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AOAC Food Allergen Community

Volume 7 | Issue 3 2016

Editorial Comment
Editorial Comment Christmas time - and some presents have already been
Christmas time - and some presents
have already been delivered delivered during the year
during the year  1
This year was special - better. A lot of progress has been made in the field
Featured Article 2 of food allergens, and many new activities have started, some milestones
Fostering Consistent Guidance on achieved. Back in 2015, AOAC started to develop a Standard Method
Food Allergen Management Performance Requirement (SMPR) document for the analysis of food allergens
Globally 2
using mass spectrometry. After numerous discussions and revisions, AOAC
News 4 SMPR® 2016.002 was approved and published in the Journal of AOAC 99(4).
Allergen analysis in support of In addition, two LC-MS/MS methods for multiple allergen detection were
consumer protection, quality submitted and discussed at the AOAC Annual Meeting. At the December
control and regulatory meeting of the European Organization for Standardization (CEN), the status of
enforcement 4 the subgroup (CEN TC 275 WG 12 AHG 4), which deals with chromatographic
1st International Conference on methods (including LC-MS/MS) was changed from ‘dormant’ to ‘active’,
Food Analysis (ICFA) 5 indicating that there will also be some developments at European level.

Scientific Developments 6 AOAC also called a thought leaders meting to start a new ISPAM activity
Allergen detection using mass for food allergens, including ELISA and PCR methods, but excluding mass
spectrometry: MRM3 as a novel spectrometry methods. During a call at eh end of November, a draft SMPR for
approach for increased specificity ELISA methods was discussed.
and sensitivity 6
During the annual meeting in Dallas, the two allergen sessions, both discussing
Major challenges for reliable LC-MS/MS based approaches and reference materials were among the best
detection of allergens in food attended sessions (you will find some more details in this edition). And we’re
products  7 very happy to report that a young scientist from Belgium, Melanie Planque,
Will allergies soon be curable?  8 won the poster award and delivered an excellent presentation.

Upcoming Events 8 In this issue, you will find some more details on the highlights from the 2016
Symposium Food Fraud Prevention Annual Meeting as well as some interesting articles about developments at
and Effective Food Allergen Codex Alimentarius regarding threshold levels. You will also find some news
Management8 from ‘Down Under’, where a research team aims to develop allergen vaccines
and therapeutic agents. Enjoy the read and best wishes for a peaceful
Christmas and a happy New Year from the Editorial Team.
Bert Popping  Editorial Board Member

