Mycotoxin screening
Bart Huybrechts
The Veterinary and Agrochemical Research Centre (CODA-CERVA)
Operational Direction Chemical Safety of the Food Chain,
Operational Unit Toxins and Natural Components
National Reference Laboratory for Mycotoxins
Leuvensesteenweg 17, 3080 Tervuren, Belgium
Introduction
Problems regarding food safety are mainly associated with the possible presence of contaminants due to human
activities, for example the use of pesticides. However, so-called natural contaminants receive a lot less attention. A
good example of this are mycotoxins, toxic substances formed by fungi. Last spring, the presence of mycotoxins
received a lot of attention due to the feeding of Eastern European imported forage maize contaminated with aflatoxin to dairy cattle, one of the most well-known and most poisonous mycotoxins. Species of the fungus families
Aspergillus, Fusarium and Penicillium are the most important mycotoxin producers in food. A first requirement to
keep the presence in food to a minimum is the prevention of fungus growth at each moment of food production
and food processing. A critical moment is the storage of the harvest; the temperature and moisture have to be
monitored continuously. Mycotoxins are very stable compounds that are resistant to most processing procedures.
This entails that the mycotoxin may still be present, even though the producing fungus can no longer be detected. Moreover, mycotoxins can enter the food chain in numerous ways. In addition to a primary contamination, a
secondary contamination can also take place in which the above-mentioned aflatoxins can enter the human food
chain via milk.
Rapid tests versus confirmatory methods
Considering that contamination with these toxins can never be completely ruled out and that a systematic
decontamination is not an acceptable option due to the inevitable olfactory degradation, it is appropriate to
monitor food for their presence. However, developing analytical methods to detect toxins reliably and routinely in
often diverse matrices (ranging from grain to meat and honey) isn’t that obvious, all the more since the norm for
several of these toxins is quite low. Whether a newly developed method for analysis is fit for commercial routine
use is determined by three practical criteria: (a) reliability, i.e. accurate and precise, (b) the speed and consequently
also to a large degree the cost of the analysis, and (c) the possibility to conduct an “on-site” analysis.
Based on the above-mentioned criteria, techniques for mycotoxin analysis can be divided into (1) rapid screening
tests and (2) confirmatory methods. The first group of tests are mainly aimed at the qualitative detection of toxins
as fast as possible, preferably already in the field, i.e. a “yes” or “no” answer. The methods in the second group are
predominantly used to confirm a positive result by means of a rapid test and/or to provide a more accurate quantitative result (“how much of the toxin is actually in there?”). Both methods have in common that they require an
extraction step: the toxin needs to be extracted from the sample by means of a solvent (e.g. alcohol). They fundamentally differ in the way in which the detection takes place.
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Rapid tests
The majority of the rapid tests rely on antibodies (immunological assays) for the detection and differ from each
other mainly in the way the antibody is used. At present, three formats are used commercially: (1) ELISAs (enzymelinked immunosorbent assay), (2) dipstick and lateral flow tests and (3) FPIAs (fluorescence polarization immunoassays). Currently, the latter tests are barely used because they are rather complicated to manipulate and timeconsuming. In the ELISA tests, the toxin in the extraction solution has to bind onto a number of antibody binding
sites, immobilized on a microtitre plate, resulting in the fading out of the signal; in other words, the less the test
provides a signal, the more the toxin is present. A disadvantage of this test is that it is relatively labour-intensive
and difficult to put into practice. Dipstick and lateral flow tests are immunochromatographic tests in which the
presence of the toxin is measured by means of a disposable test strip. These test strips contain an antibody bound
to a coloured particle; when the extraction solution has been added, the toxin present binds onto the coloured
particle and simultaneously runs down the paper strip, which demonstrates the presence of the toxin by way of
a discolouration. The main advantage of this test compared to ELISA is that it can be used in the field, which explains its recent strong rise in popularity. Both immunotests have one Achilles’ heel, however: other components
of the sample can influence the binding of the antibodies and can lead to a false negative or a false positive result.
Figure 1: ELISA-type rapid tests with a 96 well microtiter plate.
Each position requires multiple pipette steps to obtain a result.
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Figure 2: Strip test : The sample solution
can be directly applied to this strip, after
which a discolouration indicates either a
positive or a negative result.
New trends
The recent evolution in detection methods, described in detail below, can be considered to be the answer to
two conflicting questions; on the one hand there has been a demand for increasingly rapid and simple methods
which are easier to use in the field, on the other hand there’s an evolution towards more reliable reference methods which can detect the largest possible number of toxins within a single analysis. Apart from this, there is also
a demand for extraction methods that require less harmful organic extraction solvents without a loss of quality of
analysis.
