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OTA Analysis: An Important Aspect of OTA Prevention Programmes

Module 6.2. OTA Analysis: An Important Aspect of OTA Prevention Programmes. To support OTA monitoring programmes Generate data on levels and distribution of contamination Allow assessment of impact of prevention measures and optimisation of programmes

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OTA Analysis: An Important Aspect of OTA Prevention Programmes

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  1. Module 6.2 OTA Analysis:An Important Aspect of OTA Prevention Programmes

  2. To support OTA monitoring programmes Generate data on levels and distribution of contamination Allow assessment of impact of prevention measures and optimisation of programmes To certify compliance to official or buyer requirements on OTA levels To support research aimed at improving understanding and testing solutions Why do OTA analysis Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  3. To realise the benefits of OTA analysis, analytical services must have The expertise and the equipment to carry out required analyses at the levels of sensitivity and quantification demanded The capacity to perform the volume of analyses required in the required time frame Planning for OTA analysis Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  4. Suitable buildings To minimise occupational hazards; to ensure environment for good operation and maintenance of equipment Required equipment and materials To carry out screening and official methods of analysis as required Adequate and qualified staff Analysts at graduate and post graduate levels; technicians to maintain and repair equipment; support staff; regular on-the-job training Investing in OTA analysis Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  5. Promotes confidence in national coffee sector Demonstrably sound official programmes of OTA monitoring and analysis can promote confidence of importers and the country’s position in international trade Safeguards domestic consumers Protects against risk of poor quality coffees being diverted to local consumers Cost recovery from industry At least partial recovery of costs is possible Are the costs acceptable? Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  6. Main aspects of OTA analysis programmes include Sampling plans Validated methods of analysis Quality assurance system Technical issues inOTA analysis programmes Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  7. Sampling plans for OTA in coffee • Heterogeneous OTA distribution in coffee lots means that attention to sampling is necessary to obtain representative samples • Sampling plans must be designed in relation to the purpose of the sampling • To have a representative unit to estimate the ‘whole’ • To characterise a specified segment of the population • Design of official sampling plans aims to provide international methods to avoid difficulties arising from legal and technical disputes related to sampling in trade (Codex Alimentarius) Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  8. Sampling methods for OTA monitoring and analysis programmes should specify Procedures for taking and handling samples The size and number of increments for a representative sample Statistical criteria for acceptance and rejection of a lot on the basis of the sample Establishing sampling methods Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  9. Sample size and number of samples means Percentage of lots accepted at concentrations below the regulatory limit (good lots) Percentage of lots rejected at concentrations above the regulatory limit (bad lots) Uncertainty associated with the OTA test procedure Buyers’ risks Sellers’ risks Key issues in sampling forofficial OTA analysis Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  10. Validated Accurate Reproducible results (within and between laboratories) Within established minimum performance criteria Requirements forOfficial Methods of Analysis Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  11. Accuracy Applicability Detection/determination limits Linearity Precision Recovery Selectivity Sensitivity Key characteristics ofOfficial Methods of Analysis Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  12. Validated methods may be adopted as official methods by Codex Alimentarius AOAC International National and International Standards Who decides onOfficial Methods of Analysis? Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  13. In house validation – according to harmonized IUPAC Guidelines for Single Laboratory Validation of Methods of Analysis Method under ISO/IEC 17025:1999 Standard or Principles of Good Laboratory Practice Selecting methods when Official Methods are not available or applicable Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  14. Methods internationally and collaboratively validated using the International Harmonised Protocols Roasted coffee - Entwisle et al. 2001; AOAC 2000.09; CEN EN 14132:2003  Green coffee - Vargas et al. 2005; AOAC 2004.10 No official screening method for ochratoxin A in coffee has been reported on the AOAC website Official Methods Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  15. Lot SAMPLING Test sample GRINDING AND HOMOGENISATION Sample Preparation Analytical Quality Assurance (AQA) Subsampling Extraction Analysis for OTA determination Clean-up Separation & Quantification Mycotoxin test result Flow diagram of OTA analysis Diagram of general steps involved in sampling, sample preparation and analysis of mycotoxins in agricultural commodities Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  16. There are important functional criteria Accuracy and reproducibility There are significant practical considerations Speed of analysis Qualitative or quantitative Skill required for assay Initial and recurring costs Evaluating OTA analysis methods Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  17. It is a critical step in the analytical procedure Time consuming Grinding Homogenization Slurry preparation OTA analysis steps – sample preparation Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  18. Specific