It has been known for some years that cereal commodities can contain mycotoxins. With the gradual increase of human life expectancy, it has now been proven that these natural toxins contribute to certain forms of cancers in both humans and animals.

1. Digital Re-print -
March | April 2014
Understanding the threat from mycotoxins
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3. MILL MAINTENANCE
Understanding the threat from
mycotoxins
- A comparison of mycotoxin testing techniques,
regulations and best practices for flour and feed milling
by Clare Milligan, Product Manager, R-Biopharm Rhône Ltd
I
t has been known for some years
that cereal commodities can contain
mycotoxins. With the gradual increase
of human life expectancy, it has now
been proven that these natural toxins
contribute to certain forms of cancers
in both humans and animals.
Invasion of foods and feed by moulds
under certain storage or environmental con-
ditions, such as high moisture and/or specific
temperatures, can lead to the development
of these highly toxic components.
There are many different types of myco-
toxins, the most common and dangerous of
which are aflatoxins (B1, B2, G1 and G2).
However, legislation also exists for other
mycotoxins such as ochratoxin A, deoxyni-
valenol, fumonisins, zearalenone and patulin.
Aflatoxins are produced by the fungi
Aspergillus flavus and Aspergillus parasiticus
and are often associated with commodities
grown in tropical regions including various
nuts, maize, dried fruits and spices.
Mycotoxins such as ochratoxin A are
found in cold to temperate climates, but can
often be found in similar commodities to
aflatoxins including dried fruits, coffee, cocoa,
cereals, herbs and animal feed ingredients.
Fumonisins, deoxynivalenol, zearalenone and
T-2 and HT-2 toxins are produced by vari-
ous Fusarium species and are mainly found in
maize and maize-based commodities while
patulin is commonly found in apple juice
and purees.
Surveillance plans for cereals
Due to the prevalence of mycotoxins
in cereals, cereal products and animal feed
there has been growing concern for human
and animal safety resulting in increased sur-
veillance plans for cereals and retail products
to generate data on consumer exposure to
the range of Fusarium mycotoxins in the
human diet.
Legislation is currently in place
(1126/2007/EC) for most of the Fusarium
mycotoxins including DON, fumonisin, zea-
ralenone and in March 2013 recommenda-
tions were introduced for T-2 and HT-2
(2013/165/EU). See Figure 1 for further
information on current EU Legislation in
Food.
Currently there are a wide variety of tests
available for the detection of mycotoxins.
These include ‘traditional’ methods such as
thin layer chromatography and more rapid
tests such as enzyme immunoassays or
lateral flow devices commonly known as
dipstick tests.
However, for more sensitive analysis
of mycotoxins, immunoaffinity columns are
commonly used to allow legislative levels to
be easily and accurately quantified by HPLC
or LC-MS/MS.
With the increase in mycotoxin legisla-
tion there is a growing need for fast, simple
and accurate methods for both quantifying
or screening mycotoxins, which are, not only
capable of detecting the legislative levels
but also of meeting the analytical criteria
laid down by the European Commission.
R-Biopharm have developed a complete
range of test formats including ELISAs, cards,
lateral flow tests and immunoaffinity columns
to allow users to comply with both European
and International Mycotoxin regulations.
The method of choice
Immunoaffinity columns are the standard
method of choice for regulatory mycotoxin
analysis, however, there is also the need for
columns, which can offer multi-mycotoxin
analysis in conjunction with either HPLC or
LC-MS/MS using a single extraction method.
The company has therefore developed a
number of new multi-mycotoxin immu-
noaffinity columns including AFLAOCHRA
PREP®
, EASI-EXTRACT®
T-2 & HT-2 and
AO ZON PREP®
for use in conjunction with
HPLC and the most recent additions to it’s
product line are DZT MS-PREP®
and AOF
MS-PREP®
for simultaneous detection of
mycotoxins in conjunction with LC-MS/MS
(see Figure 2).
