AOAC Food Allergen Community
NEWSLETTER
Volume 2 | Issue 2
Spring 2011
Longing for a new life
The miracle of the spring time
Spring has already arrived to our gardens and parks and to contribute to this symphony of colors we would like to let our colleagues know that new blooms and refreshing airs have also come to our food allergen community. We are pleased to welcome new developments that can mark a new era in the analysis of food allergens and gluten: detection by Mass Spectrometry. The Editorial Team of the AOAC Food Allergen Community Newsletter
IN THIS ISSUE
Editorial Comment
Editorial Comment The acceptance of New Technologies 1
Ask the Expert Dr. Petra Silke Lutter
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Spotlight Mass Spectrometry: Policy and Enforcement Tool? 4 Mass Spectrometry for Food Allergen Analysis, Multiscreening 4
News Regulatory Updates - Health Canada 5 Mass Spectrometry and allergens in the Journal of AOAC international 5
Manufacture’s Corner Upcoming Meetings: AOAC Recent Publications
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Editorial Team
Editor in Chief: Carmen Diaz-Amigo Editorial Members: Terry Koerner Jupiter Yeung Michael Abbott James Roberts Bert Popping Graphic Design: Carmen Diaz-Amigo AOAC Food Allergen Community
[email protected]
The Acceptance of New Technologies
Nowadays the development of new technologies and their applications grows faster than the speed of what often our grey matter is able to comprehend. In many cases, by the time we get used to a new technology it is already obsolete. Why is the acceptance of such technologies not immediate? As we grow older, the level of skepticism also grow with us. We become more conservative regarding the unknowns and therefore we become more attached to what we already know and are comfortable with, regardless of the advantages that the new technology can offer. How many times are we amazed by 3 or 4-year old kids that can play quite complex computer games, even when their level of education is far lower than ours? Simply because they are not scared of trying. They don’t have any prejudice. However, in adulthood, the time it takes to accept a new technology can vary depending on several factors. For example: how different is the new technology from what we already know; how many disadvantages the old system has; the number and severity of problems we have had in the past when trying something new; the diversity of unknown products already in the market; the saturation of inconsistent reports in the media about the technology, just to mention a few. Mass Spectrometry (MS) is one of those complex technologies that may initially be difficult to understand by many of us, although for many years routinely used to detect many contaminants such as veterinary drug residues, mycotoxines, and forbidden dyes. However, we have to acknowledge the number of advantages and successes shown in many analytical areas along the years. The fast evolvement of this technology has allowed the detection of large molecules, including food allergens, which in fact widen our analytical options. As a community we would like to welcome mass spectrometry by dedicating this issue to it. Enjoy it! Carmen Diaz-Amigo Editor in Chief AOAC Food Allergen Newsletter
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NEWSLETTER Volume 2 | Issue 2
Spring 2011
Dr. Petra Silke Lutter Research Scientist Nestlé Research Center Switzerland What changes in the MS technology have made allergen testing possible? Methods for assessing low levels of selected proteins in a mixture with other proteins have benefited greatly from the technological evolutions in the field of mass spectrometry driven by the demands in the proteomics field. Besides the improvement of instrumentation the growing number of genes and protein sequence entries for typical food ingredients in public databases is now supporting protein identification and selection of suitable target proteins and peptides for MS based determination of food allergens. So called triple quadrupole and ion trap MS instruments that have been already used for the quantification of small molecules in the pharmaceutical and food industry were more and more used for applications on proteins and peptides. Sensitivity, but more importantly, the linear dynamic range was improved and allows now
detection and quantification of peptides down to a low mg/ kg level. The absolute quantification (AQUA) strategy was developed for the precise determination of protein levels. This approach relies on the use of a synthetic stable isotope labeled internal standard peptides that are introduced at a known concentration to the sample extract. Subsequent MS analysis of the proteolyzed sample by a multiple reaction monitoring (MRM) experiment in a tandem mass spectrometer results in the quantification and confirmation of both the native peptide and isotope labeled internal standard peptide. Calibration curves can be built from a dilution series of synthetic but nonlabeled standard peptides spiked with a known concentration of an isotope labeled peptide.
