In-house validation of an LC-MS method for the multiplexed quantitative determination of total allergenic food in chocolate.

Mass spectrometry has been widely accepted as a confirmatory tool for the sensitive detection of undeclared presence of allergenic ingredients. Multiple methods have been developed so far, achieving different levels of sensitivity and robustness, still lacking harmonization of the analytical validation and impairing comparability of results. In this investigation, a quantitative method has been validated in-house for the determination of six allergenic ingredients (cow's milk, hen's egg, peanut, soybean, hazelnut, and almond) in a chocolate-based matrix. The latter has been produced in a food pilot plant to provide a real and well-characterized matrix for proper assessment of method performance characteristics according to official guidelines. In particular, recent considerations issued by the European Committee for Standardization have been followed to guide a rigorous single-laboratory validation and to feature the main method performance, such as selectivity, linearity, and sensitivity. Synthetic surrogates of the peptide markers have been used both in native and labelled forms in matrix-matched calibration curves as external calibrants and internal standards, respectively. A two-order of magnitude range was investigated, focusing on the low concentration range for proper assessment of the detection and quantification limits (LOD and LOQ) by rigorous calibration approach. Conversion factors for all six allergenic ingredients have been determined for the first time to report the final quantitative information as fraction of total allergenic food protein (TAFP) per mass of food (µgTAFP/gfood), since such a reporting unit is exploitable in allergenic risk assessment plans. The method achieved good sensitivity with LOD values ranging between 0.08 and 0.2 µgTAFP/gfood, for all ingredients besides egg and soybean, whose quantitative markers reported a slightly higher limit (1.1 and 1.2 µgTAFP/gfood, respectively). Different samples of chocolate bar incurred at four defined concentration levels close to the currently available threshold doses have been analyzed to test the quantitative performance of the analytical method, with a proper estimate of the measurement uncertainty from different sources of variability. The sensitivity achieved resulted in compliance with the various threshold doses issued or recommended worldwide.

Multi-Allergen Quantification in Food Using Concatemer-Based Isotope Dilution Mass Spectrometry: An Interlaboratory Study.

BACKGROUND: Food allergen analysis is essential for the development of a risk-based approach for allergen management and labeling. MS has become a method of choice for allergen analysis, even if quantification remains challenging. Moreover, harmonization is still lacking between laboratories, while interlaboratory validation of analytical methods is necessary for such harmonization. OBJECTIVE: This interlaboratory study aimed to evaluate the potential of MS for food allergen detection and quantification using a standard addition quantification strategy and a stable isotope-labeled (SIL) concatemer as an internal standard. METHODS: In-house-produced test material (cookies), blank and incurred with four allergens (egg, milk, peanut, and hazelnut), allergen standards, an internal standard, and the complete methodology (including sample preparation and ultra-HPLC-MS/MS method) were provided to nine laboratories involved in the study. Method sensitivity and selectivity were evaluated with incurred test material and accuracy with spiked test material. Quantification was based on the standard addition strategy using certified reference materials as allergen protein standards and a SIL concatemer as an internal standard. RESULTS: All laboratories were able to detect milk, hazelnut, and peanut in the incurred cookies with sufficient sensitivity to reach the AOAC INTERNATIONAL Standard Method Performance Requirements (SMPR® 2016.002). Egg detection was more complicated due to food processing effects, yet five laboratories reached the sensitivity requirements. Recovery results were laboratory-dependent. Some milk and hazelnut peptides were quantified in agreement with SMPR 2016.002 by all participants. Furthermore, over 90% of the received quantification results agreed with SMPR 2016.002 for method precision. CONCLUSION: The encouraging results of this pioneering interlaboratory study represent an additional step towards harmonization among laboratories testing for allergens. HIGHLIGHTS: In this pioneering interlaboratory study, food allergens were analyzed by MS with characterized incurred and spiked test materials, calibrated with a certified reference material, and a single SIL concatemer used as an internal standard.

