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

2. Why do OTA analysis 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
Module 6.2 – OTA Analysis: An Important Aspect of OTA Prevention Programmes

3. Planning for OTA analysis
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
Module 6.2 – OTA Analysis: An Important Aspect of OTA Prevention Programmes

4. Investing in OTA analysis
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
Module 6.2 – OTA Analysis: An Important Aspect of OTA Prevention Programmes

5. Are the costs acceptable?
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
Module 6.2 – OTA Analysis: An Important Aspect of OTA Prevention Programmes

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

9. Key issues in sampling for official OTA analysis
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
Module 6.2 – OTA Analysis: An Important Aspect of OTA Prevention Programmes

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

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

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

13. Selecting methods when Official Methods are not available or applicable
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
Module 6.2 – OTA Analysis: An Important Aspect of OTA Prevention Programmes

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

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

16. Evaluating OTA analysis methods
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
Module 6.2 – OTA Analysis: An Important Aspect of OTA Prevention Programmes

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

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

19. OTA analysis steps – extraction
Recovery of the true concentration of mycotoxin is a critical point
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. OTA analysis steps – clean-up
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
Module 6.2 – OTA Analysis: An Important Aspect of OTA Prevention Programmes

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

22. OTA analysis steps – separation, detection and quantification
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)
Module 6.2 – OTA Analysis: An Important Aspect of OTA Prevention Programmes

23. OTA analysis steps – separation, detection and quantification
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)
Module 6.2 – OTA Analysis: An Important Aspect of OTA Prevention Programmes

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

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

28. Thin Layer Chromatography in OTA analysis
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
Module 6.2 – OTA Analysis: An Important Aspect of OTA Prevention Programmes

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

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

31. Screening methods for OTA analysis
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
Module 6.2 – OTA Analysis: An Important Aspect of OTA Prevention Programmes

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

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

34. ELISA disadvantages ELISA in OTA analysis 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
Module 6.2 – OTA Analysis: An Important Aspect of OTA Prevention Programmes

35. Spectrofluorimetric methods of 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. Quality Assurance of OTA analysis
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
Module 6.2 – OTA Analysis: An Important Aspect of OTA Prevention Programmes