solvent depends on matrix and/or purification/quantification steps OTA analysis steps – extraction Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  19. Recovery of the true concentration of mycotoxin is a critical point OTA analysis steps – extraction Organic solvent in the presence of acid or an extraction solvent containing aqueous sodium bicarbonate Shaker or high speed blender Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  20. Essentially involves Separation of the mycotoxin from co-extracted substances Initial concentration step Clean up methods Conventional procedures are time consuming and laborious Immunoaffinity solid-phase extraction (SPE) sorbents An important development Provide highly selective methods OTA analysis steps – clean-up Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  21. Immunoaffinity column clean-up of green coffee OTA analysis steps – clean-up Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  22. Available methods include Liquid chromatography - fluorescence detection (LC/FLD) Thin-layer chromatography (TLC) Enzyme-linked immunosorbent assay (ELISA) Fluorometry Flow lateral devices (FLD) Fluorescence polarization immunoassay (FPIA) OTA analysis steps – separation, detection and quantification Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  23. New analytical approaches Rapid, low cost screening methods Based on biosensors Dip-stick-like kits Liquid chromatography coupled with Electro-spray ionisation mass spectrometry (MS) Tandem or sequential mass spectrometry (MS/MS) OTA analysis steps – separation, detection and quantification Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  24. Main advantages Possibility of automation Separation power Selectivity Lower limits of detection (0.12-0.2 g/kg) Main disadvantages Expensive Requires skilled analysts and technicians Liquid Chromatography in OTA analysis Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  25. Chromatogram of OTA standard solution at concentration 0.0218 µg/mL. Reverse Phase 250x4.6 mm – 0.5µm. Fluorescence detector. Mobile Phase: acetonitrile: methanol: water: acetic acid (35+35+29+1). flow rate: 0.8mL/min. Sample automatic processor Liquid Chromatography in OTA analysis Detection and quantification Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  26. Thin Layer Chromatography in OTA analysis Simple, fast, cost effective, robust technique Main advantages Relatively inexpensive Developing countries have considerable expertise and experience Used in routine applications Immunoaffinity column • Sample extracts free of major matrix interferences Suitable for one dimensional TLC analysis Improving: sensitivity, separation, resolution and visualisation Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  27. Confirmation of OTA by TLC Spraying the TLC plates with aluminium chloride and sodium bicarbonate Exposing the plates to ammonia vapour RP TLC has been reported as a confirmation method for OTA in coffee. Thin Layer Chromatography in OTA analysis Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  28. TLC main disadvantages High cost of commercial fluorodensitometers Quantification is still a limiting factor Ability analysts to visually quantify the toxin Lack of resolution Poor sensitivity Thin Layer Chromatography in OTA analysis Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  29. OTA standard and naturally contaminated samples dissolvedwith toluene-acetic acid (99:1, v/v) and spotted on normal TLC plate Thin Layer Chromatography in OTA analysis Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  30. Main advantages Rapid test methods Simple to use Analysis can be undertaken in the field Screening methods for OTA analysis Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  31. Limitations Qualitative answer: yes/no contamination False negative results Need of confirmation of positive results Disadvantages High recurring cost Lack of attention with regard to sample extraction condition Tendency to disregard sample requirements Screening methods for OTA analysis Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  32. An alyte Company Kit Name Recognition Primary Matrices RIDASCREEN OTA r - Biopharm Coffee beans Ochratoxin - Column GmbH OchraTest Coffee beans Ochratoxin VICAM - Immunoscreen green Cereals, feeds , wine, OTA TECNA S.r.l. - OCHRA coffee and cocoa green Cereal, dried fruits, Tepnel BioKits OTA Assay Ochratoxin - coffee , and white wine BioSytems Ltd. green coffee, Barley, corn, AgraQuant OTA and B Romer Labs - maize, soybeans and OTAssay (2 - 40ppb) wheat AgraQuantR green Corn, maize, barley, Ochratoxin (2 - 40 OTA and B Romer Labs - coffee , wheat ppb) ELISA Test Kit Screening methods for OTA analysis Test kits for the detection of coffee Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  33. ELISA advantages Simplicity Number of samples that can be analysed at the same time ELISA in OTA analysis Popular and useful screening tool thanks to the availability of polyclonal and monoclonal antibodies against OTA Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  34. ELISA disadvantages Less accurate and sensitive Very few correlations were found (traditional) False positive or negative results Should not be used as a quantitative method Should only be used with foods for which they have been extensively tested and demonstrated to work Sufficient controls must be employed for each test ELISA in OTA analysis Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  35. Spectrofluorimetric methodsof OTA analysis This involves • The use of immunoaffinity columns coupled with fluorometer Main disadvantages • Increased false positive/false negative rates Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

  36. Analytical Quality Assurance (AQA) programmes are essential to ensure reliable results Where international acceptability of results in required, accreditation is important Detailed guidance on AQA for OTA analysis is provided in Support documentation section of Module 5.2 of this CD-Rom Quality Assurance of OTA analysis Module 6.2 – OTA Analysis:An Important Aspect of OTA Prevention Programmes

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