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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 methods that are more time-consuming but yield more detailed results. The main rapid and reference methods available are listed in Table 1:

Table 1. Different rapid and reference analytical methods

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)

The general procedure for testing consists of five main steps: sampling, grinding, extraction, purification and analysis (Figure 1). Sampling is the most critical step and needs to be accomplished according to specific guidelines due to the inhomogeneous distribution of mycotoxins in the lot. After the sample is collected, it needs to be ground using a mill specifically designed for this purpose. The next step is extraction where the molecules of interest are separated from the others. This step is accomplished using different extraction buffers in which only the substance of interest (e.g. a specific mycotoxin) is soluble. After the mycotoxin is extracted, it needs to be purified. The purification step physically separates the substance of interest from the solution. Purification methods consist of columns packed with mixtures of adsorbents. The mycotoxin goes through the column and reacts with specific substances – bond to the column - specifically designed to bind it. After the mycotoxin has been immobilized it can be analyzed.

Figure 1. Different steps prior to mycotoxin analysis.
Figure 1. Different steps prior to mycotoxin analysis.

Mycotoxin Analytical Methods

Lateral Flow Test

Lateral flow tests consist of relatively simple technology based on a series of capillary beds, such as pieces of porous paper. The first element (filter pad) acts as a sponge and sucks in the sample solution. The fluid migrates to the second element (the gold pad) where the manufacturer has installed the bio-active particles (conjugate): a special dry matrix designed to guarantee a chemical reaction between the target molecule (e.g. mycotoxin) and its chemical partner (antibody) immobilized on the gold pad surface. As the sample solution diffuses up the stripe, it comes into contact and reacts with the matrix containing the antibody on the gold pad. The target molecule (e.g. mycotoxin) binds to the antibody while migrating further through the membrane, towards the adsorbent pad. The membrane has one or more areas (referred to as strips) where a third ‘capture’ molecule has been installed by the manufacturer. By the time the sample-conjugate mix reaches these strips, the third ‘capture’ molecule binds the complex. As more and more fluid passes over the strips, particles accumulate and the strips change color. Typically, there are at least two strips. The control strip captures any particle, thereby showing that the reaction conditions and technology are working. The second strip contains a specific capture molecule designed to capture only the sample-conjugate complex (Figure 2). The advantages and disadvantages of lateral flow tests are hereby highlighted.

AdvantagesDisadvantages
+ Rapid analysis (results available in 3 – 5 minutes)- Matrix interferences
(other substances in the solution that can alter results)
+ No special equipment necessary
+ Quantitative results can be obtained using a Lateral Flow Device (LFD) reader
Figure 2. Illustration of a conventional lateral flow test.
Figure 2. Illustration of a conventional lateral flow test.

Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA is one of the most popular immunological- based methods used for the analysis of mycotoxins in foods and feeds. The reaction is carried out in 96-well microtiter plates (Figure 3).

The compound of interest (e.g. aflatoxin B1) present in the sample delivered to the laboratory, reacts with specific antibodies attached to the surface of the reaction plate wells. These antibodies are designed to bind aflatoxin B1. In the reaction, the aflatoxin B1 contained in the sample solution competes for the antibodies with a known amount of the same mycotoxin (aflatoxin B1) that is purposely added to the reaction well. This known aflatoxin B1 is labelled by the manufacturer with a molecule that produces a detectable signal, usually a color change, when properly excited by a specific liquid solution. The sample solution and the labelled mycotoxin are added to the reaction wells and allowed to compete for the antibodies for a certain period of time (usually a few minutes). Afterwards, the wells are washed to eliminate nonbound mycotoxins, and a special liquid solution that excites the molecular label is added to produce the color. As a result, the more aflatoxin B1 is present in the sample, the lighter the color will be, as only a small amount of labelled aflatoxin B1 will bind to the antibody. Vice versa, if the sample does not contain aflatoxin B1, the color will be darker, as more labelled aflatoxin B1 will bind to the antibody. The procedure is illustrated in Figure 4. The advantages and disadvantages of this method are listed below.

AdvantagesDisadvantages
+ Simple sample extraction- Matrix interferences (other substances in the solution that can alter results)
+ Good sensitivity- Only suitable for validated matrices (mainly raw commodities)
- Antibodies can react with each other and alter the results (crossreactivity).
Figure 3. A typical ELISA assay is conducted in a 96 wells microtiter plate such as the one above. The sample reacts with the antibody mixture, placed at the bottom of the wells.
Figure 3. A typical ELISA assay
Figure 4. A typical ELISA reaction. The mycotoxin contained in the sample solution competes with a known amount of labelled mycotoxin added to the reaction (steps 1 and 2). After washing away the non-bound elements, a special liquid solution that excites the molecular label is added to produce the color (step 3).
Figure 4. A typical ELISA reaction. The mycotoxin contained in the sample solution competes with a known amount of labelled mycotoxin added to the reaction (steps 1 and 2). After washing away the non-bound elements, a special liquid solution that excites

Fluorometry

Figure 5. A sample is scattered with UV light and the emitted wavelength – specific to the sample - is measured
Figure 5. A sample is scattered with UV light and the emitted wavelength – specific to the sample - is measured

Fluorometry allows the identification of small substances by excitation with a beam of ultraviolet light, followed by detection and measurement of the characteristic wavelength of the fluorescent light emitted (Figure 5).

