Multi-mycotoxin testing in food

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For as long as humans have cultivated and

stored grain we have been at risk from

mycotoxins with outbreaks of Ergots

being reported since the Middle Ages

in epidemic proportions. Humans have

linked the occurrence of mould withsickness since the 7th and 8th centuries

and hence conducted a festival to

celebrate the Roman God Robigus who

was the protector of grain and trees in order to protect from rust

and moulds.

The problem of mycotoxin occurrence has only gotten larger as

our societies have grown more complex and our ever increasing

population. The more grain and cereal we consume the greater

the chances of us coming into contact with moulds and the

mycotoxins that may be present.

Fungal growths may be present in cereal grains and can if

not detected can cause serious health issues like damage to the

immune, cardiovascular, endocrine and nervous systems. Grains

such as wheat, barley, oats and rye are extremely susceptible

and diseases like head blight can cause substantial agricultural

losses, and also lead to problems of mycotoxin contamination by

Fusarium fungi. This occurs pre-harvest on the growing crop and

can lead to the occurrence of a number of different mycotoxins

including deoxynivalenol (DON), zearalenone (ZON), T-2 and

HT-2 toxins.

Once harvested further problems can arise if the drying is

inadequate or cereals are poorly stored. This post-harvest

infection can occur with different fungal species leading to

contamination with yet other chemically different mycotoxins

such as ochratoxin A (OTA) and citrinin (CIT).

Occurrence of multi-mycotoxins in foods

Grains are not simply prone to one mycotoxin as where they are

grown and environmental conditions can leave them susceptible

to more than one toxin. For instance maize while particularly

prone to fungal contamination is also uniquely susceptible to

Fusarium species, which specically produces mycotoxins

known as the fumonisins (FB1 and FB2). In regions with higher

temperatures and moisture conditions Aspergillus infection can

also appear with subsequent formations of aatoxin B1, B2, G1

and G2.All of these mycotoxins are unlikely to contaminate the

same sample at the same time, but co-occurrence of more than

one mycotoxin is certainly common in maize or cereal grain.

These mycotoxins are chemically different in structure and

therefore each exhibit different toxicological effects, which

can be triggered at different levels of exposure. Additionally

human and animal species have signicantly different degrees of

susceptibility to the toxicological effects of these mycotoxins.

For these reasons and because of the importance of cereals in

human diet and animal feed, mycotoxins are tightly regulated in

many countries around the world by setting of maximum residue

levels (MRLs). As with many other regulations, these limits

are much lower for infant and baby foods compared to foods

intended for adults. This is because of the additional protection

needed during growth and development and the lower body

weight of infants

Regulations

In the EU aatoxins, DON, ZON, FUM and OTA are all

regulated in cereals and cereal products, with one limit applying

to unprocessed cereals and a lower limit being applied to

Cereal type

Mycotoxin EU regulatory limit µg/kg

DON ZON AFB1 OTA FB1 + FB2

Bread 500 50 2.0 3.0 -

Cereal-based baby food 200 20 0.1 0.5 200*

Maize breakfast cereals 750 100 2.0 3.0 800

by Claire Milligan, Product Manager, R-Biopharm Rhône Ltd, UK

Multi-mycotoxin

testing in food

42 | December 2015 - Milling and Grain

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cereals intended for direct human consumption. There are some

reductions in toxin levels during processing such as milling, but

as the toxins tend to concentrate in the fractions such as bran

there are consequent risks for animal feed.

The EU regulations for mycotoxins in cereals are complicated

with levels varying from one commodity to another and varying

signicantly for different mycotoxins. This can be illustrated by

the example of EU regulations for bread, processed cereal-based

foods and baby foods for infants and young children and maize-

based breakfast cereals shown in the table above. Different limits

apply to unprocessed cereals and maize, pasta, rened maize oil

and various milled fractions from maize as well as to animal feed.

Understanding the complexity of these regulations and carrying

out analytical determinations to ensure compliance is a signicant

challenge.

Analysis of multi-mycotoxins

Analysis of mycotoxins in cereals and cereal products

(including animal feed and pet food) is carried out on the raw

materials (grain and our) and on nished products. Analysis

is expensive to carry out and it therefore makes sense to target

monitoring on those mycotoxins, which are known to beassociated with specic cereal products and which are covered

by legislation. With the strong possibility of multiple occurrence

it also makes sense to determine more than one mycotoxin in an

analytical run.

Most ofcial methods, which have been rigorously validated,

stipulate the use of immunoafnity column clean-up (IACs) prior

to HPLC analysis. Recognising the importance of analysis of

multiple mycotoxins, R-Biopharm Rhône supply immunoafnity

clean-up columns specically targeted at extraction and

purication of different combinations of mycotoxins in cereals.

