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JOINT FAO/IAEA DIVISION OF NUCLEAR TECHNIQUES IN FOOD AND AGRICULTURE

INTERNATIONAL ATOMIC ENERGY AGENCY

FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS

WAGRAMER STRASSE 5, P.O. BOX 100, A-1400 VIENNA, AUSTRIA TELEPHONE: (+43 1) 2600, FACSIMILE: (+43 1) 26007, TELEX: 112645 ATO, E-MAIL: Official.Mail@iaea.org, INTERNET: http://www.iaea.org

IN REPLY PLEASE REFER TO: DIAL DIRECTLY TO EXTENSION: PRIERE DE RAPPELER LA REFERENCE: COMPOSER DIRECTEMENT LE NUMERO DE POSTE:

26058/26061

319-D6-RC-729.2 B.Doko@iaea.org

REPORT

2nd RESEARCH COORDINATION MEETING (RCM)

OF THE FAO/IAEA COORDINATED RESEARCH PROGRAMME (CRP) ON EVALUATION OF METHODS OF ANALYSIS FOR DETERMINING MYCOTOXIN

CONTAMINATION OF FOOD AND FEED

IAEA, Vienna – Austria 4 – 8 December 2000

Food and Environmental Protection Section ____________________________________________________________

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Background The impact on human and animal health on mycotoxin contamination and on national and international trade is increasingly recognized in both developed and developing countries. In most of the developing world, official monitoring of mycotoxin is uncommon. There are few regulatory guidelines and seldom are there economic incentives for high quality products without mycotoxin contamination. In order to assess the extent of consumer exposure to mycotoxins, routine screening using cost –effective, officially recognized methods is useful. This provides information on the nature and extent of contamination and permits studies on the affect of this contamination on health. Appropriate analytical methods for determining mycotoxin contamination of food will also be useful for trade. While there are methods available, these require the use of expensive reagents or considerable capital investments in equipment difficult to maintain in developing countries. The present Co-ordinated Research Programme is to complement the FAO/IAEA Training and Reference Centre (TRC) for Food and Pesticide Control under the Centre's mission "to assist Member States and their institutions to fulfil requirements to support the implementation of international standards/agreements relevant to food safety and control, the safe use of pesticides and sanitary and phytosanitary measures, by providing cost effective analytical methodologies for mycotoxins”. There is a need to develop research data on the effectiveness of the various analytical methods used by food control laboratories to monitor mycotoxin contamination in order to select and recommend cost-effective, validated procedures, suitable for use in developing countries. The overall objective of this CRP is to assist national food control authorities and institutions to improve food safety and stimulate international trade in food by identifying and validating time and cost efficient methods for detection and quantification of mycotoxins in food in order to effectively monitor the mycotoxin content of agricultural import and exports. Research Co-ordination Meeting (RCM) The second RCM of the FAO/IAEA CRP on Evaluation of Methods of Analysis for Determining Mycotoxin Contamination of Food and Feed took place at IAEA, Vienna (Austria), from 4 to 8 December 2000. As listed in Annex I, the RCM participants, including research contract/agreement holders and observers, came from Argentina, Australia, Austria, Brazil, China, Cuba, Canada, Egypt, Ghana, Germany, Indonesia, Italy, Malaysia, Philippines, South Africa, United Kingdom, USA, and Uruguay. The objective of this RCM was to review the research activities conducted by the participants under the CRP, to prepare work plan outlines and timeframe for the coming year, and determine the future activities including future research topics and the dates and location of the next RCM. In addition, some recommendations were made concerning the CRP.

The RCM was hosted by the International Atomic Energy Agency (IAEA), Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Food and Environmental Protection Section, and held at IAEA, Vienna, Austria, 4 – 8 December 2000. A total of 27 participants, including research contract/agreement holders and observers, from Argentina, Australia, Austria, Brazil, China, Cuba, Canada, Egypt, Germany, Indonesia, Iran, Italy, Malaysia, Philippines, South Africa, United Kingdom, USA, and Uruguay. The list of the RCM participants is attached as Annex I.

Mr. Paisan Loharanu, Head of Food and Environmental Protection Section, Joint FAO/IAEA

Division of Nuclear Techniques in Food and Agriculture opened the meeting. Mr. Bruno Doko of the Food

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and Environmental Protection Section was the Scientific Secretary of the meeting. Mr. P. Loaharanu welcomed the participants on behalf of Joint FAO/IAIA Division of Nuclear Techniques in Food and Agriculture and gave a brief overview of the activities of the Food and Environmental Protection Section. He indicated that expectations of the Agency were to provide Member States in the developing world with cost-effective and robust analytical methods to assess the quality of food for food safety and trade. He wished the participants a productive and successful RCM.

The Scientific Secretary, Mr. B. Doko, first apologized for the inconvenience that might have

caused the several RCM date/place changes. He then expressed his gratitude to Mrs. Maya Pineiro who had set up the CRP with a good team comprising various experts in mycotoxin research and for her efficient supervision of CRP activities. He thanked all the participants for the amount of work done, in compliance with the work plans developed at the last RCM in Kuala Lumpur, Malaysia.

The presentation of research data and discussions were completed as scheduled. Copies of the presentations and related handouts are distributed to participants. The work group activities were carried out on the basis of the previous seven work groups created. New participants, including Mr. Hassan Yazdanpanah (Iran) and Mr. Palmyre (Seychelles) are joining the CRP. Mr. Hassan Yazdanpanah joined the Work group 1 and Mr. Palmyre (Seychelles) will be joining both work groups 1 and 2, and participate in FAPAS proficiency test programme. In addition, participation in several work groups and/or move of participants from a workgroup to another was recorded.

WORKGROUP 1: Aflatoxin in maize and peanut butter Participating contract/agreement holders: Nagler (NRI/UK), Liu (China), Bhat (India), Bahri/Maryam (Indonesia), Amra (Egypt), Garcia (Cuba), Cea (Uruguay), Sola (Argentina) Gilbert (MAFF/UK), Padilla (Philippines) Park/Trucksess (USA), Stroka (EC/Italy). Coordinator: Nagler (NRI, UK) WORKGROUP 2: Fumonisin B1 in maize Participating contract/agreement holders: Amra (Egypt) Bahri /Maryam (Indonesia) Garcia (Cuba) Hamid (Malaysia) India (Bhat) Canada (R. De la Campa) Kennedy/Lee (Vietnam) Liu (China) Nagler (NRI/UK) (help) Padilla (Philippines)

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Cea (Uruguay) Park/Trucksess (USA) (help) Scott (Canada)(help) Shephard (South Africa) Sola (Argentina) Sabino (Brazil) Visconti/Pascale (Italy) (help) Coordinator: Shephard (South Africa) WORKGROUP 3: Aflatoxin M1 in milk Participating contract/agreement holders: Sola (Argentina), Guilbert (MAFF/UK), Cea (Uruguay), Padilla (Philippines) Sabino (Brazil), Stroka (EC, Italy) Dragacci (France) Coordinator: Dragacci (France) WORKGROUP 4: Ochratoxin A in coffee Participating contract/agreement holders: Pascale/Visconti (Italy), Scott (Canada) Trucksess (USA) Sabino (Brazil) + other labs (from Brazil) Bhat (India), Sola (Argentina), Cea (Uruguay) Amra (Egypt) Coordinator: Sabino (Brazil) WORKGROUP 5: Feasibility study of ELISA technology for mycotoxins in developing countries Partyicipating contract/agreement holders: Liu (China) Bahri (Indonesia ) Kennedy/Lee (Australia) Amra (Egypt ) Coordinator: Kennedy/Lee (Australia)

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WORKGROUP 6: Preparation of radiolabelled fumonisin for disposition studies in naturally contaminated corn Partyicipating contract/agreement holders: Miller (Canada), Visconti/Pascale (Italy) Coordinator: Miller (Canada) WORKGROUP 7: Multiple trichothecenes (DON & NIV) in wheat and corn Participating contract/agreement holders: Sola (Argentina) Visconti (Italy), Trucksess (USA) Gilbert (MAFF/UK) Shepard (South Africa) Amra (Egypt) Sabino (Brazil ), Cea (Uruguay Coordinator: Cea (Uruguay)

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WORKPLAN OUTLINES AND TIMEFRAMES The specific objectives, work plans, outlines, timeframes and tasks for each workgroup and each participant were decided, approved and assigned as follows: WORKGROUP 1: DEVELOPMENT AND VALIDATION OF LOW-COST, ROBUST, EASY TO PERFORM METHOD FOR AFLATOXINS IN FOODS AND FEEDS APPROPRIATE FOR USE IN DEVELOPING COUNTRIES Coordinator: Nagler (NRI, UK)

This project addresses the question of whether new developments in clean-up techniques, which are generally employed with HPLC end-determination, can be adapted for use with TLC. The project will address the question of robustness of any proposed method and will carry out a full validation study with participants from developing counties. Objectives:

• To assess the viability of adapting an existing SPE/HPLC/HPTLC method for aflatoxins to be applied with a visual TLC end-point.

