Good Hygiene Practices along the coffee chain
Module 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 2
Module 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 3
Module 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 4
Module 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 5
Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
Technical issues in
OTA analysis programmes
 Main aspects of OTA analysis programmes
include
 Sampling plans
 Validated methods of analysis
 Quality assurance system
Slide 6
Module 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 7
Module 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 8
Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
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
Slide 9
Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
Requirements for
Official Methods of Analysis
 Validated
 Accurate
 Reproducible results (within and between
laboratories)
Within established minimum performance
criteria
Slide 10
Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
Key characteristics of
Official Methods of Analysis
1. Accuracy
2. Applicability
3. Detection/determination limits
4. Linearity
5. Precision
6. Recovery
7. Selectivity
8. Sensitivity
Slide 11
Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
Who decides on
Official Methods of Analysis?
Validated methods may be adopted as
official methods by
 Codex Alimentarius
 AOAC International
 National and International Standards
Slide 12
Module 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 13
Module 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 14
Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
Flow diagram of OTA analysis
Lot
SAMPLING
Test sample
Sample Preparation
Analytical
Quality
Assurance
(AQA)
Diagram of general steps
involved in sampling,
sample preparation and
analysis of mycotoxins in
agricultural commodities
GRINDING AND
HOMOGENISATION
Subsampling
Extraction
Analysis for OTA determination
Mycotoxin test result
Slide 15
Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
Clean-up
Separation &
Quantification
Evaluating OTA analysis methods
 There are important functional criteria
 Accuracy and reproducibility
 There are significant practical considerations




Slide 16
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
OTA analysis steps – sample preparation
 It is a critical step in the analytical procedure
Grinding
 Time consuming
Homogenization
Slurry preparation
Slide 17
Module 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 18
Module 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 19
Module 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 20
Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
OTA analysis steps – clean-up
Immunoaffinity column clean-up of
green coffee
Slide 21
Module 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 22
Module 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 23
Module 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 24
Module 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.
Slide 25
Sample automatic processor
Module 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 26
Module 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 27
Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
Thin Layer Chromatography in OTA analysis
TLC main disadvantages



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
Slide 28
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
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 29
Module 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 30
Module 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 31
Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
Screening methods for OTA analysis
Test kits for the detection of coffee
An alyte
Company
Kit Name
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
Romer Labs
AgraQuant
OTAssay (2-40ppb)
Barley, corn, green coffee,
maize, soybeans and
wheat
Romer Labs
AgraQuantR
Ochratoxin (2 -40
ppb) ELISA Test Kit
Corn, maize, barley, green
coffee, wheat
OTA and B
OTA and B
Slide 32
Recognition
Primary Matrices
Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
ELISA in OTA analysis
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
Slide 33
Module 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 34
Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes
Spectrofluorimetric methods
of OTA analysis
This involves
 The use of immunoaffinity columns coupled
with fluorometer
Main disadvantages
 Increased false positive/false negative rates
Slide 35
Module 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
Slide 36
Module 6.2 – OTA Analysis:
An Important Aspect of OTA Prevention Programmes