detection need to be explored from published literature and adapted for use

• It was acknowledged that all exports to Europe rely on the CCα and CCβ values calculated on the basis of MRLs; importance of these values was stressed

• Codex Alimentarius Commission is currently reviewing its guidelines which will be adhered to.

Development of methods for aminoglycosides (LC-MS/MS and HPTLC (Guihua Liu, China)

• The LC-MS/MS method for detection of aminoglycocides was reported. Parameters have been optimized. Initial problems of splitting of peaks was resolved

• This method is yet to be validated • The major problem of the HPTLC method was matrix effect leading to poor

resolution • Following the above observations, it was recommended that: • The plates need to be carefully and properly dried after running one dimension, then,

run the second. If this is not done, there will be interference • Use commercial plates with a concentration one which allows both clean-up and

concentration of the analytes.

Development of multi-residue screening and confirmatory methods for benzmidazoles and their metabolites (Yang Shumming, China)

• LC-MS/MS method developed – using external and internal standards • Low LOD and LOQ reported • For all the tested drugs and their metabolites, very good recoveries were obtained

without internal standard; 6C13 employed as internal standard • No significant differences observed with or without internal standard • Method validated, to be applied for routine monitoring of benzmidazoles and their

metabolites • To be recommended to the MOA for national residue control plan • One manuscript has been developed for submission • During discussions, the following clarifications were sought:

a) Whether only one internal standard was used b) Whether the mean recoveries were from 5 different samples and not one

sample replicated five times c) The differences in the doses used for spiking were too large d) References should be made to existing literature; there are a lot of

publications already in this area

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Development of multi-residue LC-MS/MS method for benzmidazoles and ivermectins (Orlando Lucas, Peru)

• It was acknowledged that non-compliance with withdrawal periods of various vet drugs led to high residues in animal products

• Methodology reported was adapted from pesticide analysis procedure and optimized for benzmidazoles and ivermectines

• Two internal standards were used • Low recoveries obtained, therefore need to validate method • Used matrix matched calibrations, which may not have been appropriate • Need to address ion suppression • During discussions, the following were observed/recommended:

a) Ivermectins are very difficult to analyze; combination of the two classes of benzmidazoles and ivermectins should be very difficult

b) Consider developing two different methods, one for each class of dugs c) Spiking sample at the final step of extraction – not matrix matched d) Developing two methods may not be practical due to inadequate numbers of

personnel • The meeting was reminded that the objective of the project is to develop methods which

member countries can use. Is it possible to collaboratively develop one method? Focus should be on licensed drugs that already have MRLs

Environmental impact studies on veterinary medicines: development of methods and preparation of “incurred” soil, manure and water samples (Jin-Wook Kwon, Korea)

• Currently Korea is suffering from an outbreak of avian influenza and FMD • It is common practice to use excretions from animals as manure; therefore, it is suspected

that this is one of the sources of contamination of the environment with veterinary drugs • Methods developed by Korea for environmental monitoring reported good recoveries

except for amoxicillin • Whereas water can be analyzed directly, all other samples require extraction • False positives reported; could be as a result of either interference by stable isotopes or

instrumental • Observed that tetracyclines bind strongly to soil particles and are not easily leached even

during heavy rains; sulphonamides leach much more easily therefore high concentrations found in soils

• Is drugs adsorb strongly to soils and other biological materials, methods have to be developed of releasing these drugs in order to be analyzed in their free form; this may require contacting pharmaceutical firms for relevant data

• Data on the levels of consumption of human medicines is important • Data on CCα and CCβ needs to be reviewed in view of their importance

Pharmacokinetics of quinolines and tetracyclines in Tunisian farmed fish using labelled drugs (Aida Ben Mansour, Tunisia)

From the reports, not much has been accomplished due to difficulties in acquiring and using 14C labelled quinolones. Even after acquiring the same from the USA, the problem of

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confirming the purity of the compound still remains to be solved. Several suggestions were made on how this could be overcome including use of TLC.

There is need to undertake PK studies on different species of fish.

Investigation of possible presence of chloraamphenicol in pasture plants and in animals fed with these plants (in the absence of Ms Tserendorj Enkhtuya , Linda Stolker reported on the current state of the project).