• To establish the practicality and conditions for multiple use of affinity columns for sample clean up of aflatoxins from maize and peanuts.

• To establish the availability and ruggedness of affinity columns under conditions in developing countries.

• Subject to successful outcomes to 2 and 3 to assess the viability of adapting an existing affinity column method for aflatoxins to be applied with a visual TLC end-point.

• To assess the outcomes of 1 and 4 and decide whether to pursue full collaborative trial evaluation of either SPE/TLC or affinity column TLC methods.

Scope:

• Commodities to be covered: - maize, and peanut butter. • Performance to be achieved: -to work to a limit of 2 ng/g aflatoxin B1 • SPE column: - phenyl-bonded column

Issues: Whether to still pursue trial if it proves not possible to meet 2ng/g limit of detection, i.e. to conduct trial but with materials containing higher levels of aflatoxins. Alternatively, whether to pursue a trial with densitometry or HPLC if it proves impossible to meet 2ng/g limit of detection by TLC. First Progress Report Summary 1. The NRI SPE Method, previously optimised for quantification with HPTLC and HPLC, was modified

for use with TLC, working to 2µg/ kg. The first revision was found to have resulted in a loss of robustness, and Indonesia also found low recoveries for aflatoxins B2 and G2. This revision usually gave good recoveries of aflatoxin B1 and G1 in maize matrixes, but recoveries were not satisfactory in groundnuts. There was also a tendency towards reduced clean-up efficiency, resulting in streaking on the TLC plate, as the weight of final sample (EW) was increased in the final extract. A second revision is proposed that should restore robustness and wide application of the method across matrixes, but will require the use of a syringe to spot 20 µl onto a TLC plate, rather than the preferred use of capillary micro-caps.

2. Collaboration with partners proved to be difficult due to poor communications and loss of control due

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to an increase in variables introduced by: laboratory equipment and techniques for using it, laboratory conditions, training of staff, reagent specifications etc. Whilst operating under these conditions is a true measure of the ease of use and robustness of a method, it is not desirable when trying to develop a method.

3. A decision was made to validate the modified SPE method in a joint study with an immuno-affinity

method, in an IUPAC inter-laboratory study. For this joint study to work, it was necessary to try to find a common procedure for extraction and TLC quantification, leaving only the clean-up step as the variable. Also, in order to comply with IUPAC requirement for methods that minimise the use of reagents that pose a health risk to analysts e.g. chlorinated solvents, benzene, or reagents that are not environment-friendly, such as lead acetate, changes were sought in the SPE methodology. A straight application of all alternative reagents resulted in poor recoveries, based on TLC, indicating that further research was required.

Modifications to NRI method The effective weight(EW) of sample in the final extract was increased from 1 g to 5 g by:

• Reducing the meal: solvent ratio from 1:5 to 1:4, now 50 g sample + 200 ml solvent. Studies indicated that a ratio as low as 1:2 was efficient at extracting aflatoxin from naturally contaminated maize, using a blender, but it was considered that the extra work required to fully validate such a procedure was not justified, and also this ratio would probably limit the extraction to a blending procedure, rather than leaving a shaking option.

• It was shown that high recoveries of all four aflatoxins could be maintained whilst the loading of

crude filtrate on the SPE column was increased in volume from 5 ml to 20 ml.

• The extraction solvent was changed from aqueous acetone to aqueous methanol (80%), to allow higher loading of the SPE column, and produce a cleaner extract (acetone extracts more potential interferences on TLC).

Limit of Quantification, Assumptions : ∗ Minimum weight of aflatoxin that can be quantified visually is 0.5 ng ∗ Minimum practical dilution of final extract = 200 µl ∗ Capillary micro-cap suitable for spotting 5 and µl only, syringe needed for larger volumes As the volume of filtrate that is applied to the SPE column increases from 5 to 10 to 20 ml the weight of sample in the final extract (EW) increases from 1.25 g to 2.5 g to 5.0 g. The increasing EW allows smaller volumes of extract to be applied to the TLC plate to achieve a limit of quantification of 2 µg/ kg (ppb), see Table 1. Hence 2 ppb can be quantified by spotting 20 µl when the EW is 5 g, but 20 µl is required when the EW is 2.5 g. The original intention was to try to limit the spotting volume to 10 µl to enable the use of capillary micro-caps for plate spotting. Table 1. Limit of Quantification Parameters TLC Spotting Vol. (µl)

Limit of Quantification (µg/kg) / EW (g)

1.25 g 2.5 g 5.0 g 5 20 8 4 10 10 4 2 20 5 2 1 50 2 0.8 0.4

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Table 2. Effect of Reducing Meal to Solvent Ratio on Extraction, with Blending Meal: Solvent Ratio EW B1 B2 G1 G2 1 : 5 1 114 9.7 nd Nd 1 : 2 2.5 110 10.3 9.1 Nd Table 3. Effect of Increasing SPE Column Loading on Aflatoxin (ppb), Meal: Solvent Ratio 1: 2 Vol. Filtrate on SPE (ml)

EW B1 B2 G1 G2

5 2.5 114 10 nd Nd 10 5 113 13 9 0.6 15 7.5 117 13 10 0.6 20 10 124 13 11 0.6 Table 4. Effect of SPE Loading: Low Aflatoxin Levels (ppb) Vol. on SPE (ml) Vol. Buffer (ml) B1 G1 5 35 4.5 4.9 10 30 4.7 5.1 20 20 4.4 3.9 30 10 0.7 0.8 Table 5. IUPAC Modification Step NRI IUPAC Modification

Extraction No salt Salt (10%)

Clean-up Lead acetate Zinc acetate

TLC Spotting Solvent Toluene/ acetonitrile (9 : 1) n-hexane/ acetone/ methanol (90 : 5 : 5)

TLC Development Solvent Chloroform/ acetone (9 : 1) with EMW (96 : 3 : 1) option

Tert. butyl methyl ether/ methanol/ water (96 : 3 : 1)

First Evaluation of IUPAC Modifications

• Recovery of 5 ppb spiked maize very low • Zinc acetate tended to give less good clean-up than lead acetate. • IUPAC spotting solvent gave only slightly larger spots than toluene: acetonitrile, but may have

solubility problems. • tButyl methyl ether development solvent gave more streak than chloroform: acetone for some

samples, some OK. Table 6. FAPAS, Round 32, NRI HPLC Results, uncorrected, using the original, and 2 modified clean-up methods (TLC very low) Vol. Filtrate (ml)

Clean-up B1 B2 G1 G2

5 Lead acetate 27.6 3 3.3 1.2 20 Lead acetate 25.4 2.6 4.6 0.8 20 Zinc acetate 25.4 2.6 5.4 0.8

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Recoveries (5 ml series) for aflatoxins B1, B2, G1, and G2 were: 91%, 93%, 89%, and 83% respectively Conclusions The NRI SPE method has been successfully modified for use with TLC, working to 2 ppb levels of aflatoxin in maize. It was better to optimise clean-up rather than weight of sample in final extract (EW), at the expense of larger spotting volumes requiring syringe application. Modifications to the SPE method for IUPAC validation need further study, especially TLC procedure for spotting and development. Working group: Nagler (UK/NRI), Lui (China), Bhat (India), Bahri/Maryam (Indonesia), Amra (Egypt) Garcia (Cuba) Cea (Uruguay) Sola (Argentina), Gilbert (UK/MAFF), Park/Trucksess (USA), Stroka (EC/Italy). Time Frame and Work plan Task 1

UK (NRI) + India + Indonesia + Egypt

March 2001

Finalization of pre-collaborative protocol. Determine preferred use of: lead acetate or zinc acetate, column volume, and TLC parameters

Task 2 UK (NRI) + USA April 2001 Pre-review of final protocol for potential approval by AOAC

International (Dr. Trucksess)

Task 3 UK (NRI) May 2001 Submit final protocol for review and acceptance by AOAC

International

Task 4 USA + Italy (EC)* April – May 2001 Preparation of maize and peanut test material at three levels of

contamination (0.5 – 30 ppb range) plus blank material – test material to be ground to small particle size, mixed and packaged in 50g test portions

Task 5 USA April–May 2001 Establishment of homogeneity of test materials –(demonstrate no

difference between inter-unit CV and intra-unit CV – analysis of every 10th sample from batch)

Task 6 All + partners June 2001 Undertake preliminary collaborative trial including distribution of

samples, standards and collaborative trial protocol.