What are the advantages of MS over ELISA? Today both methods have their specific advantages and disadvantages and right to co-exist. MS in combination with liquid chromatography (LC) allows a direct determination of food allergen proteins using selected peptides and their characteristic masses (m/z). This approach is a direct measurement and not dependent on antibodies like in ELISA assays. It therefore offers short assay development time and flexibility. Allergen determination by tandem mass spectrometers allows both, the quantitative determination and a verification of the peptide identity by its typical retention time and fragments masses. So called multi reaction monitoring (MRM)-MS assays have some distinguishing features relative to immunoassays for protein determination. The target molecule detected in the MS can be characterized with very high structural specificity, which is not achieved by antibodies. Furthermore, multiple peptides of one or more proteins can be analyzed simultaneously. This offers two major advantages over ELISA. First, this allows for multiple proteins/ allergens analyses in one single experiment which can reduce time and analysis costs if one sample has to be analyzed for the presence of several allergens. Second, MS also offers the stoichiometric cross-verification of results as determined peptide amounts of the sample protein should be equimolar. Discrepancies of molar rations can give an indication of e.g. Page 2
effects from food processing or interferences from the food matrix while in ELISA only the overall absorbance is measured without any information on potential interferences and crossreactivities. This provides a significant quality advantage for MS and increases the reliability of the method while it facilitates internal control measures, especially in cases where immunoassays are subject to interferences. ELISA assays may claim recognition of native, denatured or heat-stable allergens, but especially for polyclonal antibodies, the target molecules and the affinity to native vs. denatured protein is often not known. MS is detecting selected target peptides after an enzymatic digestion of the protein and is therefore not sensitive to any structural changes of the protein. However, food processing often results in modifications of the allergen like denaturation, oxidation or hydrolyzation. All these modifications can have an impact on the structure of the protein but also on its amino acid sequence. As it is so far not well understood, how these modifications affect the recognition by the immune system of an allergic consumer, methods for allergen determination should ideally consider these possible modifications. MS offers the flexibility to determine both, unmodified and modified peptides if the modification is known.
NEWSLETTER
Volume 2 | Issue 2
Spring 2011
What are the challenges for MS? There are some specific challenges for MS but also some that are common for MS, ELISA and PCR. In regulation food allergens are defined as a whole food commodity while MS and ELISA determine individual proteins and results need to be transferred into food commodity equivalents. Without reference materials even at food commodity level, with known protein composition and solubility, reliable conversion factors cannot be established. Ideally the pure allergenic ingredient that is suspected as cross-contact is analyzed in parallel in order to determined to the amount of target protein per mass unit of the ingredient and used later for result expression of the test sample.
costs on the other hand. Stable isotope-labeled peptides can be used as calibrants and as internal standards. Added as internal standards they cannot be used to compensate for losses during extraction and digestion but for matrix suppression effects that may occur in the mass spectrometer. As calibrants they allow accurate quantification. However, it must be mentioned, that these peptides in general are synthesized on demand and require further evaluation of e.g. short and long term stability as well as a confirmation of concentration by amino acid analysis if lyophilized powders are reconstituted in order to overcome unspecific adsorption and insolubility effects.
Similar to ELISA, another major challenge for MS is the quantitative extraction of the samples. Even if MS tolerates more reagents to improve protein extraction, a 100 % extractability of the target protein is rarely achieved. Besides samples extraction, the proteolytic digestion of the proteins is a source of error due to the imperfect nature of this enzymatic reaction. Incomplete extraction and digestion of proteins can result in an underestimation of protein concentrations in screening approaches without internal standard addition, but also in peptide-based stable isotope dilution approaches. This can be overcome by the possibility to use either foreign protein or stable isotope-labeled protein surrogates as internal standards. However, such protein standards have also drawbacks regarding e.g. protein modifications that occur during food processing but may not be present in the protein standards on the one hand and isotopic impurities and high
Another challenge is the sensitivity of the method which strongly depends on the food matrix, the allergen itself and the mass spectrometer type. It is shown in recent publications, that detection and quantification down to the low the low mg/ kg level is achieved and therefore comparable with most ELISA applications. MRM-MS assays that can be highly multiplexed such that up to 20 proteins can be measured during a single analysis generally sound very attractive, however, data interpretation and treatment are time consuming and remain one of the major challenges in the near future. Furthermore, MS platforms are associated with high costs for instrumentation, the need for expert operators and finally less potential to automate and high sample throughput compared to ELISA. Depending on sample amount and laboratory conditions ELISA or PCR may remain the most appropriate screening tools while MS will establish as confirmatory method.