The multi-mycotoxin columns are pro-
duced using more than one monoclonal
antibody bound to a solid support within
Figure 1: Overview of current EU Legislation in Food
Toxin Commodity
Max Level
(ppb)
Deoxynivalenol
Unprocessed cereals (excluding durum wheat, oats and maize) 1,250
Unprocessed durum wheat and oats 1,750
Unprocessed maize 1,750
Cereals intended for direct human consumption, cereal flour (including
maize flour, maize meal, and maize grits), bran as end product intended
for direct consumption and germ
750
Bread, biscuits, pastries, cereal snacks and breakfast cereal 500
Dry pasta 750
Processed cereal based baby and infant food 200
Toxin Commodity
Max Level
(ppb)
Zearalenone
Unprocessed cereals other than maize 100
Unprocessed maize 350
Cereals intended for direct human consumption, cereal flour, bran as end
product for direct human consumption and germ
75
Maize intended for direct human consumption, maize based snacks and
maize based breakfast cereals
100
Refined maize oil 400
Bread, pastries, biscuits, cereal snacks and breakfast cereals 50
Processed cereal based foods and baby foods for infants and young children 20
Processed maize based foods and baby foods for infants and young children 20

4. a column. This enables the isolation and
concentration of target mycotoxins and the
use of monoclonal antibodies makes the test
highly specific and offers improved sensitivity.
In all cases the toxins are extracted by
blending the food or feed sample with a
solvent, the extract is then filtered, diluted
and a small volume passed through the
immunoaffinity column by the analyst. If the
target mycotoxins are present in the test
sample, they are selectively bound to the
antibodies in the column; the toxins are then
released from the column using a solvent.
The eluate is then injected onto the
HPLC or LC-MS/MS system ready for quan-
tification (see Figure 3).
The advantage of these new multi-myco-
toxin immunoaffinity columns is that only
one sample preparation method is required
for quantifying all mycotoxins in a single run
therefore having greater sample throughput
and a reduction in the use of solvents and
consumables. The development of faster,
accurate tests enables easier and more thor-
ough monitoring of foods by the supplier.
If such testing is used in conjunction with
preventative measures at all stages in the
production process, contamination risk to
the consumer will be significantly reduced.
With the new recommendations in
March 2013 for T-2 and HT-2 (2013/165/
EU) Member States, should with the active
involvement of food and feed business
operators, perform monitoring for the pres-
ence of the toxins in food and feed samples.
Member States are encouraged to test
samples simultaneously for the presence of
T-2 and HT-2 and other Fusarium toxins
such as deoxynivalenol, zearalenone and
fumonisin B1 and B2 to allow the extent of
co-occurence to be assessed.
T-2 is rapidly metabolised in a large
Toxin Commodity
Indicative Max
Level of T-2 &
HT-2 (ppb)
T-2 & HT-2
Unprocessed barley and maize 200
Unprocessed oats (with husk) 1,000
Unprocessed wheat, rye and other cereals 100
Oats for direct human consumption 200
Maize for direct human consumption 100
Other cereals for direct human consumption 50
Oat, bran and flaked oat products for human consumption 200
Cereal bran except oat bran, oat milling products other than oat bran
and flaked oats, and maize milling products
100
Other cereal milling products 50
Breakfast cereals including formed cereal flakes 75
Bread (including small bakery wares), pastries, biscuits, cereal snacks, pasta 25
Cereal-based foods for infants and young childern 15
Toxin Commodity
Max Level of B1
& B2 (ppb)
Fumonisins
Unprocessed maize 4,000
Maize and maize based foods intended for direct human consumption 1,000
Maize based breakfast cereals and maize based snacks 800
Processed maize based foods and baby foods for infants and young children 200
Figure 2: Example
Immunoaffinity Column
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5. number of cereal products, HT-2 being a
major metabolite. It is also recommended
that if the method employed by the opera-
tor allows, that the presence of masked
mycotoxins in particular the mono and
di-glycosylated conjugates of T-2 and HT-2
should also be reported by Member States.
Masked mycotoxins just as toxic
A masked mycotoxin is a compound
who's 'parent' mycotoxin is chemically
altered into a conjugated or structurally
related form of the original mycotoxin.