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Task 7 USA July–August 2001 Preparation of second set of test materials as outlined in Tasks 3

and 4 at different levels (0.5 – 30 ppb range)

Task 8 All + partners November 2001 Undertake final collaborative study including distribution of

samples, standards and collaborative trial protocol.

Task 9 UK (NRI + MAFF) March-June 2002 Collation of trial results, statistical analysis and drafting of

publication.

Task 10 All + partners Test robustness of method locally by analysing survey samples.

* Note: coordination/agreements (between USA and EC) needed with regards to preparation and distributions of test materials. ** Note: Timing of collaborative study to be coordinated with working group 2

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WORKGROUP 2: COLLABORATIVE STUDY OF TLC METHOD FOR DETERMINING FUMONISIN B1 IN MAIZE Coordinator: Shephard (South Africa) Objective:

• To establish a sensitive TLC method for fumonisin B1 in maize • To collaboratively study the proposed method according to AOAC International guidelines

Working group: Amra (Egypt) Bahri / Maryam (Indonesia) Garcia (Cuba) Hamid (Malaysia) Bhat (India) De la Campa (Canada) Kennedy /Jackson/Lee (Australia) / Le Van To (Vietnam) Kpodo (Ghana) Liu Xiumei (China) Padilla (Philippines) Cea (Uruguay) Park / Trucksess (USA) (help) Scott (Canada) (help) Shephard (South Africa) Sola (Argentina) Sabino (Brazil) Visconti / Pascale (Italy) Extra participant in study: Moreno (Colombia) Summary of Progress Report As decided at the first Research Coordination Meeting of the FAO/IAEA Coordinated Research Programme (CRP) on “Evaluation of Methods of Analysis for Determining Mycotoxin Contamination of Food and Feed” held in Kuala Lumpur, 26-30 April 1999, this project forms part of Working Group 2: Collaborative Study of TLC Method for Determining Fumonisin B1 in maize. The first progress report (September 1999) described our initial studies using various fluorescent derivatizing reagents both with normal phase and reversed-phase TLC. The best results were found using fluorescamine derivatives preformed prior to separation on reversed-phase TLC. Nevertheless, additional fluorescent derivatization reagents were investigated prior to proceeding further with this collaborative study. Further Experimental Studies The fluorescent reagents dansyl chloride and dansyl hydrazine were tested both on normal silica as well as reversed-phase TLC plates by means of prederivatization of FB1 standards. In all the experiments, no clear FB1 spot was visible. The plates in general showed a large number of reagent-derived spots present both in the standard samples and in a reagent blank. Consequently, fluorescamine as a prederivatizing agent with separation on reversed-phase TLC was chosen for the collaborative study. A study in which a single extract was separately derivatized and spotted on the plate demonstrated that, visually, the method was reproducible.

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A short investigation was performed to assess the stability of the FB1-fluorescamine derivative. The derivative kept in solution and measured by fluorimeter showed good stability over the period of several hours tested, while the derivative separated on the plate remained visible for a number of days after TLC development, although a slow decline in fluorescence after 24 hours was noted. First Round of Collaborative Study The first round of the collaborative study has been designed as a practice round to test various aspects of the method and to provide participating laboratories with practical experience prior to the main collaborative study. Laboratories will be required to set up a series of standards to test the derivatization and their ability to successfully achieve TLC separation on the reversed-phase plates down to the detection levels required for determination of FB1 in maize samples. The participants will be required to derivatize and separate two purified maize extracts prepared in the PROMEC laboratory to test the separation of FB1 in cleaned-up samples. Finally the participants will test the entire method including extraction, clean-up and TLC separation using 4 maize samples provided (including a sample below the detection limit of the method). In addition to the samples, the participants receive the required strong anion exchange (SAX) solid phase extraction cartridges for sample purification and FB1 standard. Prior to the dispatch of materials for the first round, the following actions were completed. 1. Description of Method The method (including equipment and reagent requirements) was fully described in as unambiguous terms as possible. It is the intention to make the method description as clear as possible and this description may be further amended following feed back from participants in the first round. The method description was circulated to all participants in the working group to enable them to purchase the items required and to test the method. The final method description as sent to participants in the first round is contained within Appendix 1 attached to this report. 2. Preparation of Maize Samples We are indebted to Dr Mary Trucksess, FDA, Washington DC, USA for the preparation, homogenization and supply of maize samples at a range of FB1 contamination levels. These maize samples were analyzed by HPLC at PROMEC for FB1 prior to parceling for the first round study. 3. Optimization of the Method The maize samples supplied by Dr Trucksess were tested by the proposed TLC method. A number of experiments were performed to investigate different ratios of the sample, buffer and fluorescamine solution, but the original conditions chosen during method development were eventually retained for the study. During this stage of the investigation, the importance of proper equilibration of the mobile phase within the TLC tank was observed. Since the mobile phase for the reversed-phase TLC contains water, the equilibration time is longer than the time conventionally used for normal phase TLC in which the mobile phase consists exclusively of volatile organic solvents. Investigation of alternative mobile phases (ammonium acetate, pH 6.8 and sodium phosphate buffer, pH 3.0) failed to show superior separation or enhanced fluorescent properties. 4. Dispatch of Samples The samples and test materials for the first round of the collaborative study were dispatched to participating laboratories at the end of August 2000. Submission to AOAC International As undertaken at the first RCM meeting in Kuala Lumpur, the protocol for the collaborative study of a TLC method for the determination of FB1 in maize has been carefully described in the format

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required by AOAC International for collaborative studies and has been submitted to the AOAC Int. Methods Committee on Natural Toxins for consideration. Initial Study Results: Summary of Selected Method Several spray and derivative chemicals and application were evaluated on both normal and reversed-phase TLC. A summary of the TLC method selected for further study includes:

• Extraction: shake 20g maize with 100 mL Methanol/Water (3/1) for 1hr. Extraction by shaking rather than blending or homogenization.

• Clean-up: Adjust to pH 6.0-6.1 and purify on SAX-SPE cartridges. SAX cartridge is preferred as reversed-phase (C18) is less efficient and immunoaffinity columns too expensive).

• TLC Determination: Reversed-phase TLC of preformed fluorescamine derivatives. Fumonisin quantification by TLC rather than HPLC.

Results of First Study Round Summary of Results [in (µ g/kg)] Laboratory #

Maize extract 1

Maize extract 2

Maize blank

Maize sample 1

Maize sample 2

Maize sample 3

1 *1500 *700 ND *600 *2300 *3800 2 *1000 *1000 –

2000 400 800 800 600

3 4 ND 1000 ND 400 1000 2000 5 6 ND 1000 ND 400 1000 4000 7 8 9 2000 4000 ND ND 4000è ç2000 10 700 4000 ND 1000 2000 4000 11 12 400 2000 ND 400 1000 2000 13 ND ND ND ND ND ND 14 1000 2000 ND 700 1000 4000

Mean

(positives) 1100 2025 --- 614 1638 2800

RSD (%) 52.1 64.5 --- 38.1 67.2 46.9

Expected results

2000 5000 ND 600 2500 4700

*Results recalculated from amount reported in TLC spot èç Results may have been interchanged Sample 2: Mean: 1388µg/kg; RSD: 43.3% Sample 3: Mean: 3050µg/kg; RSD: 43.6%

• 9/14 laboratories have reported results • 2 laboratories are still waiting for TLC plates • 2 laboratories have received samples, but have not reported. • 1 laboratory has not responded.