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Volume 7 | Issue 3 2016

Featured Article
Fostering Consistent Guidance on Food Allergen Management
Over the last 20 years, several actions have been taken by an allergenic ingredient, which cannot be avoided under
various players with the aim to enhance the protection of reasonable food processing conditions, there was no clear
food allergic consumers. While several measures targeting guidance as to how they should be applied in a manner that
the improvement of ingredient labelling, with emphasis is risk based.
on allergenic ingredients, have been harmonized thanks
to standards promulgated by the Codex Alimentarius Statements of different sorts are popping on food labels, with
Commission, far more needs to be done in relation to the use of no regard to what they may mean in informing the choice of
precautionary statements. Allergen precautionary statements allergic consumers and their families. Others are being used
continue to be diverse, to be used under different conditions with no particular justification, other than to “cover a potential
and are not necessarily supported by risk assessment. As a liability”. The practice has even led to witnessing precautionary
result, these labelling practices are not helpful to consumers. statements on food labels that cover the entire list of priority
allergens and that are longer than the list of ingredients itself.
When one examines the history of food allergen management, No wonder that confusion reigns, not only for consumers,
it is easy to note that most regulatory measures that were but also amongst the health professional community, whose
developed at the national level took place after the Codex guidance is sought in helping allergic consumers manage
Alimentarius Commission known as Codex and acting as the their avoidance of potentially offending foods.
international food standard setting body, developed its global
standard on food allergen labelling in 1999. Several years The scientific community acknowledged the problem and
were required, and expert advice was mobilized at the global agreed that investing in the development of the scientific
level by the World Health Organization (WHO) and the Food foundations for a risk-based approach to be applied would
and Agriculture Organization (FAO) of the United Nations to contribute to addressing this problem. A decade later, several
come-up with a set of criteria for the identification of food studies resulted in the generation of thousands of data
ingredients that have the potential to be allergenic. These points related to thresholds for food allergen reactions. Risk
ingredients were then covered by mandatory declaration on assessment methodologies have also been discussed and are
food labels. being adapted to food allergens as a food hazard. Allergen
analytical methods have been developed and are being used
Several domestic food regulatory policies were then amended to help validate sanitation practices and other quality control
and the world witnessed the development of a number of and management measures. We have even witnessed the
allergen labelling regulations and legislations in Australia/ development and adoption of allergen control programs by
New-Zealand, Europe, the United States of America, Japan, the food industry sector in an attempt to create “order and
Canada and a number of emerging economies in Latin structure” as to how allergen advisory labeling should be used.
America and Asia.
While food allergen-related recalls continue to top the list
All these requirements were based on the Codex Standard of food recalls in North America, Europe and some parts of
and ensured to prevent the omission of declaration of Australasia, there is limited to no leadership from domestic
food ingredients known to be priority allergens, when food regulatory jurisdictions to move forward with clear
these ingredients are deliberately added to the recipe of a regulatory measures focusing on allergen precautionary
prepackaged food. statements. Such measures would be the cornerstone for a
more predictable environment for industry and consumers on
This did not however address all issues allergic consumers and food allergen management.
their care givers are having with food labels. The propagation
of use of allergen precautionary or advisory labeling in the Learning from history to address this conundrum can be
form of “may contain” statements and other iterations is useful in this case. I would argue that like what happened
undermining consumers’ confidence and their trust in the in the 1990s and early 2000s, it is time for Codex and its
reliability of information offered by food labels. parent organizations to act. Attention needs to be given
to the development of global guidance related to allergen
While precautionary statements were initially meant to thresholds, allergen precautionary labeling and management
address situations of possible cross-contamination due to of cross-contamination and adventitious presence of priority

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Volume 7 | Issue 3 2016

allergens in food manufacturing. These global standards discrepancies of food allergen management policies and their
could then lead the way in driving change in domestic food impacts on international food trade. The case can also be made
regulatory requirements in this area, which could then use the that scientific data has been generated over the past decade
legitimacy of the international process. The time has come for or more and could be relied-upon for international expert
Codex to lead again on this issue. groups to be convened by FAO and WHO, to guide Codex with
the relevant scientific advice. Finally, and should there be a will
I realize that suggesting such an approach, can be met with to act, funding could be mobilized from various government
cynicism, given how long some international food standards organizations currently struggling to move forward with
can take in the making. But Codex has also shown that when addressing this issue and who would benefit from pooling
its membership wants, it can. The first Melamine standards resources under the auspices of a collective initiative.
that Codex embarked on developing in 2009, took only one
year between decision to act and adoption of the standard, It is possible for history to repeat itself. Twenty (20) years
making it the fastest agreed-upon Codex standard to date. after the initiation of the process that led to allergen labeling
Opponents to this approach would also argue that there is measures as we know them influenced by Codex leadership,
currently no interest on the part of Codex to work in this area, action leading to renewed international guidance on food
nor would there be resources made available. My argument allergen management is overdue.
back is that momentum can indeed be created. The case
can be made for Codex standards being needed, given the Samuel Godefroy  Université Laval

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Volume 7 | Issue 3 2016

Allergen analysis in support of consumer protection, quality
control and regulatory enforcement
This year, the AOAC Annual meeting felt different. Better.
What was very noticeable was the strong engagement of all
stakeholders in focal areas this year. Allergen analysis was one
of them.