Rapid tests
Multi-toxin (multiplex) flow tests
The market share of rapid tests in the form of paper test strips has increased rapidly compared to that of ELISA
tests because they’re easier to use. An important disadvantage they had in common with ELISA tests, was that
they could only detect one mycotoxin per analysis. Recently, flow tests have been commercialized that can
measure multiple toxins in a single analysis. However, the disadvantage is that, for the time being, these tests are
less sensitive than the monotoxin test strips.
Molecularly Imprinted Polymers (MIPs) & Aptamers
In the past, rapid tests were based on antibodies of biological origin (e.g. rabbits, mice), but recently rapid tests
based on non-biological binding elements were commercialized. MIPs are polymers with an embedded molecular memory, which can bind onto a specific toxin in the same way as antibodies, using a lock-key mechanism. The
stability, the reusability, the reproducibility, the low production costs and the absence of possible ethical issues
concerning the use of test animals are important commercial assets.
Aptamers (DNA-based binding elements) are already being used as an alternative to antibodies on the level of
laboratories, but their commercial future is still up in the air at present.
Rapid spectroscopy methods
Spectroscopic techniques in which for example an infra-red laser is pointed at a sample and the specific absorption of a certain wavelength of light reveals the presence of a toxin, offers a very big asset: the extraction step can
be skipped. This offers numerous new possibilities. Kernels of maize, for instance, can be screened individually
while being transported on a conveyor belt and can be removed individually without having to destroy the entire
batch. However, this technique is rather sensitive to interferences, which is an inevitable consequence of the
absence of any form of sample preparation and the technology is relatively expensive. On the other hand, once
implemented, these analyses are conducted completely automatically.
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Biosensors
A biosensor is an appliance used for the detection of molecules that combines a biological component as a
sensitive element with a physiochemical read-out. A biosensor is composed of three parts: a sensitive biological
element, a transducer and a physiochemical detector. If the toxin is present, it binds onto the element and thus
causes a physiochemical change in the detector. Although, up to this point, this technology has produced promising results on the laboratory level, for now it does not seem to yield any decisive advantages compared to the
existing technologies and it is not commercially available.
Bead-based assays
In bead-based assays, little beads coded with a colour or magnetically marked are added to a sample solution.
In addition to their identification coding, the beads are also marked with an antibody against a certain toxin. The
changes the antibody-antigen complex causes in the beads can then be measured using a laser for instance. One
of the main advantages of this technology is that it is perfectly suited for multitoxin analyses. Commercial kits are
already expected to arrive in the course of this year.
High-resolution mass spectrometry
In the field of reference methods, the tendency towards mass spectrometry as a gold standard for detection is
becoming unmistakably apparent.
In high-resolution mass spectrometry (HR-MS), a very high mass resolution is obtained. To be able to uniformly
identify a component, the mass spectrometer has to determine the mass of the component as precisely as possible; i.e. a high mass accuracy and at the same time distinguish this mass as accurately as possible from other molecule masses, i.e. the highest possible resolution. In doing so, one has to bear in mind that the highest possible
resolution may be very useful, but isn’t an essential or not even a sufficient condition to have a satisfactory mass
accuracy.
Presently, 4 types of commercially available high-resolution mass spectrometers can be distinguished, categorized
according to their operational principle: (1) magnet sector mass spectrometers, (2) Time-of-Flight mass spectrometers (TOFMS), (3) Fourier transformation ion cyclotron resonance mass spectrometers (FT-ICR) and (4) Fourier
transformation Orbitrap.
The first two types of mass spectrometers are usually considered to be continuous (magnet sector) or semicontinuous (TOFMS) scanning mass spectrometers, the latter two are so-called trap design mass spectrometers.
In the magnet sector mass spectrometer an electrically charged molecule is accelerated to a very high speed
after which it has to pass through a magnetic field that is perpendicular to its flight direction; heavier molecules
will be deflected differently than a lighter molecule due to a higher mass inertia. Up into the nineties, magnet
sectors have been the workhorse in case high resolution was needed in a routine environment. Currently, they
have largely disappeared because of a series of disadvantages; the investment cost is very high and they can
only scan mass per mass and cannot instantaneously produce a full sample profile. Moreover, they cannot be
combined with liquid chromatography, but only with gas chromatography. Time-of-flight mass spectrometers
function according to the principle that if two molecules are given the same energy, the heavier mass will move
slower through the vacuum tube than a lighter molecule. As of the nineties, these mass spectrometers have more
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or less taken over the role of magnet sectors as a high resolution workhorse because of a number of advantages:
relatively low investment costs, very high scanning speed, robustness (very simple operational principle) and they
can be combined with basically any sample preparation and sample separation method. They, however, have
two major disadvantages; according to HR-MS standards, their resolution is very low and what’s more is that their
sensitivity decreases drastically if the user asks for more resolution. Although this problem has been worked on
in these past few years, for example the reflectron technology, TOFMS-technology is currently lagging behind on
trap designs when it comes to mass resolution.