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Comments by IAEA participants General comments

• The method is simple, easy to follow, and well described • Label the last column of the dilution table better

Sample Preparation

• Centrifugation is not necessary, filtration is sufficient. • Possibility of using immunoaffinity columns rather than SAX columns for clean up of samples.

TLC Procedure

• To specify that the plate needs no activation. • Spotting the technique needs to be well described. • To specify the spotting distance from the interfac e between pre-concentration zone and silica. • Spotting time too long. • Equilibration time of TLC tank too long.

TLC Results

• Whatman LKC-18 plates give better performance than Merck HPTLC RP -18 or Merck RP -18. • Fluorescent derivatives stable during analysis (3 hours in light, longer in dark). • Trouble with spot below FB1. • Contaminant Rf 0.26 was well separated. • Extracts produced considerable backgraound fluoresecence on the plate too many

interferences. • Tailing of FB1 spot. Variation in Rf between standards and sample. • Extra spot in standards (Rf 0.6). • 1000µg/kg would be better limit as 400 µg/kg is near extinction.

Comments by AOAC Int’l.

• Supply method performance data from in-house studies at specified levels. • More details of TLC procedure should be given. • Original method was described as semi-quantitative; should this be quantitative or screening? • If screening, what statis tics does one apply to the results?

Proposal for Continuation

• Training workshop prior to final round. • Introduce a second round to further familiarize participants with the method. • Proceed with the final round: Full collaborative study (with or without AOAC International).

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Time Frame and Work plan

Task 1

South Africa (G. Shephard)

January–March 2001

Prepare collaborative study protocol and submit to AOAC International for approval. Task 2 South Africa January–March 2001 New participants to order TLC plates and laboratory supplies needed for the study. Existing

participants (who have still to receive TLC plates on order) to complete first round when plates arrive and communicate with South Africa.

Task 3 South Africa March 2001 Prepare and send out samples, SAX columns, and fumonisin B1 standards to participating

laboratory for the second round study.

Task 4 All April – May 2001 Conduct sample analyses and report back results to South Africa.

Task 5 South Africa June– July 2001 Assess study results Task 6 South Africa Aug.–September 2001 Preparation of maize test material at three levels of contamination (within contamination

range 500 to 5000 ng/g) plus blank materials acceptable for AOAC International collaborative study.

Task 7 South Africa October 2001 Send out collaborative study samples Task 8 All Nov.–December 2001 Perform collaborative round study and report results to South Africa*. Task 9 South Africa + All January – March 2002 Assess collaborative study results and communicate with participants Task 10 South Africa April 2002–May 2002 Prepare final report

*Note: Timing of collaborative study to be coordinated with working group 1

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WORKGROUP 3: DETERMINATION OF METHODS FOR AFLATOXIN M1 IN MILK. Coordinator: Dragacci (France) Objective: Select and Validate a Method for Aflatoxin M1 in Milk. Selected Methods: Immunoaffinity Column Method Following the IDF Standard 171: 1995 in procedure but changing the detection step to TLC instead of HPLC. Also perform an immunoaffinity column performance check Summary of Progress Report

An interlaboratory comparison was conducted as a pilot study to evaluate the effectiveness of an immunoaffinity clean up followed by a thin layer chromatography (IAC-TLC) for the determination of aflatoxin M1 in milk at US and European regulatory limits. AFM1-spiked milk samples were sent frozen to 13 participants in 10 different countries. The spiking level was the target value of 0.400 µg / L but after spiking and homogenization the AFM1 content was found at 0.325µg / L.

The dispatching of parcels to faraway countries by express delivery was satisfactory. The laboratories should analyse the sample in exactly following the draft of the method. A statistical analysis was run on the raw data of laboratories according t the ISO 5725 standard. The overall mean of the pilot study (0.281 µg / L) was close to the assigned value (0.325/µg / L). The relative standard deviation value for repeatability is satisfactory (8.4%), but the relative standard deviation value for reproducibility (58.7%) betrays a wide dispersion in the interlaboratory results. Several comments given by participants will be taken into account for the method which will be used for the full validation. Some technical aspects will be adapted for the full validation study to try to decrease in RSDR of this method to about 30%. Organization of the Pilot Study

Each participating laboratory received one spiked liquid milk sample, two immunoaffinity columns, the draft of the method, draft instructions, one receipt form and one report form. The aim of this pilot study was to familiarize the participants to the method and to test the dispatching and the stability of liquid milk samples in sending them by express delivery in faraway countries. Laboratory data analysed according to ISO 5725 standard by using AIL 5725 software. Participating laboratories

Thirteen laboratories (table 1) were volunteered to take part in the present pilot study and then to the full validation study. Table 1 – List of participating laboratories Country Participant Organism Argentina A. Garbini

I. Sola National Institute of Food National Institute of Industrial Technology

Brazil M. Sabino E. Azevedo Vargas

Instituto Adolfo Lutz Laboratory for Mycotoxin Analysis

Malaysia A. Hamid Food Technology Centre, MARDI People’s Rep. Of China Z. Peng

X. Liu Food Laboratory of CIQ – QingDao Institute of Nutrition and Food Hygiene

Philippines L. Padilla National Food Authority Senegal A. Kane Institut de Technologie Alimentaire Spain M.A. Moreno Facultad de Veterinaria, UCM Switzerland A. Cominoli Service de Protection de la Consommation United Kingdom M.J. Nagler Natural Resources Institute Uruguay M. Pineiro Laboratorio Tecnologico del Uruguay

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Preparation of samples

Samples for the pilot study were prepared by the AESSA-LERHQA as follows: one batch of blank milk containing less than 0.005f pg AFM1/L was selected. This milk was an UHT skimmed one purchased in a local shop. The spiking level for the AFM1 contamination of milk was targeted at 0.400µg / L.

Contamination was done in progressively diluting AFM1 standard solution in 5 L of blank milk

under continuous magnetic stirring. The contaminated batch was stabilized with a 15% sodium azide (NaN3) solution to inhibit the growth of micro-organisms.

The AFM1-contaminated batch was divided into 250 mL-Nalgen bottles labelled “SAMPLE FOR THE PILOT STUDY – AFM1 in liquid milk” and frozen at 30o C until the distribution. Frozen samples with ice plates were sent to the laboratories by express delivery.

The assigned value was determined by AFSSA-LERHQA in analyzing samples by High

Performance Liquid Chromatography after immunoaffinity clean up according to the EN ISO 11501 standard. The measured content of AFM1 was 0.325µg / L. Reagents

Each participant received two immunoaffinity columns (Aflaprep MTM) containing anti-AFM1 antibodies from Rhone Diagnostics Technologies (Galsgow, UK). All immunoaffinity columns (IAC) came from the same batch. No FM1 standard was provided to participants. They had to analyse sample with their own standard. Draft of the method and instructions for participants: The draft of the method and the instructions were sent to each participant. Interlaboratory study

On the 31st July 2000, one milk sample from the AFM1 spiked batch with an unknown level to participants was sent to all laboratories. Participants were required to carry out the analysis in duplicate in following strictly the provided draft of the method, taking a test portion of 100 mL and using their own AFM1 standard. Results should be sent back to AFSSA-LERHQA before the 4th September 2000. Results of the Pilot Study Dispatching of parcels

A receipt form should be sent back to AFSSA-LERHQA as soon as receipt by the laboratory. The table 3 summarizes the information on the progress of the dispatching given by the participants. For two laboratories (1 and 4), the immunoaffinity columns attached to the parcel were loosen during the travel. These laboratories used their own immunoaffinity columns for the pilot study; they were Aflalprep MTM from Rhone Diagnostics Technologies (laboratory 1) and AflaTestTM from VICAM (laboratory 4). Laboratory raw data