Development and Validation of a Quantitative Method for Multiple Allergen Detection in Food Using Concatemer-Based Isotope Dilution Mass Spectrometry.

BACKGROUND: Accurate food labeling is essential to protect allergic consumers. However, allergen contaminations may occur during the whole food production process. Reliable, sensitive, and robust methods for detecting multiple allergens in food are needed. OBJECTIVE: This work aims to develop and validate an LC coupled to tandem mass spectrometry (MS/MS) method for the detection and quantification of hazelnuts, peanuts, milk, and eggs in processed food products. METHODS: In-house-produced incurred test materials, cookies and chocolates, were used for the method development and validation. The quantification was based on the standard addition strategy using qualified reference materials as allergen protein standards and an innovative stable isotope-labeled concatemer as an internal standard. RESULTS: A method targeting 19 allergen-specific peptides was developed and validated in two laboratories, which strengthens its robustness. The AOAC INTERNATIONAL performance requirements for repeatability, intermediate precision, reproducibility, and recovery were reached for at least one peptide per allergen across both matrixes, and quantification limits complied with the action levels of the Food Industry Guide to the Voluntary Incidental Trace Allergen Labelling (VITAL®) Program Version 3.0. CONCLUSION: The combination of incurred test materials, standard addition strategy, and stable isotope-labeled concatemer as an internal standard allowed us to develop and validate a robust method for detecting and quantifying multiple allergens in food with sufficient sensitivity to protect allergic consumers. HIGHLIGHTS: The combination of characterized incurred test material, calibration with certified reference material, a single stable isotope labelled concatemer and cross-lab validation result in the required standardization and harmonization in food allergen detection according to the stakeholders' group to assess the robustness of our method.

Assessing human urinary clomiphene metabolites after consumption of eggs from clomiphene-treated laying hens using chromatographic-mass spectrometric approaches.

The anti-estrogen clomiphene is prohibited in sports at all times. Yet, adverse analytical findings (AAFs) have increased since 2011. This is possibly due to improved analytical sensitivity, but also contamination of food of animal origin needs to be taken into consideration as a potential source of drug exposure. For instance, studies with laying hens that received orally administered clomiphene have shown a significantly increased egg production rate but, as a consequence, eggs were found to incorporate residues of clomiphene. In order to evaluate if the consumption of clomiphene-contaminated eggs can cause an AAF of a doping control sample, eggs obtained from an animal administration study with clomiphene were consumed by human volunteers. Each volunteer ate two eggs, and urine samples were collected and analyzed using routine doping control procedures. Subsequently, additional volunteers received a microdosed clomiphene capsule to compare the excretion profiles. Maximum urinary concentrations of hydroxy-clomiphene (HC) between 80 and 300 pg mL-1 were detected following the consumption of clomiphene-containing eggs, which would constitute AAFs if observed in athletes' doping control samples. In order to support the differentiation of potential routes of drug exposure, a method was developed which allows for the chromatographic separation of (E)-3-, (Z)-3-, (E)-4-, and (Z)-4-HC using a derivatization step. By comparing the peak areas of these metabolites, characteristic relative distribution patterns were found that assist in identifying AAFs resulting from clomiphene ingested via contaminated eggs and, thus, enable to distinguish clomiphene intake via contaminated eggs from the intake of microdoses or therapeutic dosages, e.g. for doping purposes.

Development of incurred chocolate bars and broth powder with six fully characterised food allergens as test materials for food allergen analysis.