Why food industry has opted to invest in MS technology for allergen analysis? To comply with allergen labeling laws and to protect consumers but also reputation and business, food producers need reliable analytical methods to monitor the presence of allergens during production in order to avoid crosscontamination in production lines and related products. This requires that relevant analytical methodology for detection of traces of allergens be established. But how to chose the most appropriate method? Without certified reference material for food allergens and incurred reference samples it is difficult to judge and compare the performance of currently available ELISA and PCR methods. As mentioned above, commercial ELISA kits are often not fully transparent in terms of specificity of the antibodies and translation factors to express measured protein concentration into mass fraction of the allergenic food commodity. Additionally, users of proprietary kits are forced into a reactive position in terms of dealing with batchto-batch variability, modifications or discontinuation of such assays. Therefore, much effort has to be made to validate the performance of such kits in food products. It is well known, that ELISA assays from different manufacturers - even if validated
properly - do not always report comparable results. This fact may cause disputes or even costly and image damaging product recalls if the same sample is analyzed by different ELISA kits by e.g. governmental labs and food producers and obtained results are inconsistent. Today, threshold or action levels are not set for all regulated food allergens. However, such levels would help industry to know exactly how to perform allergen management related to threshold levels and would lead to a harmonization of warning labels for consumers. From a technical point of view reliable and especially confirmatory methods for a quantitative determination of allergens are indispensable to comply with regulatory labeling requirements once thresholds are established. The capability of MS technology to develop and validate confirmatory methods for food allergen quantification has been demonstrated in recent publications and makes it the most promising technique for fully transparent future standardizations and further harmonization of alternative methods. Page 3
NEWSLETTER Volume 2 | Issue 2
Spring 2011
Spotlight Mass Spectrometry: A Policy and Enforcement Tool? In many jurisdictions information on the potential risk for hidden food allergens is disseminated using some type of labelling, but not all products contain an allergen warning and some may be labelled incorrectly. If cross-contamination is suspected in a product the level of risk to the consumer and subsequent level of enforcement needs to be assessed. This process is complex and many factors are involved, but in a protein based regulatory system a measurement of the level of allergenic, or associated protein, is usually required. ELISA methods are used to collect this information, but mass spectrometry methods of protein analysis are becoming important in the support of food allergen policy and enforcement. These methods can confirm positive results obtained from an ELISA and they can be used to collect information on samples in which immunological methods have produced false negative results. Modern multiple stage mass spectrometry (MS/MS) techniques have the ability to determine the amino acid sequence of peptide fragments present in a
sample. The mass of the peptide and sequence is typically enough information to positively identify the protein in which the peptide originates. This data, in conjunction with a positive ELISA result, provides little ambiguity in the positive result and subsequent enforcement. The specificity of information collected from the mass spectrometer can also be used in cases where the identification of the plant source is important, for example the gluten source (wheat, barley, rye). Mass spectrometry can also be used to collect information on samples that have caused an allergic reaction, but have produced negative results using ELISA methods due to some processing or matrix effect. Mass spectrometry is a powerful technique and will continue to be an important tool in food allergen policy and enforcement into the future. Terry Koerner Health Canada
Mass Spectrometry for Allergens Analysis: Multiscreening Different analytical techniques are available for allergen detection. Currently two are used routinely: enzyme-linked immunosorbent assays (ELISA) based on antibodies and, to a significantly lesser extend, polymerase chain reactions (PCR) detecting DNA. The latest addition to this suite of methods are mass spectrometry (MS) based applications. These methods have a higher degree of technical complexity than ELISA, but have a number of advantages over both, ELISA and PCR.
protein itself and not the DNA. As DNA is a different substance class, distribution may not be the same as for proteins and it is therefore a poor indicator for possible presence or absence. In the worst case scenario, PCR can deliver a negative result while lifethreatening amounts of proteins for the allergic individual can be present, like in the case of egg white, milk or some proteins concentrates. DNA may be used as an allergen indicator but should only be used to confirm protein-method based results.
