**1. Introduction**

Food allergies have increased significantly, affecting between 3 and 4% of adults and at least 6% of children [1]. According to the European Academy of Allergology and Clinical Immunology (EAACI), the prevalence of food allergy has doubled over the past 10 years [2]. After an adverse reaction to a foodstuff, which may range from mild to severe (e.g.

anaphylaxis) [3, 4], allergic patients have to exclude that foodstuff from their diet. Each year in the United States, some 100 deaths are caused by anaphylaxis due to food allergy [5], the main culprits being allergens from peanut, tree nuts, fish, shellfish and milk [6]. Currently, there exist no treatments for food allergy, but clinical trials have been performed to test subcutaneous immunotherapy and oral immunotherapy used to desensitize patients [7]. The high level of adverse reactions observed in these trials has led clinicians to find safer alternative therapies, such as sublingual and epicutaneous immunotherapy. These approaches consist, respectively, in placing allergens (drops or tablets) under the tongue or in using a skin patch to induce sustained protection against anaphylaxis [8]. Although they do not treat allergic disease, they improve considerably the quality of life of highly allergic patients and constitute a real hope for them [9, 10]. The number of potentially allergenic ingredients that must appear on food labels differs in different parts of the world [11]. In Europe, regulation (EU) 1169/2011 imposes indicating the following 14 ingredients: milk, peanut, egg, soybean, fish, crustaceans, cereals containing gluten, tree nuts, celery, lupin, mustard, sesame, molluscs and sulfur dioxide [12]. This regulation fails to take into account the accidental introduction of allergens during production, transportation or storage, even though allergens introduced in this manner can trigger severe reactions [13–15]. To protect food consumers, the industry has widely used precautionary allergen labelling (PAL) (i.e. statements such as 'may contain', 'may contain traces of…') [16]. Yet, the lack of correlation between the presence of allergens and precautionary labelling has led customers to lose trust in food labels [17–20]. In a study of food product recalls over a four-year period in the European Union, the United States, Canada, Hong Kong, Australia and New Zealand, 42–90% of the recalls, depending on the country, were justified by the presence of allergens not indicated on the label [21]. Between 2007 and 2012, the Food and Drug Administration (FDA) recalled 732 products because of allergen contaminations [22] and allergic reactions are due to five foods: milk, egg, peanuts, wheat and soybean (**Figure 1**).

The distribution of these recalls in the European Union, reported in **Figure 2**, shows that the products most commonly involved in food recalls are cereals and bakery products.

**Figure 1.** Number of food recalls per allergen category in the United States, Europe, Canada, and Australia between 2012 and 2015 [23–26].

anaphylaxis) [3, 4], allergic patients have to exclude that foodstuff from their diet. Each year in the United States, some 100 deaths are caused by anaphylaxis due to food allergy [5], the main culprits being allergens from peanut, tree nuts, fish, shellfish and milk [6]. Currently, there exist no treatments for food allergy, but clinical trials have been performed to test subcutaneous immunotherapy and oral immunotherapy used to desensitize patients [7]. The high level of adverse reactions observed in these trials has led clinicians to find safer alternative therapies, such as sublingual and epicutaneous immunotherapy. These approaches consist, respectively, in placing allergens (drops or tablets) under the tongue or in using a skin patch to induce sustained protection against anaphylaxis [8]. Although they do not treat allergic disease, they improve considerably the quality of life of highly allergic patients and constitute a real hope for them [9, 10]. The number of potentially allergenic ingredients that must appear on food labels differs in different parts of the world [11]. In Europe, regulation (EU) 1169/2011 imposes indicating the following 14 ingredients: milk, peanut, egg, soybean, fish, crustaceans, cereals containing gluten, tree nuts, celery, lupin, mustard, sesame, molluscs and sulfur dioxide [12]. This regulation fails to take into account the accidental introduction of allergens during production, transportation or storage, even though allergens introduced in this manner can trigger severe reactions [13–15]. To protect food consumers, the industry has widely used precautionary allergen labelling (PAL) (i.e. statements such as 'may contain', 'may contain traces of…') [16]. Yet, the lack of correlation between the presence of allergens and precautionary labelling has led customers to lose trust in food labels [17–20]. In a study of food product recalls over a four-year period in the European Union, the United States, Canada, Hong Kong, Australia and New Zealand, 42–90% of the recalls, depending on the country, were justified by the presence of allergens not indicated on the label [21]. Between 2007 and 2012, the Food and Drug Administration (FDA) recalled 732 products because of allergen contaminations [22] and allergic reactions