Besides the partially controversial discussions about which
information is needed to accept mass spectrometry based
methods as First Action Method, AOAC also launched
an initiative to establish an ISPAM working group for
immunological and molecular biological analysis of food

The allergen sessions were extremely well attended, and
the session organized by Romer Labs and co-chaired by Dr. Panellists of the allergen session at the AOAC Annual Meeting.
Carmen Diaz-Amigo and Prof. Sabine Baumgartner had the From left to right, first row: Scott Radcliffe (Romer Labs), Vincent
highest attendance of all sessions during this years’ AOAC Paez (Sciex), Jerry Zweigenbaum (Agilent), Steve Gendel (IEH
Annual Meeting. Labs), Markus Lacorn (R-Biopharm), Tony Lupo (Neogen), Bert
Popping. Back row: Melanie Dawns (University of Nebraska), Sabine
Baumgartner (BOKU University)
The session featured three parts: the first part reported about
advances in reference materials for food allergens, reported by
Prof. Roland Poms (MoniQA). Prof. Poms even showed some established if a method is sufficiently reliable and robust to
sachets of reference materials. In the second advances of LC- become a reference method.
MS/MS analysis of food allergens were discussed, presented
by Prof. Sabine Baumgartner, Prof. Melanie Downs and Dr. Bert The discussion continued to the calibrants used for the
Popping. The third, not scheduled part, was a wider discussion individual methods. It became clear that currently all method
with method and equipment providers, which were asked on providers - in the absence of certified reference materials - use
stage, moderated by the previously mentioned presenters. their own material, which introduced variability.

As an introduction to this discussion, the evolution and lively All method and equipment providers agreed that commutable
dynamics of European directives and regulations for food reference material – if used as calibrant and quality control
allergen labelling were shown, followed by a comparison of material, as it is currently in production and distribution
results in proficiency tests (PT). It became clear that quality by MoniQA, would help eliminate some of the variabilities
control and enforcement of food allergens were currently between the methods and contribute to the harmonisation of
challenged by the differences in results between methods. results, ultimately benefiting consumers, food manufacturers,
enforcement authorities and ultimately affected consumers.
It was mentioned that contrary to other PT where the assigned The panellists agreed that the use of a common reference
value for the sample is reported at the conclusion of the test, material would be a good step forward. Further contributions
for food allergen analysis, assigned values are per applied to this discussion were made by Covance and IEH.
method (e.g. per ELISA kit used by the participants of the PT).
This is mainly due to the variability of results between the The participating companies, namely Agilent, Neogen,
different methods used. R-Biopharm, Romer Labs and SCIEX were applauded by the
audience and thanked for their engaging discussion.
Dr Håkan Emteborg from the EC JRC Institute for Reference
Materials and Measurement (IRMM) explained that this is a Bert Popping  Editorial Board Member
‘catch 22’ situation. Since there is no reference method, no
certified reference material can be manufactured. And since
there is no certified reference material, it cannot be

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Volume 7 | Issue 3 2016

First International Conference on Food Analysis (ICFA)

The 1st International Conference on Food Analysis (ICFA) and A significant emphasis was given to food allergens including
the workshops on proficiency testing and food allergens were plenary presentation by Prof. Andreas Lopata from James
held in Melbourne, Australia (21st - 24th November 2016) Cook University providing insight on the current research in
with the theme “Where food analysis meets industry and food allergy. Other presenters included Dr. Martina Koeberl
regulation”. The conference brought together the themes on (National Measurement Institute, Australia), on identification
food quality and food safety, strengthening the partnerships of common allergenic legume protein by mass spectrometry,
between food industry, food trade, food legislation and as well as Adrian Rodgers and Dr. Alice Lee. The topics discussed
regulation, public health and the science community. in the workshop on food allergens included, new technologies
for allergen detection, accreditation and proficiency testing,
Approximately 180 delegates consisting of research scientists, low level gluten detection, lupin as possible mandatory
those involved in analytical sciences, and those associated allergen in Australia, allergen detection problems in processed
with public health, environment and agriculture, regulation food products and how to report analytical allergen detection
and trade participated for the event. There were over 25 results. It is expected that the conference to be held two yearly
international delegates representing Asia, Europe, North in Melbourne.
America and other parts of the world.
Martina Koeberl
The conference was organised by The National Measurement National Measurement Institute, Australia
Institute – Australia, ACS Laboratories, National Testing
Authorities Australia, James Cook University, Victoria
University, Melbourne University and Queensland Health and
Forensic and Scientific Services.