An example for mass spectrometric detection of allergens is the screening method developed by Heick, Popping and Fischer [Abstract]. After the extraction of the allergenic protein and its enzymatic cleavage into smaller peptide fragments these are separated with liquid chromatography. Mass spectrometric detection is then performed in multiple reaction mode (MRM). The method is capable of detecting seven allergens in a single run (milk, egg, soy, peanut, walnut, hazelnut, almond), allowing for a more general screening than risk-based ELISA kits which can detect one allergen at a time only. There is also a clear advantage over PCR as this DNA-based methodology neither detects egg white which contains major allergens nor is PCR very sensitive for milk, another major allergen. An additional significant advantage of the MS method over both, ELISA and PCR, is its specificity since not only one data point is monitored, but eight per (allergenic) target analyte. MS is highly sensitive with limits of detection in the same range as ELISA’s. Both, MS and ELISA methods, can be used for quantitative detection.
Another important issue all allergen detection methods have to deal with is the influence of processing on the target allergen. Methods based on ELISA techniques may be heavily influenced by processing as the antibodies usually target the secondary and tertiary structure of the allergenic protein which can easily be destroyed during processing. The primary structure, which is targeted by MS, usually remains intact. An example is the detection of egg in flour and bread samples with MS and ELISA. The method based on MS was capable of detecting egg in the unprocessed and in the processed samples, whereas three out of four commercially available ELISA kits were only able to detect egg in flour, but not in the processed sample, the bread [Abstract]. And this makes the MS technology much better suitable for processed products which are typically found on supermarkets shelves.
Comparing MS methods to PCR approaches, the most important advantage is that MS is a direct method detecting the allergenic Page 4
In summary all these advantages make MS based methods a very valuable tool for allergen detection with significant advantages of existing methods. Julia Heick & Bert Popping Eurofins
NEWSLETTER
Volume 2 | Issue 2
Spring 2011
News Regulatory Updates - Health Canada Health Canada has taken action to protect Canadians with food allergies, sensitivities and celiac disease by passing a new regulation which strengthens existing labelling regulations. It is estimated that approximately five to six per cent of young children and three to four per cent of adults in Canada suffer from food allergies. Nearly one per cent of the population is affected by celiac disease, for whom the consumption of foods containing gluten can lead to long term complications. The new regulations on food labelling for priority allergens, gluten sources and sulphites were published in the Canada Gazette Part II, on February 16, 2011 and will benefit Canadians in a number of ways. They will provide a clearer ingredient label by: yy prescribing the use of common names for priority allergens (almonds, Brazil nuts, cashews, hazelnuts, macadamia nuts, pecans, pine nuts, pistachios and walnuts, peanuts, sesame seeds, wheat, triticale, eggs, milk, soybeans, crustaceans, shellfish or fish, mustard) yy requiring the declaration of gluten sources from the grains of the following cereal: barley; oats, rye; triticale and wheat) yy including mustard seed in the definition of priority food allergen, and yy ensuring that hydrolyzed plant proteins sources are declared in the list of ingredients.
The new regulations will also require food allergen, gluten sources or sulphites that are ingredients of other ingredients such spices, seasonings and flavours, to be declared when added to pre-packaged foods. Once the regulations are inforce manufacturers will be required to declare the presence of priority allergens, gluten sources and sulphites above 10 ppm in the ingredients list or in a separate “contains” statement. Because of the complexity of the changes and the shelf-life of foods, industry has been given 18 months to implement the new allergen labelling regulations, which will come into force on August 4, 2012. In the meantime, industry continues to be strongly encouraged by both Health Canada and the Canadian Food Inspection Agency to always declare priority allergens, gluten sources and sulphites on the labels of prepackaged foods. Health Canada is confident that this latest step will help vulnerable consumers to have the information they need to make informed decisions about the food they buy. [More information] Jean-Marc Gelinas Health Canada
Mass Spectrometry and Allergens in J. AOAC Int. With the title “Allergen Detection by Mass Spectrometry - The New Way Forward” Guest Editors Bert Popping (Eurofins) and Samuel Benrejeb Godefroy (Health Canada) introduce the Special Section of the Journal of AOAC Int. Five scientific papers from experts in the field are presented in this issue [See introduction] The manuscripts show not only the advantages of mass spectrometry (MS) over other technologies currently used for the detection of food allergens and gluten, but also the fact that quantification and multi-allergen detection are real options and not just potentials. Specific examples of target analytes include milk and gluten. Moreover, one of the manuscripts describe a best practice guideline for allergen detection by MS. These papers have very recently been published on-line and the printed version will become available this summer.