So-called trap design mass spectrometers, like the ICR or the Orbitrap mass spectrometer offer very high resolutions, up to 100 times higher than the resolution of other types. In these fourier transformation mass spectrometers
the ions are caught in a metal cage (a trap), after which they are subjected to a magnetic field (ICR) or a direct
current electrical field (Orbitrap). The way they react to the exposure to this field is dictated by their mass and only
by their mass. Any energy the ions might receive during, for instance, their introduction in the detector, does not
influence their resolution. This results in their most important advantage: an extremely high resolution. Since they
store ions in a trap and thereby, as it were, concentrate them, they usually have an excellent sensitivity which does
not go down if a higher resolution is asked for. On the other hand, they will begin to scan more slowly if higher resolution is needed, a disadvantage the TOFMS does not have, which complicates their integration with the newest
ultra-fast chromatography techniques. Moreover, not only do they concentrate the desired analyte molecules in
their trap, but all molecules that come from the sample. This way the trap can be overcharged, which results in a
incorrect mass accuracy (matrix effect). This can be prevented by, for instance, allowing only 1% of the ions in the
trap, which of course reduces the sensitivity. Therefore, the following rule goes for mass spectrometry: sensitivity
comes at the cost of resolution in the case of continuous (quadrupole, sector) or semi-continuous (TOFMS) scan
mass spectrometers or resolution comes at the cost of scan speed for trap designs.
The most important distinction between the ICR and the orbitrap is the field the ions are subjected to. For an ICR,
this is an extremely powerful magnetic field. This results in the highest possible resolutions that are currently available in a commercial mass spectrometer. The magnets are, however, very expensive (a typical ICR costs more than
€ 2,000,000) and very fragile. In addition, the ICR is too slow to be combined with the new rapid chromatography
techniques. For routine analyses it is consequently irrelevant for the most part.
The orbitrap is the newest addition to the high resolution mass spectrometers and subjects the ions to a direct
current electric field instead of to a magnetic field. Since a direct current electric field is “purer” than a magnetic
field, it has a higher resolution than an ICR, albeit from a theoretical point of view; currently there are still technical
limitations to the production of the actual trap which impede higher resolutions. The typical relatively slow scanning speed of fourier transformation mass spectrometers also comes into play here. Fourier transformation is a
mathematical operation in which the time-related signal of the detectors is converted into a mass read-out. Their
scanning speed can be increased without lowering the resolution by making the trap physically smaller, but this
makes them more susceptible to overload effects in the trap, which leads to mass inaccuracy (and false-positive
or false-negative results). In any case, the current generation can be combined with chromatographic separation
techniques.
Orbitraps are expected to overtake ICRs in terms of resolution in a few years’ time, at only a fraction of the cost of
ICRs. Add to the equation a sensitivity that is currently better than that of TOFMs and almost as good as that of
Quadrupole mass spectrometers, and it shouldn’t be surprising that many consider this technology as the future
in HR-MS.
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High resolution mass spectrometry opens up one very interesting perspective: so-called generic screening or
non-targeted screening in which the detector continuously scans a large area of molecular masses during the
sample analysis. This way, a total contamination profile of the sample can be drawn up, which contains not only
mycotoxins, but also plant toxins, pesticides, fungicides, etc. Retro-active analysis is also one of the possibilities,
when a client, for instance, requests an additional analysis (as a response to new regulations for example). This
information can then be retrieved by means of software for samples that have already been analysed. Moreover,
this technology not only allows to have a look at the toxins, but also at their metabolites. Toxins, such as fungi
toxins are often converted by the plant into substances that are less harmful to them. When the latter substances
enter into the human digestive system, they can however be reconverted into their initial (probably more toxic)
form. However, these metabolites aren’t detected by means of classical analysis techniques (masked toxins). With
HR-MS, these can be screened for routinely. The detection limits of this approach are still somewhat high, so it still
has to be determined application by application whether this approach works or not. An additional limitation is
the development of adapted software, which is temporarily lagging behind; manual screening for hundreds of
components in each sample is not feasible in a routine environment.
Figure 3: Thermo Fisher Scientific Orbitrap mass spectrometer.
Figure 4: Mass spectrum with a resolution of 100,000
(graph at the top) and a resolution of 10,000 (graph at
the bottom). It is clear that the higher mass resolution
results in narrower peaks. This means that the mass
spectrometer can determine the exact mass more
accurately with a lower risk of false-positive or falsenegative results.
[email protected]
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