Mycotoxin Analysis

A variety of testing solutions exist for mycotoxin analysis in food and feed. These solutions range from rapid tests that are easy to conduct, to reference methodology tests that are more time consuming but yield more detailed results. Results obtained can be qualitative, semi-quantitative, or quantitative.

Table 1. Rapid Testing - Reference Testing
Rapid TestingReference Testing
Lateral Flow TestThin Layer Chromatography (TLC)
Enzyme-Linked Immunosorbent Assay (ELISA)Gas Chromatography
FluorometryHigh Performance Liquid Chromatography (HPLC)
Liquid Chromatography – Mass Spectrometry (LC/MS)
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supported by Romer Labs

The general procedure for testing consists of five main steps:

Sampling – sophisticated due to heterogenic distribution of mycotoxins in samples


Grinding
– mills specifically designed for grinding of samples


Extraction
– different extraction buffers depending on mycotoxin and analysis method


Purification
– columns are packed with mixtures of adsorbents, interferences stick to packing material, mycotoxins go through unaffected (in case of immunoaffinity-columns, the columns contain antibodies that bind the toxin and interferences pass through)


Analysis

Lateral Flow Test

  • one-step lateral flow immunochromatographic assay
  • based on a competitive immunoassay format
  • typically the test strip is composed of a sample pad, a conjugate/gold pad, a membrane, an adsorbent pad, and an adhesive backing

Procedure:

  • antibody-particle complex (conjugate) is mixed with sample extract in a microwell
  • strip placed into the well
  • mixed content wicked to a membrane containing a test zone and a control zone
  • test zone captures free conjugate visible line
  • intensity of the line inversely proportional to the concentration of mycotoxin in the sample
  • control line is always visible, regardless of mycotoxin in the sample
Table 2. Lateral Flow Test Pros - Cons
ProsCons
rapid analysis (3-5 minutes)matrix interferences


no specialist equipment necessary
quantitative results can be obtained using a Lateral Flow Device (LFD)

Enzyme-Linked Immunosorbent Assay (ELISA)

  • one of the most popular immunologically based methods used in test kits for the analysis of mycotoxins in foods and feeds
  • direct competitive ELISA most commonly used

Procedure:

  1. known amount of labeled toxin competes with any possible toxin in the sample for the specific antibodies attached to the reaction vessel
  2. unbound toxin is washed from the vessel, enzyme labeled toxin remains in the vessel
  3. reaction with the substrate for the enzyme
  4. addition of stop solution, resulting colored product is measured optically 
  • qualitative results when only visually assessed
  • quantitative results in 10-20 minutes when using a reader
  • ideally for checking commodities for compliance with EU regulatory limits
Table 3. ELISA Pros - Cons
ProsCons
simple sample extraction matrix interferences
no clean up steps requiredonly for validated matrices (mainly raw commodities)
highly sensitivecross-reaction of antibodies

Fluorometry

  • measures fluorescence using a fluorometer
  • quantitative results
  • optimally used when single samples have to be tested within a short period of time, e.g. testing of incoming truck loads during harvest season
Table 4. Fluorometry Pros - Cons
ProsCons
rapid – less than five minutes
only for total aflatoxin

very robust
can be used by untrained personnel
no laboratory required
Fluorometer

Thin Layer Chromatography (TLC)

  • plate coated with adsorbent matrix such as silica gel (stationary phase)
  • solvent system containing sample (mobile phase) migrates through the matrix towards the top of the plate
  • compounds are separated based on the interactions between mobile phase and stationary phase
  • spots visible under ultraviolet light
  • standard samples for comparison are spotted in the same manner in different concentrations
  • estimation of sample concentration via interpolation
Table 5. TLC Pros - Cons
ProsCons
simple, cheap, rapidseparation may not be satisfactory
a number of samples can be run  simultaneously poor precision
insensitive for some toxins

Gas Chromatography

Gas Chromatography

Specific compounds of an injected sample are separated by a gas (mobile phase) flowing through a heated glass column coated with a stationary non-volatile liquid (stationary phase). Separated analytes coming off the column are detected by a chemical or physical detection system. 

Table 6. Gas Chromatography Pros - Cons
ProsCons
sensitive derivatization is time-consuming and prone to errors
low variabiltyused less frequently

High Performance Liquid Chromatography (HPLC)

  • small portion of a sample injected into a stream of solvent (mobile phase)
  • pumped through a column of adsorptive matrix (stationary phase)
  • components are separated based on interactions between stationary and mobile phase • components are then eluted from the matrix
  • a detector gives results that are compared to selected concentrations of standards
  • variety of detectors: spectrophotometric detectors (UV-VIS, diode array), refractometers (RI), fluorescence detectors (FLD), electrochemical detectors, radioactivity detectors and mass spectrometers (MS)
  • most commonly utilized with HPLC-UV or –FLD and -MS/MS systems.
Table 7. HPLC Pros - Cons
ProsCons
sensitive
only small amounts of sample neededtime consuming
applicable in complex matricescompounds must have UV absorption or fluorescence properties or require derivatization
minimum variability
highly accurate

Liquid Chromatography – Mass Spectrometry (LC/MS)

  • liquid chromatography coupled to mass spectrometry (LC/MS) or tandem mass spectrometry (LC/MS/MS)
  • reference method of choice in laboratories
  • mass spectrometry as detector for liquid chromatography (e.g. HPLC)
  • multi-toxin analysis: can detect all regulated mycotoxins simultaneously
  • powerful tool for the analysis of highly complex mixtures
Table 8. LC/MS Pros - Cons
ProsCons
low detection limitsexpensive
quantitative resultshighly trained personnel needed
ability to generate structural informationtime consuming, compared to rapid tests
minimal sample treatment
possibility to cover a wide range of analytes differing in their polarities
applicable in complex matrices
RomerLabs
supported by Romer Labs