The design and production of incurred test materials are critical for the development and validation of methods for food allergen analysis. This is because production and processing conditions, together with the food matrix, can modify allergens affecting their structure, extractability and detectability. For the ThRAll project, which aims to develop a mass spectrometry-based reference method for the simultaneous accurate quantification of six allergenic ingredients in two hard to analyse matrices. Two highly processed matrices, chocolate bars and broth powder, were selected to incur with six allergenic ingredients (egg, milk, peanut, soy, hazelnut and almond) at 2, 4, 10 and 40 mg total allergenic protein/kg food matrix using a pilot-scale food manufacturing plant. The allergenic activity of the ingredients incurred was verified using food-allergic patient serum/plasma IgE, the homogeneity of the incurred matrices verified and their stability at 4 °C assessed over at least 30-month storage using appropriate enzyme-linked immunosorbent assays (ELISA). Allergens were found at all levels from the chocolate bar and were homogenously distributed, apart from peanut and soy which could only be determined above 4 mg total allergenic ingredient protein/kg. The homogeneity assessment was restricted to analysis of soy, milk and peanut for the broth powder but nevertheless demonstrated that the allergens were homogeneously distributed. All the allergens tested were found to be stable in the incurred matrices for at least 30 months demonstrating they are suitable for method development.

Selecting Processing Robust Markers Using High-Resolution Mass Spectrometry for the Detection of Milk in Food Products.

BACKGROUND: Cow's milk allergy is one of the most reported food allergies in Europe. To help patients suffering from food allergies it is important to be able to detect milk in different foods. An analytical method that is gaining interest in the field of allergen detection is ultrahigh performance liquid chromatography-tandem mass spectrometry, where the analyte is a target peptide. When these peptide biomarkers are selected, the effect of food processing should be taken into account to allow a robust detection method. OBJECTIVE: This work aims at identifying such processing stable peptide markers for milk for the ultrahigh performance liquid chromatography-tandem mass spectrometry based detection of food allergens in different food products. METHOD: Milk-incurred food materials that underwent several processing techniques were produced. This was followed by establishing tryptic peptide profiles from each matrix using ultrahigh performance liquid chromatography-high resolution mass spectrometry. RESULTS: A careful comparison of peptide profiles/intensities and the use of specific exclusion criteria resulted in the selection of eight peptide biomarkers suitable for application in ultrahigh performance liquid chromatography-tandem mass spectrometry based milk detection methods. One of these markers is an α-lactalbumin specific peptide, which has been determined to be stable in different incurred materials for the first time. CONCLUSIONS: To our knowledge, this is the first systematic and experimentally based approach for the selection of suitable milk peptide biomarkers robust toward multiple, often applied food processing techniques for milk. Ensuring the exact knowledge of the food processing circumstances by starting from well-defined raw material and using fully controlled settings to produce incurred test material allowed the construction of a peptide database with robust markers. These robust markers can be used for the development of a robust detection method for milk in different food matrixes. HIGHLIGHTS: To facilitate food allergen detection in processed food, processing stable peptide markers for the detection of milk in food products were determined using Ultra-High Performance Liquid Chromatography-High Resolution Mass Spectrometry on well-defined raw materials which were processed in accordance with often used processing techniques.

Determination of colistin in luminal and parietal intestinal matrices of chicken by ultra-high-performance liquid chromatography-tandem mass spectrometry.

Justification for continued use of colistin in veterinary medicine, for example medicated water, relies on pharmacokinetic/pharmacodynamic (PK/PD) studies that require accurate measurement of colistin content in the digestive tract. A method for the detection and quantification of colistin in poultry intestinal material was developed and validated. Colistin is not absorbed after oral administration, and the biophase is the gastrointestinal tract. Extraction of colistin from the matrix was achieved using solid-phase extraction with a methanol:water (1:2; v/v) solution. Polymyxin B was used as an internal standard. Colistin A and colistin B, the main components of colistin, were separated, detected and measured using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). The method was validated for linearity/quadraticity between 1.1 (LOQ) and 56.7 mg/kg. Mean accuracy was between 82.7% and 107.7% with inter- and intra-day precision lower than 13.3% and 15% respectively. Freeze-thaw, long-term and bench storage were validated. Incurred samples following colistin treatment in poultry at the approved clinical dose of 75,000 IU/kg in drinking water and oral gavage were quantifiable and in line with expected intestinal transit times. The method is considered appropriately accurate and precise for the purposes of pharmacokinetic analysis in the gastrointestinal tract.