Participants of the 1st International Conference on Food Analysis.

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Volume 7 | Issue 3 2016

Scientific Developments
Allergen detection using mass spectrometry: MRM3 as a novel
approach for increased specificity and sensitivity
Food allergy is one of the major challenges in consumer To ensure highest sensitivity, a targeted proteomics method
protection in industrialized countries. It is estimated that using triple-quadrupole instrumentation was developed.
150 million people worldwide suffer from allergic reactions In addition, the analytical performance of multiple reaction
to food and that about 5% of the population in westernized monitoring (MRM) and MRM3, available on QTrap instruments,
countries is affected. The “may contain” labelling of potential was compared. In MRM3-experiments, the precursor ion
allergen contaminations has been shown to provide only is isolated in Q1, fragmented in the collision cell and the
limited protection for allergic consumers, partially because fragment ion of interest is accumulated in the linear ion trap
of its extensive use. An alternative strategy would be the (Q3) and subjected to fragmentation, resulting in a secondary
monitoring of allergen trace contaminations using sensitive MRM transition (Fig. 1).
and specific analytical tools on the basis of clinically defined
allergen threshold doses. As the availability of these clinical For the targeted method development 38 MRM3 experiments
data has strongly increased in recent years, the analytical specific to 18 proteotypic peptides (three per nut species)
surveillance of allergen contaminations is now within reach. were optimized and in parallel respective MRM transitions
There is however still a need for the development of sensitive were recorded for all peptides. All nut species were spiked at
and quantitative analytical methods. different concentrations ranging from 0.3 µg/g to 100µg/g
in three different matrices (bread, ice cream, muesli). Limits
Targeted proteomics using mass spectrometry is a promising of detection of the MRM3 approach were approximately 1
alternative technique that offers increased specificity and μg/g or below in fortified matrix samples. Compared to the
reproducibility compared to currently used methods. As the MRM-based detection, the MRM3 method showed increase in
available clinical data indicate that the detection of minute sensitivity of up to 30-fold. Regression analysis demonstrated
trace levels of food allergens is required for efficient consumer high linearity of the MRM3 signal in spiked matrix samples
protection, highest sensitivity is needed for the MS-based together with robust intersample reproducibility, confirming
detection. that the method is highly applicable for quantitative purposes.
Notably, matrix interferences observed for some peptides in
We therefore employed a bottom-up proteomics approach MRM-mode were not present in MRM3 data demonstrating
for the identification of allergen-specific proteotypic peptides increased specificity.
from six different nut species (peanut, hazelnut, walnut,
pistachio, almond, cashew). Following thorough experimental To the best of our knowledge, this is the first study that
validation of specificity and sensitivity, 44 suitable tryptic systematically compares MRM3 with MRM for the analysis of
marker peptides for the six nut species were identified. complex foods.

Jens Brockmeyer  |  University of Stuttgart
Figure 1. Principle of the MRM3 experiment.
Korte R, Brockmeyer J. (2016) MRM3-
based LC-MS multi-method for the
detection and quantification of nut
allergens. Anal Bioanal Chem, 408(27):
7845-7855. Abstract 