• In a recent publication, it was reported that plants in Mongolia have high levels of chloramphenicol

• It was observed that these samples had been collected and not analyzed for a long time, therefore need to use more recently collected plant samples from the same region

• Further validation using samples from Mongolia and the USA did not reveal high levels of chloramphenicol and there were no differences between the countries

• Controlled experiments in the laboratory on the uptake of chloramphenicol through the soil water did not show any preference for chloramphenicol residues

• Investigations are ongoing to assess the effect of seasons on chloramphenicol levels in selected plants

CROSS-CUTTING ISSUES

1. Antibody production a. Protocols – develop new and/or adapt existing b. Selection of appropriate carrier proteins c. Purification of polyclonal antibodies

2. Sample preparation a. Spiking before or after extraction

3. Assay validation a. Reference should be made to EU regulations on validation of screening

assays b. Rodrigo to share publication

4. Importation of reagents a. Can agency assist countries? b. Each country to examine its own regulations pertaining to export/import

of biological materials 5. Calculations of CCα and CCβ values and their appropriate application (see EU

regulations on MRLs) 6. PK data to be generated in all food producing animal species; drug companies to

be contacted to avail some of this information that may be in their dossiers 7. Environmental assessment of veterinary drugs as an emerging isse should be

addressed

Discussion issues from Group work

Group One: Immunoassays

In addition to the above cross-cutting issues:

• there is need to develop immunization protocols that are fit for purpose • Reference should be made to existing guidelines on validation of screening

methods

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• We need to re-focus the research work in order to deliver the project objectives e.g. if reagents cannot be obtained within the expected period, what happens?

• Each country to examine its regulations to facilitate acquisition of reagents in time

• It is expected that research materials will be shared between CRP holders

Group 2: Chromatographic methods (HPLC/TLC/MS)

A number of emerging issues need to be considered:

• Naturally occurring compounds found veterinary drugs • Food and environmental contamination • Multi-residues methods preferred due to low inputs (cost and labour) • Proficiency testing recommended, however, issues of transport need to be

addressed • How can information be communicated? (existing website, email, skype) • Study on environmental contamination has high regulatory impact on regulation

The CRP system addresses the availability of methods to member countries to meet Codex Standards/Regulations

Conclusions and Way forward

Following a though peer review of all the projects, it was agreed that a lot of work has been accomplished, however, this needs to be refocused to meet the original objective. The following were therefore agreed upon:

1. Develop appropriate protocols for production of polyclonal and purification of antibodies

2. Reference should be made to EU regulations regarding validation of sreening assays as well as the CCαβ values

3. Each country to examine its own regulations pertaining to export/import of biological materials

4. Emerging issues on food and environmental contamination by vet drugs be addressed, including generating PK data in various animal and fish species where this is not available

5. Efforts should be made to develop multi-residue methods that are fit for purpose (easy to use and cost-effective)

20

Annex IV: Work plans for 2nd Phase April 2011 to De cember 2012. BRAZIL: Contract No. 15596 Project Title Development of a Multi-Dot ELISA for

enrofloxacin and ciprofloxacin residue in chicken

tissues

Lead Partner Guilherme de Paula Nogueira

Start month April 2011

End month March 2012

Interaction with other partners Terence Fodey, Heinrich Meyer, Rodrigo Granja,

Yang Shuming

Technical support required Antibody purification, assay validation

OBJECTIVES

Rapid and simple screening device for antibiotic residue in poultry meat:

- Production of antibody, purification and characterization

- Development and validation of an immuno-assay (ELISA, RIA)

- Residue quantification of the tissue and blood samples collected at the in vivo experiment.

DESCRIPTION OF WORK

Task 1: Conjugation with protein, another immunization

Task 2: Affinity purification of the previous produced antibody

Task 3: Hire a company (Rea antibodies) to produce new antibody against Ciprofloxacin

Task 4: Antibody characterization: cross reaction, specificity

Task 5: Develop an enzyme immune assay from the obtained

Task6: Compare with other sources antibodies (China and Belfast) and test samples from in vivo study.

Task 7: Write and send an annual report to be evaluated by IAEA scientific board.