Evaluation of the discriminatory potential of antibodies created from synthetic peptides derived from wheat, barley, rye and oat gluten.

Celiac disease (CD) is triggered by ingestion of gluten-containing cereals such as wheat, barley, rye and in some cases oat. The only way for affected individuals to avoid symptoms of this condition is to adopt a gluten-free diet. Thus, gluten-free foodstuffs need to be monitored in order to ensure their innocuity. For this purpose, commercial immunoassays based on recognition of defined linear gluten sequences are currently used. These immunoassays are designed to detect or quantify total gluten regardless of the cereal, and often result in over or underestimation of the exact gluten content. In addition, Canadian regulations require a declaration of the source of gluten on the label of prepackaged foods, which cannot be done due to the limitations of existing methods. In this study, the development of new antibodies targeting discrimination of gluten sources was conducted using synthetic peptides as immunization strategy. Fourteen synthetic peptides selected from unique linear amino acid sequences of gluten were bioconjugated to Concholepas concholepas hemocyanin (CCH) as protein carrier, to elicit antibodies in rabbit. The resulting polyclonal antibodies (pAbs) successfully discriminated wheat, barley and oat prolamins during indirect ELISA assessments. pAbs raised against rye synthetic peptides cross-reacted evenly with wheat and rye prolamins but could still be useful to successfully discriminate gluten sources in combination with the other pAbs. Discrimination of gluten sources can be further refined and enhanced by raising monoclonal antibodies using a similar immunization strategy. A methodology capable of discriminating gluten sources, such as the one proposed in this study, could facilitate compliance with Canadian regulations on this matter. This type of discrimination could also complement current immunoassays by settling the issue of over and underestimation of gluten content, thus improving the safety of food intended to CD and wheat-allergic patients.

Multi-Class UHPLC-MS/MS Method for Plant Toxins and Cyanotoxins in Food Supplements and Application for Belgian Market Samples.

The presence of plant toxins and/or cyanotoxins in food supplements implies consumer health risks. Therefore, a targeted ultra-high performance liquid chromatographic-tandem mass spectrometric method to detect/quantify 25 toxins simultaneously in food supplement formulations was developed and validated. Full validation for tablets/powders and secondary validation for a liquid and soft gel capsule indicated that most compounds were efficiently extracted (≥ 75%), while others were only partly extracted (18 - 61%). Trueness was fulfilled (70 - 120%), with some exceptions (mostly at the lowest validation level). Intralaboratory repeatability, intra- and interlaboratory reproducibility values of ≤ 20%, ≤ 25%, and ≤ 25% were obtained for most, respectively. Matrix effects were found to be significant for most compounds. Good sensitivity (µg/kg level) was observed for galegin(e), lycopsamine, lycorine, rubiadin, skimmiamine, and vascin(e), in contrast to helveticoside, lucidin, lucidin-3-primveroside, plumbagin(e), and thujone, which were detected at the mg/kg level. The other compounds were characterized by a sensitivity between 10 to 1000 µg/kg. The validated methodology was applied for 52 food supplements (tablets, capsules, liquids/syrup, etc.) purchased from the Belgian market. In more than 25% of the samples, one or more toxins were detected (concentrations determined using standard addition). Lycopsamine, microcystin LR, solamargine, thujone, and vasicin(e) were the most frequently detected toxins. A clear link between the toxins detected and the plant species on the food supplement ingredient list could not always be established. This generic "dilute-and-shoot" procedure can be used for further research on toxins in food supplements and by extension other plant/algae-based food/feed commodities (herbs, edible flowers, etc.).

Depletion of clomiphene residues in eggs and muscle after oral administration to laying hens.