are due to five foods: milk, egg, peanuts, wheat and soybean (**Figure 1**).

The distribution of these recalls in the European Union, reported in **Figure 2**, shows that the

**Figure 1.** Number of food recalls per allergen category in the United States, Europe, Canada, and Australia between 2012

and 2015 [23–26].

8 Allergen

products most commonly involved in food recalls are cereals and bakery products.

**Figure 2.** Percentage distribution of food allergen recalls in the European Union (according to the Rapid Alert System for Food and Feed) [24].

The widespread use of PAL can be explained by the lack of regulatory thresholds and the complexity of food allergen management through the supply chain. To counter this lack, the Voluntary Incidental Trace Allergen Labelling (VITAL) system has been developed in Australia and New Zealand to assist food producers in managing cross-contaminations during food production [27]. This system sets allergen thresholds, based on clinical studies, for the protection of 95–99% of the allergic population. Other referentials for allergen thresholds are the European Academy of Allergy and Clinical Immunology (EAACI) and the Netherlands Food and Consumer Product Safety Authority (NVWA) [28] (**Table 1**).

While the systems just mentioned have no regulatory value, food laboratories use them in evaluating method sensitivity. To obtain a concentration expressed in 'mg proteins per kilogram', a food portion size must be considered in order to compare the analytical method with VITAL thresholds (e.g. a portion size of 50 g, **Table 1**). Yet while VITAL thresholds are expressed in 'mg proteins', laboratories express their results in 'mg ingredients' [29, 30] or may refer either to soluble proteins [31, 32] or total proteins [33] per kg. To compare method performances, a conversion factor must thus be applied (e.g. 25% proteins in whole peanuts [34]). Moreover, VITAL action levels have been determined from clinical studies, mostly on the basis of the allergenicity of raw ingredients, although studies have demonstrated a major decrease in allergenicity in baked products. For example, 50–85% of allergic children are able to tolerate baked egg [35] and a study published in 2015 found 63% to tolerate 3.8 g egg-white protein in baked-egg products [36].

Nevertheless, the prevalence of baked product recalls confirms that laboratories must develop sensitive methods for detecting allergens in processed foodstuffs. The most widely used methods are based on the recognition of allergen proteins by antibodies, notably lateral flow device methods and enzyme-linked immunosorbent assays (ELISAs) [39]. DNA-based meth-


**Table 1.** VITAL (http://allergenbureau.net/vital/), EAACI (http://www.eaaci.org/) and NVWA (https://www.nvwa.nl/) reference doses for different food allergens.

ods such as those exploiting the real-time polymerase chain reaction (PCR) [40] are also used to detect the presence of allergens. Currently, mass spectrometry is becoming an alternative to these methods, as heat-processing induces protein denaturation and structural modifications that might result in non-recognition of the target protein by conformational antibodies and thus in the case of ELISAs, lead to false negatives or at least major underestimation of allergen content [41–44]. Mass spectrometry has the advantage of permitting simultaneous analyses for several allergens in food, including processed food products, with high sensitivity and specificity.

This chapter highlights the important improvements made over the last 10 years in mass spectrometry applied to the development of allergen detection methods. It covers and discusses the mass spectrometry methods currently used to detect and quantify allergens in processed food products, including their validation.
