ota analysis: an important aspect of ota prevention programmes
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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 - PowerPoint PPT PresentationTRANSCRIPT
Good Hygiene Practices along the coffee chain
OTA Analysis:An Important Aspect of OTA
Prevention Programmes
Module 6.2
Slide 2Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
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
Slide 3Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
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
Slide 4Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
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
Slide 5Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
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
Slide 6Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
Technical issues inOTA analysis programmes
Main aspects of OTA analysis programmes include Sampling plans Validated methods of analysis Quality assurance system
Slide 7Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
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)
Slide 8Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
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
Slide 9Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
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
Slide 10Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
Requirements forOfficial Methods of Analysis
Validated Accurate Reproducible results (within and
between laboratories)
Within established minimum performance criteria
Slide 11Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
Key characteristics ofOfficial Methods of Analysis
1. Accuracy
2. Applicability
3. Detection/determination limits
4. Linearity
5. Precision
6. Recovery
7. Selectivity
8. Sensitivity
Slide 12Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
Who decides onOfficial Methods of Analysis?
Validated methods may be adopted as official methods by
Codex Alimentarius AOAC International National and International Standards
Slide 13Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
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
Slide 14Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
Official Methods
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
Slide 15Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
Flow diagram of OTA analysis
Diagram of general steps involved in sampling, sample preparation and
analysis of mycotoxins in agricultural commoditiesSample Preparation
SAMPLING
Test sample
Lot
Extraction
Separation & Quantification
Clean-upAnalysis for OTA determination
GRINDING AND HOMOGENISATIO
N
Subsampling
Mycotoxin test result
Analytical
Quality
Assurance
(AQA)
Slide 16Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
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
Slide 17Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
OTA analysis steps – sample preparation
It is a critical step in the analytical procedure
Time consuming
Grinding
Homogenization
Slurry preparation
Slide 18Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
OTA analysis steps – extraction
Specific solvent depends on matrix and/or purification/quantification steps
Slide 19Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
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
Slide 20Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
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
Slide 21Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
OTA analysis steps – clean-up
Immunoaffinity column clean-up of green coffee
Slide 22Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
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)
Slide 23Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
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)
Slide 24Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
Liquid Chromatography in OTA analysis
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
Slide 25Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
Liquid Chromatography in OTA analysis
Detection and quantification
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
Slide 26Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
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
Slide 27Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
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.
Slide 28Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
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
Slide 29Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
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
Slide 30Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
Screening methods for OTA analysis
Main advantages
Rapid test methods Simple to use Analysis can be undertaken in the field
Slide 31Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
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
Slide 32Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
Screening methods for OTA analysis
Test kits for the detection of coffeeAnalyte Company Kit Name Recognition Primary Matrices
Ochratoxin r-Biopharm GmbH
RIDASCREEN OTA Column - Coffee beans
Ochratoxin VICAM OchraTest - Coffee beans
OTA TECNA S.r.l. Immunoscreen OCHRA -
Cereals, feeds, wine, green coffee and cocoa
Ochratoxin Tepnel BioSytems Ltd.
BioKits OTA Assay - Cereal, dried fruits, green coffee, and white wine
OTA and B Romer Labs AgraQuant OTAssay (2-40ppb) -
Barley, corn, green coffee, maize, soybeans and wheat
OTA and B Romer Labs AgraQuantR Ochratoxin (2-40 ppb) ELISA Test Kit
- Corn, maize, barley, green coffee, wheat
Slide 33Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
ELISA in OTA analysis
ELISA advantages Simplicity Number of samples that can be analysed
at the same time
Popular and useful screening tool thanks to the availability of polyclonal and monoclonal
antibodies against OTA
Slide 34Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
ELISA in OTA analysis
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
Slide 35Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
Spectrofluorimetric methodsof OTA analysis
This involves The use of immunoaffinity columns
coupled with fluorometer
Main disadvantages Increased false positive/false negative
rates
Slide 36Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
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