The DZT MS-PREP® immunoafnity columns are intended

for clean-up of DON, ZON and combined T-2 and HT-2 toxins,

whereas the AOF MS-PREP® columns are aimed at the analysis

of aatoxins, OTA and fumonisins.

The DZT MS-PREP® columns are particularly relevant for the

analysis of cereals and cereal products from wheat, oats and rye,

whereas the AOF MS-PREP® columns have more relevance to

the analysis of maize and maize based products where aatoxins

and fumonisins are more likely to be a problem.

By a simple procedure of coupling two IACs in tandem, i.e.

DZT MS-PREP® and AOF MS-PREP® it is possible to detect

all six mycotoxins that need to be monitored in processed cereal

based foods and baby foods for infants and young children which

include maize.

When single mycotoxins such as aatoxin B1 or OTA are

determined, it is often better to use HPLC with uorescence

detection to reach lower limits of detection. However, DON,

fumonisins, T-2, HT-2 toxins require different analytical

strategies, and when they are brought together into one method

the use of more sophisticated analytical systems such as LC-MS/

MS is the preferred approach.

Analysis without clean up?

Some laboratories will argue that with the specicity of mass

spectrometric detection systems it is unnecessary to carry

out immunoafnity column clean-up and crude extracts from

cereals can be directly analysed. This approach is acceptable for

screening but it has been demonstrated that without adequate

clean-up, co-extractives from the matrix can cause interference

which impacts adversely on identication and quantication.

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Milling and Grain - December 2015 | 43

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Ensuring that the peak being measured is in fact the mycotoxin

in question, and accurately measuring at the very low levels like

that are required for baby food and argues strongly for carrying

out adequate sample preparation and clean-up. Interferences

can lead to false positives and wrong decisions about rejecting

commodities, whilst ion suppression can lead to under estimates

of true concentrations and the risks of accepting a batch of

material that should be rejected. These are both unnecessary

risks to the food industry and to the food control laboratories

which can be readily overcome with minimal additional work in

preparing samples prior to instrumental analysis.

The EU Rapid Alert System for Food & Feed (RASFF)

The EU Rapid Alert System for Food and Feed (RASFF)

provides good intelligence information on the extent of cereals

being rejected by the authorities and as containing multiple

mycotoxins exceeding EU limits. Aatoxins and fumonisins

have been frequently found to co-occur at levels exceeding EU

limits for both groups of toxins in the same sample. For example,

popcorn from Argentina containing aatoxins and fumonisins,

DON and OTA in cornour from Poland, aatoxins and OTA

in maize from Ukraine were all rejected by the EU. Along withthe notications sent out by each designated national contact to

the EU Commission, the RASFF portal has a searchable online

database open to members of the public. This system of alerts has

helped avert many food safety risks.

Many consignments of cereals and cereal products are rejected

by the EU as containing high levels of individual mycotoxins

such as DON, ZON, fumonisins and aatoxins but it is unlikely

these consignments would have been identied as a potential risk

to human health without an initial multi-mycotoxin screening.

A solution to the problem

Immunoafnity columns targeted at DZT and AOF analysis

meet the needs of mycotoxin laboratories engaged in ensuring

compliance of cereals with regulatory limits. These columns

have been used for the development of validated methods by

R-Biopharm Rhône, where the method performance has been

demonstrated to exceed the minimum requirements set out both

by the EU and standardisation bodies such as CEN. Methods can

be supplied to customers in a format compatible with method

SOPs to enable ready adoption into an accredited environment.

With products that are manufactured to ISO 9001 and employing

an ISO 13485 quality management system R-Biopharm Rhône

products are widely used by 17025 accredited laboratories in the

EU and elsewhere, and are widely appreciated as being reliable

products of consistently high quality.

Final thoughts

Although application of good agricultural practice can reduce

the risks of fungal infection of cereals and can minimise

mycotoxin levels, the co-occurrence of mycotoxins in cereals is

an inevitable fact of life.

There is considerable variability in levels of Fusarium toxinsfound in cereals from year to year as climate can have a

signicant impact on toxin formation. Wet conditions during

the growing season and during harvest have a major impact

on fungal infection, and climate change resulting in less

predictable conditions is leading to increased risks of mycotoxin

contamination. Testing for the presence of mycotoxins will

continue and as trade between countries across the globe grows

we can expect legislation to be tightened beyond the EU and its

current trading partners.

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Multi-mycotoxin testing in food