All laboratories sent back their results to AFSSA-LEHQA before the 4th September 2000 as required (except the laboratories 2,3,5 and 11). The raw data not corrected for recovery are reported on the table 4

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Table 4 – Laboratory raw data (uncorrected for recovery) Laboratory # Assay 1 Assay 2 Mean 1 0.113 0.162 0.138 2 0.333 0.250 0.292 3 0.006* 0.388 - 4 0.500 0.500 0.500 5 0.150 0.160 0.155 6 0.18 0.18 0.18 7 0.100 0.100 0.100 8 0.444 0.400 0.422 9 0.314 0.285 0.300 10 0.260 0.250 0.255 11 0.600 0.600 0.600 12 0.148 0.142 0.145 13 0.077** - 0.077 *Laboratory 3 explained that the 0.006 value was clearly related to a technical problem during the elution step from the immunoaffinity column (too fast and incomplete elution). ** Laboratory 13 explained that the 0.077 value was the mean of four values (spots of 10 and 20 µ L, each in duplicates) Consequently:

- for the laboratory 3, the 0.006* was not included in the statistical analysis; only the remaining value was used in the statistical calculation.

- For the laboratory 13, the single value (0.077) was included in the statistical analysis. Statistical analysis

A statistical evaluation was carned out on uncorrected data according to ISO 5725 standard by using AIL 5725 software. No straggling nor outlying data were found. Results of this statistical analysis are reported in the table 5. Table 5 – Results of the statistical analysis

Assigned 0.325 Value µ g / L

Number of laboratories 13 Overall mean (M, µg / L) 0.281 Standard deviation for repeatability (SDr, µg / L)

0.0236

Repeatability value at 95% (r, µg / L)

0.067

Relative standard deviation for repeatability

(RSDR, %)

8.4

Standard deviation for reproducibility (SDR,/ µg / L)

0.1648

Reproducibility value at 95% (r, µg / L)

0.466

Relative standard deviation for reproducibility (RSDR, %)

58.7

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Detection means

Two kinds of detection could be used by the laboratories to quantify the content of AFM1: visual detection with UV-lamp or densitometer detection. After the receipt of the raw data, AFSSA-LERHQA contacted laboratories to know what kind of detection they used.

8 laboratories used the densitometer detection (laboratories 3, 9, 10 and 13). The laboratory 11 did not yet answer to this question. Comments from the laboratories

Comments about the method, which were sent back by the laboratories with their own data, are summarized in the table 6. Table 6 – Comments from the laboratories Comments Laboratories The method is clear to understand and easy to perform 4,12 There were different kinds of editorial amendments 2,9 Interest of heating the liquid milk sample before centrifugation 2 20 mL of water should be enough to wash IAC (instead of 40 mL) 2 Other solvents are proposed to develop the TLC-plates (tertiary butyl methyl ether instead of diethyl ether)

2

It is difficult to spot the sample again if the first spots are outside the calibration curve

3

It is difficult to detect the lowest standard spot even at 20 µg

4

It is proposed to give results with only two decimal places 9 To specify only one way of detection: visual detection with UV-lamp or densitometer

2

Laboratory 8 used the solvent recommended for the bi-directional TLC (chloroform/acetone/isopropanol: 87/10/3) instead of the one recommended for the unidirectional TLC. Conclusion

The dispatching by express delivery was satisfactory: no sample was curdled. A specific attention is required to the laboratory in sending back their receipt form as soon as receipt to allow a good interpretation of results by AFSSA-LERHQA. Laboratories should make an effort in sending their results on due time. The method must be exactly followed by the laboratories to have the best final validation. The recommendation of two calibration curves was not easy to understand. Two laboratories used their own immunoaffinity without relevant discrepancy. The overall mean of the pilot study (0.281 µg / L) is closed to the assigned value of 0.325 µg / L. The relative standard deviation value for repeatability is satisfactory (8.4%). The relative standard deviation value for reproducibility (58.7%) betrays a wide dispersion in the inter-laboratory results. However, some explanations could be given as follows:

- this method is a semi-quantitative one because of its relative difficulty to compare a sample spot to wide range of calibration curve.

- All participants used their own standards - The laboratory recoveries were not taken into account.

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All these aspects will be studies although the full validation study; nevertheless, if compared to the validation study of a IAC-HPLC method where the RSDR was less than 30%, it is possible to hope to decrease the RSDR for the IAC-TLC method from 57.8% to about 30%. Work group: Sola (Argentina), Guilbert (MAFF/UK), Cea (Uruguay), Padilla (Philippines) Sabino (Brazil), Stroka (EC, Italy) Dragacci (France) Time Frame and Work plan

Task 1

IAEA (B. Doko)

December 2000

Provide participants with Aflatoxin standards (M 1; B1; B2; G1 and G2) Task2 FRANCE (AFSSA) + UK (MAFF) January–May 2001 Procure materials for second collaborative trial.

Raw milk naturally contaminated at 2 ng/mL to be obtained from AFSSA (France) for dilution to target levels of contamination for collaborative trial purposes.

Task 3 FRANCE (AFSSA) February 2001 Draft (or revise) protocol for second collaborative trial Task 4 URUGUAY (LATU ) + ARGENTINA (INTI) + EC (ISPRA) April 2001

Establish affinity column re-use conditions, Column to be tested to establish the maximum number of times it can be re-used before there is a reduction in recovery of aflatoxin M1 and optimum conditions for re-use to be established.

1. Rhone Poulenc column re-use tests for M1 by J. Stroka (EC) 2. Vicam column re-use tests for M1 by J. Cea (Uruguay) 3. Compare the performance of columns Cea (Uruguay)

Task 5 FRANCE (AFSSA) June 2001 Distribute samples, IAC columns (Rhone) and protocol to participants Task 6 FRANCE (AFSSA) July 2001 Compile sample results

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WORKGROUP 4: OCHRATOXIN A IN COFFEE Coordinator: Sabino (Argentina)

Objective:

• Select analytical method for Ochratoxin A (OTA) in coffee.

• Evaluate and validate method Visconti (for test material from Nestle and of BRC) Brazil. Miller (to help provide test materials) Working Group: Pascale/Visconti (Italy), Scott (Canada) Sabino (Brazil) + several other labs (from Brazil) Bath (India) Sola (Argentina) Cea (Uruguay) Trucksess (FDA) Amra (Egypt) Time Frame and Work plan

Task 1

Brazil (Sabino) + Italy (Pascale)

February 2001

Prepare protocol and review by Pascale (Italy), Pre-review of final protocol for potential approval by AOAC International (Dr; Trucksess).

Task 2 UK (MAFF/Gilbert) + Italy (Visconti) February March 2001 Provide known and unknown tests materials Task 3 All April – May 2001 Perform the tests Task 4 Italy (Pascale) + India (Bhat)+ USA (Park) April – May 2001 Perform column regeneration tests

Columns (from VICAM) for regeneration tests to be provided by Pascale/Visconti (Italy)

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WORKGROUP 5: FEASIBILITY STUDY OF ELISA TECHNOLOGY FOR MYCOTOXINS IN DEVELOPING COUNTRIES Coordinator: Kennedy/Lee (Australia) Objective: To conduct a feasibility study on the immunological and Elisa technology for the analysis of mycotoxins in developing countries. Summary of Progress Report The following activities have been carried out in this project in the past 12 months: 1. November 1999: ELISA Workshop (mycotoxin and pesticides, Nov 15-17) and ASEAN Seminar

on Post Harvest Quality Assurance (Nov. 7-12) at the PHTI in Ho Chi Mink City, Vietnam. The ELISA Workshop trained Vietnamese personnel (10) and other five Asian scientists in the use of immunodiagnostic techniques for aflatoxins, including Dr. S. Bahri, a member of the CRP (RC 10488).

