OTA Analysis: An Important Aspect of OTA Prevention Programmes

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



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



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



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



Technical issues inOTA analysis programmes

Main aspects of OTA analysis programmes include Sampling plans Validated methods of analysis Quality assurance system



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)



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



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



Requirements forOfficial Methods of Analysis

Validated Accurate Reproducible results (within and

between laboratories)

Within established minimum performance criteria



Key characteristics ofOfficial Methods of Analysis

1. Accuracy

2. Applicability

3. Detection/determination limits

4. Linearity

5. Precision

6. Recovery

7. Selectivity

8. Sensitivity



Who decides onOfficial Methods of Analysis?

Validated methods may be adopted as official methods by

Codex Alimentarius AOAC International National and International Standards



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



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



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)



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



OTA analysis steps – sample preparation

It is a critical step in the analytical procedure

Time consuming

Grinding

Homogenization

Slurry preparation



OTA analysis steps – extraction

Specific solvent depends on matrix and/or purification/quantification steps



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



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



OTA analysis steps – clean-up

Immunoaffinity column clean-up of green coffee



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)



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)



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



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



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




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.




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




OTA standard and naturally contaminated samples dissolved with toluene-acetic acid (99:1, v/v) and spotted on normal TLC plate



Screening methods for OTA analysis

Main advantages

Rapid test methods Simple to use Analysis can be undertaken in the field




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




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



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



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



Spectrofluorimetric methodsof OTA analysis

This involves The use of immunoaffinity columns

coupled with fluorometer

Main disadvantages Increased false positive/false negative

rates



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

Documents

OTA Analysis: An Important Aspect of OTA Prevention Programmes