DELIVERABLES

No. Description Month

1 Antibody purification April 2011

2 New antibody production (Own production and Rhea) April-August 2011

3 Antibody characterization (Belfast and China) September-November 2011

4 Assay development and standardization November2011–January 2012

5 Quantify hormone of samples from in vivo assay February- March 2012

6 Validate the assay by LC-MS at Thomson (Campinas) April 2012

EXPECTED RESULTS

Validated assay for enrofloxacin and ciprofloxacin with necessary sensitivity

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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 H M M Granja



Interaction with other partners Terence Fodey, Heinrich Meyer, Guilherme de Paula Nogueira

Technical support required Radio-immunoassay development and validation

OBJECTIVES

To develop of a Radioimmunoassay kit for the determination of Florfenicol in Fish tissue Samples.

DESCRIPTION OF WORK

Task 1: Contact tritium labeled compound suppliers to obtain the tritium labeled florfenicol;

Task 2: Determine the best route for sending this material (antibodies and florfenicol amine standard);

Task 3: Develop RIA method for florfenicol residues in fish tissue samples using the thiamphenicol antibody provided by Queen´s University in Belfast, QUB and the florfenicol amine metabolite standard..

DELIVERABLES


1 Supplier of tritium labeled florfenicol identified July 2011

2 Reagents from Queens University, Belfast (QUB), obtained. August 2011

3 RIA using QUB reagents assay developed and validated. July 2012

4 Report prepared for IAEA. August 2012

EXPECTED RESULTS

A RIA for rapid screening of florfenicol and its metabolite florfenicol amine in fish tissue samples developed and validated.

22 22

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



Interaction with other partners Terence Fodey, Orlando Lucas, Guilherme de Paula Nogueira

Technical support required Organizing inter-laboratory test and joining PT to validate the established LC-MS/MS method for detecting benzimidazoles.

Obtaining CAM and more sensitive antibodies to bezimidazoles and making ELISA kits with accepted sensitivity for most MRL in food, validate the established ELISA kits.

OBJECTIVES

1. Optimization of a LC-MS/MS screening/confirmatory method for the determination of Aminoglycosides(AGs) drug residues.

2. Validation the optimized LC-MS/MS confirmatory method.

3. To explore the applicability of HPTLC by using LC-MS/MS extracts to obtain an additional screening method for AGs drug residues.

DESCRIPTION OF WORK

Task1: Checking the documents for transport antibodies from Europe to China.

Task2: Trying to get a cam (a methyl 5(6)- [(carboxypentyl)-thio]-2-benzimidazolecarbamate derivative) by the help of Mr. Terry Fedey.

Task3: Continuing to sythenize new antigens in order to get monoclone antibodies with higher specification and sensitivity.

Task4: Establishing ELISA kit with the antibodies I had with the sensitivity good enough to meet mostly MRL in the world.

Task5: Validating a established ELISA kits

Task6: Transporting SOP, standards and samples among inter-laboratories partners.

Task7: Finding official or commercial PT for benzimidazole testing, and joining in them.

DELIVERABLES


1 Monoclonal antibodies for enroflaxin and benzimidazole with low sensitivity

August 2011

2 SOP of LC-MS/MS for detecting 12 bezimidazoles in animal food

August 2011

3 Standard substances including labeled internal standards. August 2011

23 23

4 Benzimidazoles incurred samples. December 2011

5 ELISA kits for screening benzimidazoles March 2012

EXPECTED RESULTS

1. ELISA kit with accepted sensitivity and specification to meet MRL of Codex in most of food

2. Confirmatory methods for detecting multi- benzimidazoles, Pro-benzimidazoles and their metabolites in animal products with validation of inter-laboratory and PT testing.

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CHINA: Contract No. 15598

Project Title Project Title: Development of methods for aminoglycosides (LC-ESI-MS/MS and HPTLC)

Lead Partner Guihua LIU



Interaction with other partners Linda Stolker, Hubert de Brabander and Phil Kijak

Technical support required Methods optimization and validation

OBJECTIVES

1. Optimization of a LC-MS/MS screening/confirmatory method for the determination of Aminoglycosides(AGs) drug residues.

2. Validation the optimized LC-MS/MS confirmatory method.

3. To explore the applicability of HPTLC by using LC-MS/MS extracts to obtain an additional screening method for AGs drug residues.

DESCRIPTION OF WORK

Task 1: Use of different SPE approaches (HLB and CAX exchange) for sample extraction and purification and evaluation the use of internal standards.

Task 2: To validate the quantitative LC-MS/MS based on the confirmatory method according to the 2002/657/EC guideline for meat.

Task3: To write a SOP for the confirmatory LC-MS/MS method for AGs.