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Volume 7 | Issue 3 2016

Major challenges for reliable detection of allergens in food products
Food allergies have increased impressively over the last few Despite the emergence of incurred and standard materials
decades. In order to protect customers with food allergies, (from producers such as the National Institute of Standards
allergens must appear clearly on food labels. Labelling food Technology and LGC Standards), there is a lack of standards
products adequately can be hard, however, because of for most allergens. Unfortunately, this leads to the use of
potential cross-contaminations during food production. To different materials (walnuts, roasted walnuts, walnut milk…).
help food producers in this tedious task, the food community Furthermore, different members of the food community report
has developed analytical methods for food allergen detection. their results in different units: ingredients, soluble proteins
Nonetheless, there remain issues that must be addressed in (BCA quantification), total proteins (theoretical content),
order to harmonize food allergen control. or protein (casein, ovalbumin…). The lack of standards and
guidelines makes it very hard to compare methods, so that
Recently, although the number of food allergen analyses has results are almost useless for laboratories and even worse for
increased dramatically, regulatory thresholds still have to be food producers. Concerned about this lack of harmonization,
established. Systems proposing thresholds in food products the AOAC Stakeholder Panel on Strategic Food Analytical
(VITAL, EAACI…) have been developed, but these thresholds Methods (SPSFAM) has recently established requirements
have not yet acquired regulatory force. for the evaluation of mass-spectrometry-based methods for
allergens (SMPR 2016.002 “Standard method performance
Method sensitivities are mostly determined by spiking requirements for detection and quantification of selected food
solvents or matrix extracts with allergens. This approach allergens”). In its guidelines, this panel recommends reference
makes methods attractive, but creates a significant gap standards, target matrices, ranges of quantification, etc.
between declared and real thresholds, especially for heat-
processed food products. Some laboratories, aware of this The publication of validation guidelines and the development
shortcoming, have produced and commercialized incurred of reference standards and materials are first steps towards
materials (FAPAS, MoniQA, LGC…). Another problem is that harmonization and method performance testing. Yet the food
sensitivity determinations based on measurements in only community still has a long way to go for full harmonization of
one matrix cannot guarantee sensitive detection of allergens methods.
in processed matrices or in ones with a high fat content or
high acidity. That a method is fit for purpose should thus be Melanie Planque | CER Group
established carefully in each case. Nathalie Gillard | University of Namur

Summary of the lack of harmonization encountered by the food community and possible solutions.
Identified problems

Proteins Protein
Expression of results Ingredients Soluble proteins (BCA Total proteins (Theoretical
Casein αS1, β-lactoglobulin
quantification) content estimation)

Determination of
Spiked Incurred
method sensitivity

Reference materials and
Solution for Guidelines Mass Spectrometry
standards Regulatory Regulatory thresholds
harmonization methods (SMPR 2016.002)

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Volume 7 | Issue 3 2016

Will allergies soon be curable?
The team of professor Lopata from the Australian James Cook
University is looking to develop vaccines or therapeutic agents
for cure or lessen the effect of food allergies. The Queensland
based group analyses the molecular structure of proteins with
the aim to modify them so they can be used for treatment of
allergies and/or diagnostic tools. Professor Lopata pointed out
however, that these therapeutic agents would not be used for
acute treatment of allergies like an epi-pen. The development
is oriented more in the direction of desensitisation.

At present, the group focus on fish. According to the working
group, they have blood samples from more than 200 children
who are allergic to different species of fish. Professor Lopata
pointed out that there are already some diagnostic tools
Professor Andreas Lopata (Left) and Dr Aya Taki (right).
available for cod, but that exiting diagnostics to identify
cod allergy are not fail-safe on one hand and that cod is not
frequently consumed in Australia on the other hand. Their
research will focus on fish species more frequently consumed
in Australia. According to the group, fish diagnostic tools and
therapeutic tools could be ready in 2 years and other foods for
which allergies exists in four years. The research is funded by AOAC Food Allergen Community Newsletter
the National Health and Medical Research Council.
Contribute with articles, news items or suggestions.
James Roberts 
Submission deadline for the 1st issue of 2017: March 24
Australian National Measurement Institute
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January 26-27, 2017
Bari, Italy
* All articles are subject to review by the Editorial Board.
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More Information 

The AOAC Food Allergen Community is a forum serving the scientific community working on Food
Allergens: The community aims to help AOAC INTERNATIONAL in its consensus-based scientific and
advisory capacity on methods of analysis for allergens in foods and other commodities. It is also
meant to serve the broader Stakeholder Community whose objectives it is to enhance the protection
of food allergic consumers worldwide.
Contact us at

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