2. Preparations for fumonisins, ochratoxin A and aflatoxin B1 proficiency trials have been made by

Ms. Karen Jackson of AgriFood Technology (ACIAR collaborative research programme). The fumonisins trial is currently completed. This follows a HPLC training workshop held at the PHTI in Vietnam, which included a range of mycotoxin analyses. Its also incorporated successful analysis of aflatoxin samples using immunoaffinity columns (IAC) based on the anti-aflatoxin antibody prepared by our Vietnamese collaborators. It is envisaged that in 2001 in the final stages of this project, IAC will be included in validation trials.

3. Aflatoxin B1 hapten conjugated to three carrier proteins and to an enzyme have been prepared

for formatting a competitive ELISA. Immunization of rabbits and collection of antisera was carried out according to the schedule. The initial characterization of purified antibodies showed very promising results. Further characterization is being conducted.

The rate of development of the immunological materials and ELISA for aflatoxin B1 (and others aflatoxins) has been regulated to suit the work the plan of the 3-year ACIAR project (1999-2002) and the training needs of personnel as well as building of facilities. The aim is to allow Vietnam to produce its own kits at a cost structure suiting that country. The highest priority is now being given to aflatoxin B1, the first candidate to be developed and it is anticipated that a prototype ELISA kit will be available by mid-2001. Workgroup: Liu Xiumei (China) Bahri (Indonesia) Kennedy/Lee (Australia)

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Time Frame and Work plan

Task 1

Australia (Lee)

May – June 2001

Multi -toxin immunoaffinity methods Validate antibody assay in microtiter 96 well plates in several matrix:

1. Feed, Copra, 2. Groundnuts, 3. Peanuts extractions, 4. Maize, 5. Pistachio,

Note: Provide Dr. Lee with FAO document on sampling (B. Doko)

Task 2 Australia (Lee) + Egypt (Amra) June 2001– Jan. 2002 Evaluate dipstick technology Task 3 China (Liu) June 2001– Jan. 2002 Evaluate antibodies from China (using samples from South Africa, i.e. from the same

samples used for FB1 collaborative studies).

Task 4 Indonesia (Bahri) June 2001– Jan. 2002 Evaluate antibodies from Indonesia on samples obtained from UK (Nagler).

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WORKGROUP 6: TECHNIQUES FOR EVALUATING DISPOSITION OF FUMONISINS IN NATURALLY CONTAMINATED CORN DURING FOOD PROCESSING Coordinator: Miller (Canada) Objectives: To develop a method to produce corn kernels infested with Fusarium verticillioides and contaminated by fumonisin produced in vivo enriched by C14 at sufficient activity to permit the material to be ground into meal and used to evaluate the disposition of fumonisins in food products made from corn meal in comparison to data from chemical analysis. Progress Report This is additional to the progress on the project given in 1999. Irradiated kernels (13; 10 g) were placed in pre-sterilized large glass Petri dishes containing filter paper and 30 g of distilled water with (3 replicates plus controls). After equilibration (3 days), a small piece (1 mm2) of agar culture of MRC 826 taken from the leading edge of a 2% malt extract agar plate culture was placed on the filter paper. After 2 days incubation at 28 C, 14 C1- C2 sodium acetate in ethanol (ICN Ltd.), aliquots were placed directly on the tip cap of each kernel with a 50 µL syringe under aseptic conditions. Three treatments were done such that either 170, 330 or 500 µCi 14C1-C2 acetate were added over a 5 day period. The cultures were incubated a further three days and then freeze dried and ground. For ergosterol and fumonisin anaylses, the samples were washed first with 0.01% sodium acetate in water to remove residual acetate. Extensive studies had demonstrated that this did not affect the recoveries of either compound. Consistent with the results from the trials in 1999, approximately 1 ppm fumonisin was produced under these conditions. As has been found in studies involving additions of acetate precursor, there was an effect of acetate on fungal biomass but there were no significant differences in the specific activities of these acetate-derived metabolites. The specific activities were in the desired range. uCi ppm uCi/mg uCi/mg acetate added ergosterol ergosterol fumonisin 170 56 3.6 3.0 330 27 5.5 2.0 500 21 3.4 2.4 Corn meal muffins (after Scott and Lawrence, J AOAC International 77:541, 1994) were made using corn meal and corn meal spiked with unlabelled pure fumonisin B1. These were analysed by the EU (Visconti) method using fumonitest immune affinity columns followed by HPLC analysis using the OPA derivative. Consistent with other studies, there was a significant reduction in fumonisin recovered. The remaining ground corn kernels containing in vivo labelled fumonisin was used to make additional corn meal muffins by substituting about 20% of the commercial corn meal. After baking, the muffins were extracted by the Visconti method. Aliquots of the material eluting from the column were analysed by HPLC and also analysed on a scintilation counter. No sign of FB2 nor AP1 could be seen in the HPLC chromatogram. The amount of fumonisins stripped off the immune-affinity column was much greater by the calculation using 14C than by analysis. Further studies are needed to understand exactly what this means. Working group: Miller (Canada) & Visconti (Italy) Time Frame and Work plan

Task

Canada (Miller) + Italy (Visconti)

In 2001

Study the recovery of fumonisins from corn-meal muffins

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WORKGROUP 7: MULTIPLE TRICHOTHECENES (DEOXYNIVALENOL AND NIVALENOL) IN WHEAT AND CORN Coordinator: Cea (Uruguay) The studies preferably focused mainly deoxynivalenol (DON) and nivalenol (NIV) Objective: To Select, evaluate and validate a TLC method for wheat and maize. Working group: Sola (Argentina) Visconti/Pascale (Italy), Trucksess (USA) Gilbert (MAFF/UK), Shepard (South Africa) Amra (Egypt), Sabino (Brazil), Cea/Pineiro Time Frame and Work plan

Task 1

Uruguay (J. Cea)

Set up the protocol; send out the draft protocol for review to advisors Task 2 Uruguay (J. Cea) + Italy (Pascale) + UK (Gilbert) October 2002 Develop working group research plan (By next meeting)

Trucksess (USA), Gilbert (UK) and Scott (Canada) are selected for backup and guidance.

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MEETING RECOMMENDATIONS To maintain close communications through e-mail and other media between the participants, the research contract/agreement holders and the technical officer (Bruno Doko) as well as the members of each workgroup and FAO contact (Maya Pineiro). A system for e-mail reporting at fixed dates should be established. The technical officer was asked to make use of e-mail to keep each participant current with the assigned tasks and timeframes. Additional participants from developing countries, specially African countries, should be incorporated to the CRP. Seek support on provision and distribution of standards and test materials from FAO/IAEA specially in view of the new chemical warfare classification of some mycotoxins. Where possible, the group asked the Joint FAO/IAEA Division to procure and distribute analytical supplies (columns and certain special reagents) to developing country participating laboratories in order to conduct the assigned CRP research task. Have technical backup visits from advanced country agreement holders to developing country contract laboratories to assist in performance of CRP tasks. Asian and Latin American laboratories were identified for backup visits. Training courses and fellowships for training at excellence centers in advanced country agreement holders’ laboratories or at Seibersdorf on Quality Assurance for Mycotoxins. A video/CD-ROM of the methods chosen on this CRP for collaborative or ring testing should be prepared. DR John Gilbert with the FAPAS group offered the possibility of undertaking the task as part of the conclusions of the CRP. As tasks and collaborative/ring testing are finished by participating laboratories survey samples (>25) should be analyzed employing the new method evaluated in their CRP workgroup, with comparison to standard/official method. *Study of multitoxin methods for aflatoxins, zearalenone and ochratoxin A as well as multiple trichothecene and T-2 methods in grains should be included in the next assignments for the rest of this CRP, starting in 2001 Inclusion in Web Page of CRP documents as portable files (PDF format) The next RCM should be in South Africa in 2002. The retained dates should be know ahead of time; knowing that period ranging from November to January has been excluded. Future research topics: Effect of food processing on fumonisisn B1. Fate of radiolabelled fumonisin in local food production processes in countries with large corn consumption (J. D. Miller). Development and evaluation of local technology for preparation of immunoaffinity columns (multifunctional: aflatoxin B1 and fumonisin B1) with monoclonal antibodies (A. Lee). Development and validation of simple and rapid methods for detection of aflatoxin in food and feed appropriate for use in developing countries (Ramesh V. Bhat) Status report on HACCP for mycotoxins (M. Nagler) Status report on decontamination for mycotoxin decontamination (B. Doko and D. Park) *P. Scott (Canada) to provide guidance on the selected methods obtained from Sola (Argentina), Amra (Egypt) and Cea (Uruguay)