The selective oestrogen receptor modulator (SERM) clomiphene is therapeutically used to induce ovulation. While prohibited as a doping agent in sports, it is frequently detected in sports drug testing urine samples. Few reports exist on clomiphene's (illicit) use in the farming industry to increase the egg production rate of laying hens, which creates a risk that eggs as well as edible tissue of these hens contain residues of clomiphene. To investigate the potential transfer of clomiphene into eggs and muscle tissue, laying hens were orally administered with clomiphene citrate at 10 mg/day for 28 days. To determine clomiphene residues in eggs, chicken breast and chicken thigh, the target analyte was extracted from homogenised material with acetonitrile and subjected to ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. The test method reached a limit of quantification (LOQ) of 1 µg/kg and was characterised concerning specificity, precision, trueness and linearity. Analyses were performed on whole egg, egg white and yolk separately, and chicken muscle from breast and thigh. Clomiphene was detectable in eggs two days after the beginning of the drug administration period. The drug concentrations increased to 10-20 µg per egg within one week, and after withdrawal of clomiphene, residues decreased after 4 days, but traces of clomiphene were still detectable until the end of the study (14 days after the last administration). In the chicken's muscle tissue, clomiphene levels up to 150 µg/kg (thigh) and 36 µg/kg (breast) were found. Six days after the last dose, tissue clomiphene concentrations fell below the LOQ. Overall, these results underline the concerns that clomiphene may be transferred into animal-derived food and future research will therefore need to focus on assessing and minimising the risk of unintentional adverse analytical findings in doping controls.

Suitability of High-Resolution Mass Spectrometry for Routine Analysis of Small Molecules in Food, Feed and Water for Safety and Authenticity Purposes: A Review.

During the last decade, food, feed and environmental analysis using high-resolution mass spectrometry became increasingly popular. Recent accessibility and technological improvements of this system make it a potential tool for routine laboratory work. However, this kind of instrument is still often considered a research tool. The wide range of potential contaminants and residues that must be monitored, including pesticides, veterinary drugs and natural toxins, is steadily increasing. Thanks to full-scan analysis and the theoretically unlimited number of compounds that can be screened in a single analysis, high-resolution mass spectrometry is particularly well-suited for food, feed and water analysis. This review aims, through a series of relevant selected studies and developed methods dedicated to the different classes of contaminants and residues, to demonstrate that high-resolution mass spectrometry can reach detection levels in compliance with current legislation and is a versatile and appropriate tool for routine testing.

Discovery based high resolution MS/MS analysis for selection of allergen markers in chocolate and broth powder matrices.

Peptide marker identification is an important step in development of a mass spectrometry method for multiple allergen detection, since specificity, robustness and sensitivity of the overall analytical method will depend on the reliability of the proteotypic peptides. As part of the development of a multi-analyte reference method, discovery analysis of two incurred food matrices has been undertaken to select the most reliable peptide markers. Six allergenic ingredients (milk, egg, peanut, soybean, hazelnut, and almond) were incurred into either chocolate or broth powder matrix. Different conditions of protein extraction and purification were tested and the tryptic peptide pools were analysed by untargeted high resolution tandem mass spectrometry and the resulting fragmentation spectra were processed via a commercial software for sequence identification. The analysis performed on incurred foods provides both a prototype effective and straightforward sample preparation protocol and delivers reliable peptides to be included in a standardized selected reaction monitoring method.

Comparative study of concatemer efficiency as an isotope-labelled internal standard for allergen quantification.

Mass spectrometry-based methods coupled with stable isotope dilution have become effective and widely used methods for the detection and quantification of food allergens. Current methods target signature peptides resulting from proteolytic digestion of proteins of the allergenic ingredient. The choice of appropriate stable isotope-labelled internal standard is crucial, given the diversity of encountered food matrices which can affect sample preparation and analysis. We propose the use of concatemer, an artificial and stable isotope-labelled protein composed of several concatenated signature peptides as internal standard. With a comparative analysis of three matrices contaminated with four allergens (egg, milk, peanut, and hazelnut), the concatemer approach was found to offer advantages associated with the use of labelled proteins, ideal but unaffordable, and circumvent certain limitations of traditionally used synthetic peptides as internal standards. Although used in the proteomic field for more than a decade, concatemer strategy has not yet been applied for food analysis.

