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 Testing	Reference Testing
Lateral Flow Test	Thin Layer Chromatography (TLC)
Enzyme-Linked Immunosorbent Assay (ELISA)	Gas Chromatography
Fluorometry	High Performance Liquid Chromatography (HPLC)
	Liquid Chromatography – Mass Spectrometry (LC/MS)

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
Pros	Cons
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:

known amount of labeled toxin competes with any possible toxin in the sample for the specific antibodies attached to the reaction vessel
unbound toxin is washed from the vessel, enzyme labeled toxin remains in the vessel
reaction with the substrate for the enzyme
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
Pros	Cons
simple sample extraction	matrix interferences
no clean up steps required	only for validated matrices (mainly raw commodities)
highly sensitive	cross-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
Pros	Cons
rapid – less than five minutes	only for total aflatoxin
very robust
can be used by untrained personnel
no laboratory required

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
Pros	Cons
simple, cheap, rapid	separation may not be satisfactory
a number of samples can be run simultaneously	poor precision
	insensitive for some toxins

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
Pros	Cons
sensitive	derivatization is time-consuming and prone to errors
low variabilty	used 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
Pros	Cons
sensitive
only small amounts of sample needed	time consuming
applicable in complex matrices	compounds 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
Pros	Cons
low detection limits	expensive
quantitative results	highly trained personnel needed
ability to generate structural information	time consuming, compared to rapid tests
minimal sample treatment
possibility to cover a wide range of analytes differing in their polarities
applicable in complex matrices