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ANNEX: I

Names and contact addresses of the 2nd RCM participants

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TC / RC / RA # Contract / Agreement holders Country RC/10486 Dr. Hassan Amra

National Research Center, Mycotoxins Central Laboratory Food & Dairy Technology Department Dokki, Cairo, EGYPT, Tel (office): +202-3371499; Tel (home): 415-3028 Fax: +202-3370931 E-mail: amraha7@yahoo.com

EGYPT

RC/10488 Dr. Sjamsul Bahri / Dr. Romsyah Maryam

Research Institute for Veterinary Science (RIVS), Department of Toxicology Jl. R. E. Martadinata 30, P.O. Box 151, Bogor 16114, INDONESIA Fax: +62-251-336425; Tel:+62-251- 331048, 334456 E-mail: balivet@indo.net.id rmaryam@indo.net.id r_maryam@hotmail.com

INDONESIA

RC/10689 Dr. Abidin Bin Hamid

Food Technology Centre, MARDI, GPO Box 12301 50774 Kuala Lumpur, MALAYSIA Tel: 60-3-8943 7266; Fax: +60-3-8942 2906; E-mail: abh@mardi.my

MALAYSIA

RC/10494 Dr. Jacqueline Cea

Laboratorio Tecnologico del Uruguay (LATU), Departamento de Micotoxinas Avenida Italia 6201, Montevideo, URUGUAY Fax: +598-2 601-8554; Tel: 5982 601-3724 E-mail: jcea@latu.org.uy or micotox@latu.org.uy

URUGUAY

RA/10492 Dr. Raymond D. Coker / Dr. Martin J. Nagler

Food Systems Department, Natural Resources Institute, The University of Greenwich, Central Avenue, Chatham, Kent ME4 4TB, UK Fax: +44-1634-880066; Tel: : +44-1634-883459; E-mail: r.d.coker@gre.ac.uk ; M.J.Nagler@gre.ac.uk

UK

RC/10485 Dr. Miguel O. Garcia

Instituto de Nutricion e Higiene de los Alimentos, Departmento de Quimica y Toxicologia, Infanta #1158, La Habana 10300, CUBA Fax: +53-7-338 313; Tel: 537 785919 E-mail: hugoluis@cubarte.cult.cu or mogoroc@yahoo.com

CUBA

TC/10852 Prof. John Gilbert

Central Science Laboratory (CSL), Sand Hutton, York YO41 1LZ, UK Fax: +44-1904-462 11; tel. +44-1904-462 000; E-mail j.gilbert@csl.gov.uk ; science@csl.gov.uk

UK

RC/10787 Mrs. Kafui Kpodo*

Food Research Institute, Analysis Division, P.O. Box M.20, Accra, GHANA Fax: +233-21-777 647; Tel. +233-21-7010 557, E-mail: kpodofri@ghana.com

GHANA

RC/10578 Dr. Alice Lee

Dept. of Agricultural Chemistry & Soil Science, Faculty of Agriculture, University of Sydney, A03 Bldg., Ross Street, Sydney, AUSTRALIA Fax: +61-2-9351 5108, E-mail: a.lee@acss.usyd.edu.au

AUSTRALIA

RC/10484 Dr. Liu Xiumei

Institute of Nutrition and Food Hygiene, Chinese Academy of Preventitive Medicine, CHINA

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Department of Microbiology and Natural Toxins 29 Nan Wei Road, Beijing 100050, PEOPLE’S REPUBLIC OF CHINA Fax: +86-10-83150677; Tel: +86-10-83150677 E-mail: xmliu@public.east.cn.net

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TC/10573 Dr. J. David Miller

Carleton University, Department of Chemistry Ottawa, Ontario, CANADA K1S 5B6 Tel. +1-613-520-2600, x-1053; Fax: +1-613-520 2710/3749 E-mail: davidmiller@pigeon.carleton.ca

CANADA

RC/10490 Dr. Luz Padilla

National Food Authority, Food Development Center, FTI Complex, South Superhighway, Taguig, Metro Manila, PHILIPPINES Fax: +63-2-838 4016; Tel: (632) 838 4478 E-mail: infofdc@pacific.net.ph

PHILIPPINES

RA/10495 Dr. Douglas L. Park

Director, Division of Natural Products, Center for Food Safety & Applied Nutrition, Department of Health & Human Services, Food and Drug Administration (FDA), 200 C Street SW, Washington, DC 20204, USA Fax: +1-202-205 4422 ; Tel : +1 202 260 2694 E-mail: dpark@cfsan.fda.gov

USA

RC/10788 Dr. Ramesh V. Bhat

Indian Council of Medical Research, Food & Drug Toxicology Research Centre National Institute of Nutrition, Division of Food Safety Hyderabad - 500 007, INDIA Fax: +91-40-701 9074; Tel: +91-40-701 8909 or 701 8910 E-mail: icmrnin@ren.nic.in or nin@ap.nic.in

INDIA

RC/10482 Dr. Myrna Sabino

Instituto Adolfo Lutz, Biological Chemistry Section, Bromatology and Chemistry Division, Av. Dr. Arnaldo 355, Sao Paulo, SP, BRAZIL, 01246-902 Fax: +55 11 3085 3505; Tel: +55 11 3068 2921 or 2922 E:mail: myrna@sti.com.br or mysabrino@ial.sp.gov.br

BRAZIL

RA/10483 Dr. Peter Scott

Food Research Division, Health Products & Food Branch Health Canada, Ottawa, Ontario, CANADA K1A 0L2 Fax: +1-613-941 4775; Tel: +1-613-957 0981 Email: Peter_Scott@hc-sc.gc.ca

CANADA

RA/SAF/10491 Dr. Gordon Shephard

Medical Research Council, Programme on Mycotoxins and Experimental Carcinogenesis (PROMEC) P.O. Box 19070, Tygerberg 7505, REPUBLIC OF SOUTH AFRICA Fax: +27-21-938-0260; Tel: +27-21-938-0279 E-mail: gordon.shephard@mrc.ac.za

SOUTH AFRICA

RC/10480 Dr. Ines Sola

National Institute of Industrial Technology, Food Technology and Research Center, Avenida General Paz o/Constituyentes y Albarellos, CC157-1650 San Martin, Pcia de Buenos Aires, ARGENTINA, Tel: and Fax: +54-11 4753-5743, E-mail: ines@inti.gov.ar

ARGENTINA

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RA/10489 Dr. Angelo Visconti / Dr. Michelangelo Pascale Consiglio Nazionale delle Ricerche (CNR), Instituto Tossine e Micotossine da Parassiti Vegetali, Viale Luigi Einaudi 51, I-70125 Bari, ITALY Fax: +39-80-548 6063; Tel. +39-80-548 6073 E-mail: visconti@area.ba.cnr.it or itmpmp21@area.ba.cnr.it

ITALY

IAEA Scientific Secretary Dr. Bruno Doko

International Atomic Energy Agency, Food & Environmental Protection Section, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Wagramerstrasse 5, P.O. Box 100, A-1400 Vienna, AUSTRIA, Tel: +43 1 2600 26058; Fax: +43 1 26007 E-mail: Bruno.Doko@iaea.org

AUSTRIA

Head Section Dr. Paisan Loaharanu

International Atomic Energy Agency, Food & Environmental Protection Section, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Wagramerstrasse 5, P.O. Box 100, A-1400 Vienna, AUSTRIA, Tel: +43 1 2600 21638; Fax: +43 1 26007 E-mail: P.Loaharanu@iaea.org

AUSTRIA

Section Secretary Mrs. Gertraude Strnadl International Atomic Energy Agency, Food & Environmental Protection Section, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Wagramerstrasse 5, P.O. Box 100, A-1400 Vienna, AUSTRIA, Tel: +43 1 2600 26061; Fax: +43 1 26007 E-mail: G.Strnadl@iaea.org