Effects of chronic exposure to a pharmaceutical mixture on the three-spined stickleback (gasterosteus aculeatus) population dynamics in lotic mesocosms.

Pharmaceutical substances are ubiquitous in the aquatic environment and their concentration levels typically range from ng/L up to several µg/L. Furthermore, as those compounds are designed to be highly biologically active, assessing their impacts on non-target organisms is important. Here, we conducted a mesocosm experiment testing a mixture of five pharmaceuticals (diclofenac, carbamazepine, irbesartan, acetaminophen and naproxen) on fish, three-spined stickleback (Gasterosteus aculeatus). The mixture concentration levels were chosen on the basis of the contamination of the Meuse river in Belgium which had been measured previously during a monitoring campaign undertaken in 2015 and 2016. Three nominal mixture concentration levels were tested: the lowest concentration level mixture was composed by environmentally-relevant concentrations that approximate average realistic values for each pharmaceuticals (Mx1); the two other levels were 10 and 100 times these concentrations. Although no impact on stickleback prey was observed, the mixture significantly impaired the survival of female fish introduced in the mesocosms at the highest treatment level without causing other major differences on fish population structure. Impacts on condition factors of adults and juveniles were also observed at both individual and population levels. Using a modelling approach with an individual-based model coupled to a bioenergetic model (DEB-IBM), we concluded that chronic exposure to environmentally-relevant concentrations of five pharmaceuticals often detected in the rivers did not appear to strongly affect the three-spined stickleback populations. Mechanisms of population regulation may have counteracted the mixture impacts in the mesocosms.

Critical review on proteotypic peptide marker tracing for six allergenic ingredients in incurred foods by mass spectrometry.

Peptide marker identification is one of the most important steps in the development of a mass spectrometry (MS) based method for allergen detection, since the robustness and sensitivity of the overall analytical method will strictly depend on the reliability of the proteotypic peptides tracing for each allergen. The European legislation in place issues the mandatory labelling of fourteen allergenic ingredients whenever used in different food formulations. Among these, six allergenic ingredients, namely milk, egg, peanut, soybean, hazelnut and almond, can be prioritized in light of their higher occurrence in food recalls for undeclared presence with serious risk decision. In this work, we described the results of a comprehensive evaluation of the current literature on MS-based allergen detection aiming at collecting all available information about proteins and peptide markers validated in independent studies for the six allergenic ingredients of interest. The main features of the targeted proteins were commented reviewing all details available about known isoforms and sequence homology particularly in plant-derived allergens. Several critical aspects affecting peptide markers reliability were discussed and according to this evaluation a final short-list of candidate markers was compiled likely to be standardized and implemented in MS methods for allergen analysis.

High-resolution mass spectrometry-based selection of peanut peptide biomarkers considering food processing and market type variation.

To protect allergic patients and guarantee correct food labeling, robust, specific and sensitive detection methods are urgently needed. Mass spectrometry (MS)-based methods could overcome the limitations of current detection techniques. The first step in the development of an MS-based method is the identification of biomarkers, which are, in the case of food allergens, peptides. Here, we implemented a strategy to identify the most salient peptide biomarkers in peanuts. Processed peanut matrices were prepared and analyzed using an untargeted approach via high-resolution MS. More than 300 identified peptides were further filtered using selection criteria to strengthen the analytical performance of a future, routine quantitative method. The resulting 16 peptides are robust to food processing, specific to peanuts, and satisfy sequence-based criteria. The aspect of multiple protein isoforms is also considered in the selection tree, an aspect that is essential for a quantitative method's robustness but seldom, if ever, considered.

Selection of universal peptide biomarkers for the detection of the allergen hazelnut in food trough a comprehensive, high resolution mass spectrometric (HRMS) based approach.