This method is of particular interest when testing single samples within a short period of time, e.g. testing of incoming truck loads during the harvest season. The advantages and disadvantages are listed below:

AdvantagesDisadvantages
+ Rapid – results available in less than five minutes- Only total aflatoxin measurement is possible
+ Can be used by untrained personnel
+ No laboratory required

Thin Layer Chromatography (TLC)

Thin-layer chromatography is a separation technique where the substance of interest (e.g. mycotoxin) is trapped by some binding material, known as the stationary phase, which is immobilized in a matrix (e.g. silica gel). The method is performed on a plate (e.g. glass or aluminum), coated with a thin layer of adsorbent material. The analysis sample consists of a solvent containing the analyte (the solvent + analyte complex is called the mobile phase). When the mobile phase is applied to the plate, the latter adsorbs it through capillary action. At this point, the substances contained in the mobile phase react with the stationary phase. As different substances ascend the TLC plate at different rates, separation is achieved (Figure 6). The advantages and disadvantages of this technique are listed below.

AdvantagesDisadvantages
+ Simple, cheap, fast- Separation may not be precise
+ Multiple samples can be run simultaneously- Does not work for all mycotoxins
Figure 6. The different steps in the TLC. Different compounds move at different rates in the stationary phase. The circles represent the different samples and the different colors in the circles represent different chemical compounds that compose the samples. The left picture represents the starting point and the right picture represents the end point, where the different compounds in the samples are separated according to their rates of ascension of the TLC plate.
Figure 6. The different steps in the TLC.

Gas Chromatography (GC)

This method requires specific equipment and trained technicians. It works as follows: a gas carries the compounds of interest contained in an injected sample (mobile phase). The gas carrying the sample flows through a heated glass column coated with a stationary non-volatile liquid (stationary phase) (Figure 7). Substances will separate according to their ability to cross the stationary phase (a process known as elution). Separated compounds coming off the column are detected by a chemical or physical detection system. The advantages and disadvantages are hereby listed.

AdvantagesDisadvantages
+ High sensitivity- Data analysis is time-consuming and prone to errors
+ High specificity (low interference)- Expensive
- Requires highly skilled technicians to carry out the analysis
Figure 7. A sample is carried by a gas into a heated glass column coated with a non-volatile liquid. Different substances will cross the column at different rates. The different substances will generate peaks that are read by the detector and shown on the computer.
Figure 7. A sample is carried by a gas into a heated glass column coated with a non-volatile liquid.

High Performance Liquid Chromatography (HPLC)

The method relies on pumps that circulate a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material (Figure 8). Different components in the sample, e.g. mycotoxins, interact with the adsorbent material in different ways (differences in affinity). Therefore crossing the column at different rates, allowing a separation as they flow out of the column. Afterwards, a detector gives results that are compared to selected standards. A variety of detectors are available such as spectrophotometric detectors (UV-VIS, diode array), refractometers (RI), fluorescence detectors (FLD), electrochemical detectors, radioactivity detectors and mass spectrometers (MS). The advantages and disadvantages of the method are hereby listed.

AdvantagesDisadvantages
+ High sensitivity- Time consuming
+ Only small amounts of sample are needed- Compounds must have UV absorption or fluorescence properties
+ Applicable to complex matrices- Expensive
+ Great reliability- Highly skilled technicians needed to carry out the analysis
+ Highly accurate
+ Fulfills legal requirements
Figure 8. Typical HPLC reaction where a carrier liquid carries the sample of interest into the column. Different components separate at different rates.
Figure 8. Typical HPLC reaction

Liquid Chromatography – Mass Spectrometry (LC/MS)

This technique combines the physical separation proprieties of the HPLC with the mass analysis capabilities of the mass spectrometer (MS). The two analytical methods work synergistically. Chromatography separates mixtures with multiple components (e.g. mycotoxins), before the mass spectrometer then provides the structural features of individual components with high sensitivity and specificity. The most common variations of the method are either liquid chromatography coupled to mass spectrometry (LC-MS) or tandem mass spectrometry (LC-MS/MS) (Figure 9). In LC-MS/MS, the mass to charge ratio of the ions belonging to individual mycotoxins are measured before then being fragmented. Each fragment is remeasured in the second mass spectrometry step for extra specificity. Due to the extreme sensitivity, this method is the reference method of choice in many laboratories and it currently represents state-of-the-art of analytical chemistry. The advantages and disadvantages are listed below:

AdvantagesDisadvantages
+ Low detection limits- Expensive
+ Qualitative and quantitative results- Highly trained personnel required to carry out the analysis
+ Generate structural information- Time consuming compared to rapid tests
+ Minimal sample treatment required
+ Can cover a wide range of analytes
+ Applicable to complex matrices
Figure 9. The LC/MS-MS combines the separation capabilities of the HPLC with the ability to detect the mass and other structural features of a specific sample. In the tandem mass spectrometry (MS/MS) the ions of the individual components are measured at first, then fragmented and each fragment is re-measured in the second mass spectrometry step, for extra molecular specificity and sensitivity.
Figure 9. The LC/MS-MS combines the separation capabilities of the HPLC with the ability to detect the mass and other structural features of a specific sample.



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