Task 4: Apply the extracts obtained from LC –MS/MS to the HPTLC plates to investigate the use of HPTLC for screening.

Task 5: To use the developed method to survey the residues of AGs in meat sold in Shenzhen area.

DELIVERABLES


1 Procedure for sample preparation August 2011

2 Validation the LC-MS/MS confirmatory method March 2012

3 SOP available March 2012

4 Methodology for AGs on HPTLC December 2012

EXPECTED RESULTS

Methodology for the analysis of AGs in meat

Screening /Confirmatory method by LC-MS/MS

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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



Interaction with other partners Yang Shumming; Terry Fodey

Technical support required multi-residue antibody production and characterization; training in LC-MS

OBJECTIVES

1. To produce novel conjugates for tetracyclines and antihelmintics

2. To develop and validate immunoassays for a range of anthelmintics and antibiotics

3. Determine necessary documentation required for receiving reagents from other partners

DESCRIPTION OF WORK :

Task 1: To prepare/acquire conjugates and/or antibodies of interest

Task 2: To undertake immunizations in a range of animal species to obtain large volumes of antibody

Task 3: To monitor antibody development through test bleeds

Task 4: To characterize antibodies

Task 5: Sample preparation: Undertake literature review on target analytes, including PK data. Information synthesized and documented for use to develop future strategies

Task 6: To develop methods and protocols

Task 7: To undertake in-house validation of developed assays

DELIVERABLES


1 A range of Conjugates June 2011

2 Characterized antibodies Dec 2011

3 Prototype immunoassays Feb 2012

4 A summary of sample preparation techniques March 2012

EXPECTED RESULTS

Approved protocols for multi-residue antibody production

Validated, sensitive, specific, affordable, easy to use assays for the detection and monitoring of selected antibodies and antihelmintics of economic importance to Kenya

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Republic of KOREA: Contract No. 15578.

Project Title The 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

Start Month March 2011

End Month March 2012

Interaction with other partners

AAM Stolker, Guihua Liu, Orlando Lucas, Hubert De Brabander, if necessary, other member countries

Technical support required Coordination of member countries for special purpose

OBJECTIVE(S). To apply and develop the modern LC-MS-MS techniques for veterinary medicine residue in the environmental samples

DESCRIPTION OF WORK

Task 1: Identification of hindrance effect induced by stable isotope

Task 2: Development of sample preparation and LC-MS-MS technique for following VDs in the environmental samples; cephalexin, tylosin, erythromycin, streptomycin, enrofloxacin, and fluorophenicol)

Task 3: Application of development method to real environmental samples

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),

Nov. 2012.

2 SOP and Guideline for this purpose. Dec. 2012

3 Holding a workshop/seminar of relevant research group July 2011-

Dec. 2012

EXPECTED RESULTS

1. Publications of the results in a special issue of a scientific journal 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 Guideline for this purpose 4. Technical coordination with the member countries (Netherlands, China, Peru, Belgiun, IAEA

Seibersdorf Lab.)

27 27

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.




Yang Shuming (China), Guihua Liu (China), Hubert De Brabander (Belgium), Philip Kijak (USA) and Linda Stolker (Netherland).

Technical support required.

EU legislation (2002/657) about validation procedures for veterinary drug residue analysis in foods. Revision of the scientific publication about the analysis of avermectin residues by LC with florescence detector. Chromatographic tandem-mass analytical method

OBJECTIVE(S). 1. To optimize the analytical strategy for ten prioritized representative target analytes of benzimidazoles and avermectins for milk

2. To validate the final method as screening method.

DESCRIPTION OF WORK

Task 1: To investigate the use of fluorescence detection for the analysis of avermectin residues in bovine milk. Task 2: Optimization of the LC-MS/MS method for the analysis of benzimidazol residues in bovine milk. Task 3: To validate the optimized LC-fluorescence / LC-MSMS method as according to EU legislation (2002/657). Task 4: Use the validated method for interlaboratory testing in cooperation with other member of the CRP.

DELIVERABLES


1 Applicability of the florescence detection for the analysis of avermectin residues in bovine milk

June 2011

2 LC-fluorescence / LCMSMS validated method for the analysis of avermectin and benzimidazol residues in bovine milk.

August 2011

3 Interlaboratory validated method for the analysis of avermectin and benzimidazol residues in bovine milk.

December 2011

EXPECTED RESULTS

To provide to the national member states laboratories a fast and relatively simple interlaboratory validated LC-fluorescence / LC-MSMS method for the analysis of avermectin and benzimidazol residues in bovine milk.