AUSTRIA

FAO Dr. Maya Pineiro

Nutrition Officer (Food Control), Food Quality and Standards Service Food and Nutrition Division, Food and Agriculture Organization of the United Nations (FAO), Viale delle Terme di Caracalla 00100 Rome ITALY, Fax No.: 0039 06 570 54593/53152 Tel. No.: 0039 06 57053308, Email: Maya.Pineiro@fao.org

ITALY

OBSERVERS EC Dr. Elke Anklam

European Commission (EC), DG Joint Research Centre, Institute for Health and Consumer Protection, Food Products Unit, TP 260, I-21020 Ispra, Italy Fax: 0039-0332-785930; Tel: 0039-0332-785390 E-mail: elke.anklam@jrc.it

ITALY

EC Dr. Jörg Stroka

European Commission, DG Joint Research Centre, Institute for Health and Consumer Protection, Food Products Unit, TP 260, I-21020 Ispra, Italy Fax: 0039-0332-785930; Tel: 0039-0332-785170 E-mail: jeorg.stroka@jrc.it

ITALY

CARLETON UNIVERSITY

Dr. Regina de la Campa Carleton University, Department of Chemistry Ottawa, Ontario, CANADA K1S 5B6 Fax: +1-613-520 2710 or 3749 E-mail: ReginaDeLaCampa@pigeon.carleton.ca

CANADA

UMSHS Mr. Hassan Yazdanpanah IRAN

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Pharmacology & Toxicology Department, School of Pharmacy Shaheed Beheshti University of Medical Sciences & Health Services (UMSHS), P.O. Box 14155-6153, Tehran, I.R. IRAN Fax: +98-21-879 5008; Tel: +98-21-877 4283 E-mail: hsyazdan@tco.ac.ir

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ANNEX II

Tentative Agenda Monday, 4 December 2000 (Day 1) 08:30 am Registration 09:30 am Opening address: Meeting Room: B0442 - Paisan Loaharanu (IAEA) - Maya Pineiro (FAO) 10:00 – 10:15 Appraisal of CRP activities Bruno Doko (IAEA, Scientific Secretary) 10:15 – 10:30 Coffee break, Administration/domestic arrangements Presentation of Research data and discussions 10:30 – 11:00 Evaluation and Methods of Analysis for Determining Mycotoxins Contamination Of Food and Feed: Simple and Rapid Method for Analysis of Aflatoxins (Dr. B. Ramesh, Indian Council of Medical Research, Hyderabad, India) 11:00 – 11:30 Evaluation and Methods of Analysis for Determining Mycotoxins Contamination Of Egyptian Food and Feed: Recovery tests for Aflatoxin M1 and Fumonisin B1 (Dr. H. Amra, National Research Center, Cairo, Egypt) 11:30 – 12:00 Evaluation of Methods of Analysis for Determining Aflatoxin M1 Contamination in Milk (Dr. I. Sola, National Institute of Industrial Technology, Buenos Aires, Argentina) 12:00 – 12:30 TLC method for the Determination of Fumonisin B1 in maize: Participation in the Collaborative study. Improvement of the Analytical Method for the Determination of Ochratoxin A in Wine (Dr. M. Pascale, CNR, Bari, Italy) 12:30 – 13:45 Lunch Break 13:45 – 14:15 Monitoring Mycotoxins in Grain and Food Production Systems for Risk Management: Collaborative Development of an ELISA Test Kit for Aflatoxin B1 (Dr. Lee, Nanju, University of Sydney, Sydney, Australia) 14:15 – 14:45 The Development of a McAb-based Enzyme Linked Immunosorbent Assay for Detecting Fumonisin B1 from Food and Feed in China (Dr. Liu Xiumei, Institute of Nutrition and Food Hygiene, Beijing, China) 14:45 – 15:15 Evaluation of Methods for Aflatoxin and Fumonisin Determination in Foods and Feeds in Indonesia (Dr. S. Bahri, Research Institute for Veterinary Science, Bogor, Indonesia)

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15:15-– 15:45 Evaluation and Development of Rapid Monitoring Methods for Aflatoxin Determination in Food and Fed (Dr. A. Bin Hamid, Food Technology Centre, MARDI, Kuala Lumpur, Malaysia) 15:45 – 16:15 Evaluation of analytical methods for determination of mycotoxin in food (Dr. M. O. Garcia, Instituto de Nutricion e Higiene de los Alimentos, La Habana, Cuba) 16:15—16:30 Coffee Break 16:30 – 17:00 Evaluation of the Practical Applicability of Methods of Analysis for Aflatoxin In Imported and Exported Philippine Foods (Dr. L. Padilla, National Food Authority, Taguig, Metro Manila, Philippines) 17:30 – 18:00 Liquid Chromatographic Determination of Ochratoxin A in Green Coffee Beans, Roasted Coffee Beans and Soluble Coffee (Dr. M. Sabino, Instituto Adolfo Lutz, Sao Paulo, Brazil) 18:00 – 18:30 Methodology for the Determination of Fumonisins in Foods (Dr. P. Scott, Health Canada, Food Research Division, Ottawa, Canada) Tuesday, 5 December 2000 (Day 2) Meeting Room: B0442 09:00 - 09:45 The IDF/IUPAC/IAEA Validation of the Aflatoxin M1 Immunoaffinity Column/TLC method (Dr. I. Sola National Institute of Industrial Technology, Buenos Aires, Argentina) 09:45 – 10:30 Evaluation of Methods and Column Capacity and Recovery Checks for Determination of total Aflatoxins, Aflatoxin M1 and Fumonisins (Dr. J. Cea, LATU, Uruguay) 10:30 – 11:15 Results of Validation Studies for Aflatoxin Determination in Various Matrices and Outline of a Method Based on TLC to be Validated. (Dr. E. Anklam and J. Stroka, European Commission, Joint Research Centre for Health and Consumer Protection, Ispra -Italy) 11:15 – 11:30 Coffee breaks 11:30 – 12:15 Production of 14C Fumonisins in Corn Kernels for Studies of Recovery From Food Products Made From Milled Corn (J.D. Miller, Carleton University, Dept. of Chemistry, Ottawa, Canada) 12:15 – 13:45 Lunch Break 13:45 – 14:15 Rapid Response to Mycotoxicoses of Unknown Etiology, Validation of Appropriate Technology Mycotoxin Methods for International Trade; International Workshop for Mycotoxins – 2002 (Dr. D. L. Park, Food and Drug Administration, Washington DC, USA) 16:00 – 16:15 Coffee break

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16:15 – 17:30 Collaborative Study of and Analytical Method for the Determination of Fumonisin In Maize and/or Patulin in Apple Juice Concentrate (Dr. G. Shephard,Medical Research Council, PROMEC, Ty gerberg, South Africa) 17:30 - 18:00 Inter-laboratory Validation of Analytical Method for Determining Aflatoxin in Food and Feeds by Phenyl-Bonded Solid Phase Extraction/HPLC (Dr. J. Nagler, NRI, Food Security Department, Kent, UK) 18:00 – 18:30 Food Analysis Performance Assessment Scheme: Proficiency Tests For Participants of The CRP on Evaluation of Analytical Methods for Mycotoxin Contamination of Food and Feed (Dr. J. Gilbert, CSL, UK) Wednesday, 6 December 2000 (Day 3) Meeting Rooms: B0442 & A0431 09:00 – 12:15 Working group Activities 12:15 – 13:45 Lunch Break 13:45 – 17:30 Visit of Seibersdorf IAEA Laboratories (the bus will leave VIC at 14:00) 18 – 20:00 Welcome Receptions (The bus will depart from Seibersdorf, after the visit of the Laboratories) Thursday, 7 December 2000 (Day 4) Meeting Room: B0442 09:00 – 10:30 Work plan outlines and Timeframes - for each contract/agreement holder - for each workgroup 10:30 - 12:15 Forthcoming events - Future research topics - Next RCM (proposed/selected place and dates) 12:15 – 13:45 Lunch Break 13:45 – 18:00 Conclusion and Recommendations Friday, 8 December 2000 (Day 5) Meeting Room: B0442 09:00 – 12:00 Conclusion and Recommendations (continue) 12:30 Closure of the RCM