Masked mycotoxins are thought to be just
as toxic as the original toxin but may behave
very different chemically and can be more
complex to analyse. R-Biopharm Rhone's
immunoaffinity columns have been tested
and have been found to cross react with
the many of the masked mycotoxins and are
suitable for use with LC-MS/MS allowing the
analysis of all toxins in a single run.
With more commodities being covered
by legislation comes the need for large and
small food companies and importers to test
for mycotoxins. R-Biopharm Rhone have
developed a range of test formats to meet
the growing demands of the market. For
those customers who require quantitative
analysis of mycotoxins, but who do not have
an HPLC or LC-MS/MS, a wide range of
ELISAs have been developed for a range of
mycotoxins.
For port health authorities and small
laboratories who need a fast, qualitative test,
there are a range of test kits that can be used
to screen cereals and cereal products. The
AFLACARD, OCHRACARD and RIDA®
QUICK tests allow for visual screening of the
samples. These kits allow users to analyse
samples quickly and easily yet be assured
that they are keeping in line with current
legislation.
Lateral flow dipsticks
RIDA®
QUICK lateral flow dipsticks are
available for screening for deoxynivalenol,
aflatoxins, zearalenone and fumonisins.
Interpretation of results is easy; a visible test
band appears for positive samples and no
line is visible for negative samples. RIDA®
QUICK SCAN is available to read the test
bands in order to give customers a quantita-
tive result (see Figure 4).
Enzyme linked immunosorbent assay
(ELISA) can also be used for screening
various mycotoxins. The RIDASCREEN®
ELISAs use the high specificity of antigen
and antibody interaction to detect and
quantify the mycotoxin of interest by pho-
tometric detection. RIDASCREEN®
T-2 /
HT-2 R3805) has been recently added
to the range and is a competitive enzyme
immunoassay for the quantitative analysis of
T-2 and HT-2 toxins in oat, corn, barley and
wheat. The mycotoxins are extracted from
the cereal samples with methanol / water
and the concentration of mycotoxin present
is determined by means of the standard
curve (range: 1 - 36 ppb (μg/kg)). The limit
of detection is approximately 30 ppb (30 μg/
kg) and the recovery in natural contaminated
cereal samples (Trilogy®
reference mate-
rial, TR-MT100) was determined to be 105
percent ± 15 percent and in spiked samples
was found to be 95 percent ± 15 percent.
The performance, stability and quality
of RIDASCREEN®
ELISA kits are validated
both internally and externally before being
officially released to the market for use.
Accreditations from recognised bodies such
as AOAC, UKAS and AFNOR are also
sought. It is important to stress that the
research and development of a product
does not end with its launch into the market.
Work continues and is carried out on a
regular basis at laboratories in Germany and
Scotland to ensure products continue to
meet ever changing legislation and the differ-
ent types of matrices that these regulations
cover. Customer feedback is very much an
integral part in ensuring that the company
continues to develop successful products to
be a leading supplier in this sector.
Drastically reducing
contamination
The development of faster, accurate tests
enables easier and more thorough monitor-
ing of foods by the supplier. If such testing is
used in conjunction with preventative meas-
ures at all stages in the production process;
this will drastically reduce contamination and
lower the risk to the consumer.
Since the formation of the company
in the late 1980s R-Biopharm Rhone has
established itself as a leading developer of
test solutions for food and feed analysis,
manufacturing test kits that offer high pre-
cision and accuracy. With over 30 years
experience in the development of various
test formats, methods and services for the
detection of mycotoxins, the company plans
to continue to develop and offer exciting
prospects in the field of mycotoxins ahead
of legislation and to provide complete myco-
toxin solutions for both large and small food
companies.
The company is well positioned to
advance the development of innovative
and versatile fast tests necessary to obtain
accurate results from a wide range of new
challenges.
More inforMation:
Claire Milligan
Product Manager, R-Biopharm Rhône Ltd
Tel: +44 141 945 2924
Fax: +44 141 945 2925
Email: info@r-biopharmrhone.com.
Website: www.r-biopharm.com
Figure 3: Overview of
Immunoaffinity Column Principle
 Mycotoxins  Other materials
Figure 4: Overview of RIDA®
QUICK
DON Method
GRAIN&FEED MILLING TECHNOLOGY24 | March - April 2014
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