Haraszi et al., Analytical Methods for Detection of Gluten in Food—Method Developments in Support of Food Labeling Legislation [Abstract] Johnson et al., Current Perspectives and Recommendations for the Development of MS Methods for the Determination of Allergens in Foods [Abstract] Monaci et al., Reliable Detection of Milk Allergens in Food Using a HighResolution, Stand-Alone MS [Abstract] Lutter et al. Development and Validation of a Method for the Quantification of Milk Proteins in Food Products Based on LC with MS Detection [Abstract] Heick et al. Application of a LC Tandem MS for the Simultaneous Detection of Seven Allergenic Foods in Flour and Bread and Comparison of the Method with Commercially Available ELISA Test Kits [Abstract] Carmen Diaz-Amigo, Eurofins
September 18 - 21, 2011 Sheraton New Orleans New Orleans, Louisiana www.aoac.org Present your Scientific Work to your colleagues Submit your Poster Take advantage of the AOAC member rates Find out more about AOAC membership benefits online
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NEWSLETTER Volume 2 | Issue 2
Spring 2011
Manufacturer’s Corner
Conferences and Workshops
R-Biopharm® has launched the RIDASCREEN®FAST Milk Test Kit to detect casein and ß-lactoglobulin in equal parts. The Sandwich ELISA has three 10 minute incubations with standards ranging from 2.5 to 67.5 ppm milk powder NIST RM 1549. An extensive validation found a recovery of milk protein in spiked heated and non-heated sample of 97.5 %. For more information visit www.r-biopharm.com
Food Allergen Sessions & AOAC Food Allergen Community Meeting - 125th AOAC Annual Meeting and Exposition
Undeclared milk allergens have been responsible for 43% of FAAN Allergy Alerts so far this year. Improved detection in the laboratory can now be achieved down to 0.2 ppm casein using the AgraQuant® Casein ELISA and in the factory to 2 ppm in products and 400 ng on surfaces using the new AgraStrip® Allergen tests. For more information visit www.romerlabs.com
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New Orleans, Louisiana (USA), September 18-21, 2011 The upcoming AOAC Annual Meeting will include 1 scientific session on international perspectives on reference materials for food allergens and gluten. In addition to this there will be 2 roundtables on validation of allergen sanitation SOP in food establishments and food allergen multi-screening by mass spectrometry. [More information] AOAC International is also inviting to submit posters related to allergens. Submission deadline is June 30. [More Information] As every year , the AOAC Food Allergen Community will hold its annual meeting during the event to discuss ongoing and future projects. The community meeting is public and we welcome anyone who wishes to participate and share information with the rest of the community. Additional information will be provided in the next issues of the Newsletter.
AOAC Food Allergen Community Newsletter You can contribute with articles or news items Submission deadline for the July 2011 issue - June 2, 2011 Send your articles to
[email protected]
Recent Publications
Your suggestions HERE
Zeleny and Schimmel (2010) Towards comparability of ELISA results for peanut proteins in food: a feasibility study. Food Chem. 123(4), 1343-1351 [Abstract]
We welcome suggestions for topics, information about publications, new developments, meetings, etc. Send us an e-mail to
[email protected] or complete the following electronic form: Name: Affiliation: E-mail: Suggestion: [Select from the list] Comment:
Simonato et al. (2011) Immunochemical and mass spectrometry detection of residual proteins in gluten fined red wine. J. Agric. Food Chem. 59(7):3101-10 [Abstract] Monaci et al. (2010) Identification of allergenic milk proteins markers in fined white wines by capillary liquid chromatography-electrospray ionization-tandem mass spectrometry. J. Chromatogr. A 1217(26):4300-5 [Abstract]
SUBMIT The AOAC Food Allergen Community is a forum serving the scientific community working on Food Allergens: The community is aimed 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
[email protected] Find us on Facebook under AOAC Food Allergen Community Page 6