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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

Start month May 2011

End month April 2012

Interaction with other partners Guihua Liu, Orlando Lucos, Thomas Kuhn, Yang Shuming, Hubert De Brabander

Technical support required Improve the established basic method

OBJECTIVES

To expand the laboratory capabilities to develop secondary screening and confirmatory testing for antibiotic residues using HPTLC and HPLC respectively

DESCRIPTION OF WORK

Task1: Currently developed method for detecting sulphonamides (5) in chicken and shrimp will be further improved by:

a) Using HPTLC plates or plates without of fluorescence indicators b) Sample application using a spotter or spotting on HPTLC concentrated zone plates c) Enhance the fluorescence by on spot reagent or using the stabilizers d) Instead of using D2 lamp use Hg lamp in the TLC scanner e) The final reconstitution solution will be changed from MeOH to an appropriate solvent/solvent

mixture for better sample application

Task 2: The specificity of the method will be tested using two dimensional chromatography and also by transfer TLC spot to HPLC

Task 3: Conduct method validation

Task 4: Preparation of the SOP and report on validation data. Prepare manuscript for publication

DELIVERABLES


1 Further improvement of the analytical technique November 2011

2 Assess the specificity of the method December 2011

3 Validated method Feb 2012

4 SOP of the TLC/HPTLC method April 2012

EXPECTED RESULTS

Prepare protocol for sample preparation and analysis by the TLC/HPTLC

A validated TLC/HPTLC method for the detection of sulphonamide residues which will be used for routine residue monitoring program.

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THILAND: 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 2011

End month April 2012

Interaction with other partners Jin-Wook Kwon, Terry Fodey, Chris Elliot

Technical support required

OBJECTIVES

To develop and validate the microbiological screening method for the detection of multi-residues of antimicrobial substances.

DESCRIPTION OF WORK

Task1: Study on sample extraction for different matrices including meat, milk, serum, honey and milk.

Task 2: Produce a prototype assay for routine use with a selection of matrices including sample preparation procedures.

Task 3: Conduct method validation

Task 4: Submit a report to IAEA including a SOP and validation data. Prepare potential manuscript for publication.

DELIVERABLES


1 Sample extraction technique July2011

2 A prototype assay for routine use with a selection of matrices including sample preparation procedures

October 2011

3 Validated method Feb 2012

4 SOP of the screening method April 2012

EXPECTED RESULTS

1. Approved protocols for sample extraction and sample preparation

2. A validated 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 simple residue monitoring plan.

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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




1. Philip Kijak, Division of Residue Chemistry, FDA, USA. 2. Raj Patel, International Agency for Atomic Energy, NAFA, Vienna. 3. Tor Enar Holsberg ,Norwegian Veterinary School, Norway. 4. Heinrich Myer, Weihenstephaner Berg 5 Freising, Germany. 5. Hammadi Guerbej, National Institue for Marine Sciences &

Technologies,Tunisia.

Technical support required 1. Protocols for pharmacokinetics studies in fish using whole body autoradiography and liquid scintillation counting

2. Protocol & antibody for RIA Method

OBJECTIVE(S). 1. Pharmacokinetics studies on sea bass of an active flumequine. 2. Implementation and validation of RIA Screening method for

flumequine detection. 3. Publication of results in appropriate journals.

DESCRIPTION OF WORK

Task 1: Purity test of 14C-flumequine using liquid scintillation counting. Task 2: Perform Pharmacokinetics studies on sea bass fish: -Oral administration -Analysis using whole body autoradiography & liquid scintillation counting. Task 3: Set up and validate RIA method for flumequine detection

DELIVERABLES


1 Report to the agency containing the results of the pharmacokinetics studies of flumequine performed on Sea Bass

September 2011

2 Report to the agency containing the results collected from the development of the RIA technique as a screening technique for flumequine in fish tissue.

January 2012

EXPECTED RESULTS

Tissue distribution & Elimination rate of the selected active flumequine in sea bass (September 2011).

RIA screening method for flumequine detection in fish (March 2012)

Coordinated Research Project (CRP): D5.20 · on the development of radiometric and allied...

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