The interest of using LC-MS/MS as a method for detection of allergens in food is growing. In such methods, peptides are used as biomarkers for the detection and quantification of the allergens. The selection of good biomarker peptides is of high importance to develop a specific, universal and sensitive method. Biomarkers should, for example, be robust to food processing. To evaluate robustness, test material incurred with hazelnut having undergone different food processing techniques was produced. Proteins of these materials were extracted, digested and further analyzed using HRMS. After peptide identification, selection was carried out using several criteria such as hazelnut specificity and amino acid composition. Further selection was done by comparing peptide MS intensities in the different food matrices. Only peptides showing processing robustness were retained. Eventually, eight peptides coming from three major hazelnut proteins were selected as the best biomarkers for hazelnut detection in processed foods.

Development of a sensitive polyclonal antibody-based competitive indirect ELISA for determination of citrinin in grain-based foods.

A sensitive competitive indirect enzyme-linked immunosorbent assay (ciELISA) was developed for the detection and quantification of citrinin (CIT) in grain-based food samples. The limit of quantification (IC20) of the established method was 0.10 ± 0.02 ng mL-1, with the limit of detection (IC10) being 0.04 ± 0.007 ng mL-1 in wheat and corn flour matrices with a coefficient of variation (CV) less than 20%. The assay was very specific to CIT and showed no cross-reactivity with other mycotoxins (OTA, T-2 toxin, HT-2 toxin, DON, patulin and zearalenone). In spiked wheat and corn flours, the recoveries ranged from 86.6% to 115.6% with CVs of less than 20%. The effectiveness of this method was verified by participating in a proficiency test (PT) from the Food Analysis Performance Assessment Scheme (FAPAS) 17181 corn flour. A successful z-score (-0.6) for this PT sample showed that the present method is comparable to the instrumental methods used by other laboratories in the PT testing scheme. A small survey of grain-based foods was conducted using this method and CIT was detected in 43% of the samples up to a concentration of 17.7 ng g-1. This method is suitable for sensitive and rapid quantitation of citrinin in wheat and corn matrices.

Detection and Quantification of Allergens in Foods and Minimum Eliciting Doses in Food-Allergic Individuals (ThRAll).

Risk-based approaches to managing allergens in foods are being developed by the food industry and regulatory authorities to support food-allergic consumers to avoid ingestion of their problem food, especially in relation to the traces of unintended allergens. The application of such approaches requires access to good quality data from clinical studies to support identification of levels of allergens in foods that are generally safe for most food-allergic consumers as well as analytical tools that are able to quantify allergenic food protein. The ThRAll project aims to support the application of risk-based approaches to food-allergen management in two ways. First, a harmonized quantitative MS-based prototype reference method will be developed for the detection of multiple food allergens in standardized incurred food matrices. This will be undertaken for cow's milk, hen's egg, peanut, soybean, hazelnut, and almond incurred into two highly processed food matrices, chocolate and broth powder. This activity is complemented by a second objective to support the development and curation of data on oral food challenges, which are used to define thresholds and minimum eliciting doses. This will be achieved through the development of common protocols for collection and curation of data that will be applied to allergenic foods for which there are currently data gaps.

Gluten Analysis in Processed Foodstuffs by a Multi-Allergens and Grain-Specific UHPLC-MS/MS Method: One Method to Detect Them All.

Background: Celiac disease, a complex, long-term autoimmune disorder and gluten intolerance, is estimated to affect from 1 to 5% of the world's population. Objective: As a consequence, to protect gluten-sensitive consumers, the development of reliable analytical methods allowing the detection of gluten in various food products is needed. Methods: Currently, ELISA is probably the most widespread used methodology. The method based on the R5 antibody has received type I status in Codex Alimentarius. However, the ELISA method suffers from some limitations, especially concerning quantification of nonwheat gluten. As a consequence, the development of another complementary methodology such as LC-tandem MS (MS/MS) is considered to be essential. Furthermore, this method could also be used for the simultaneous detection of gluten with other allergens, which will constitute a great additional benefit for producers of "free-from" food products and/or having a management policy integrated for several allergies and/or intolerances. Results: A multi-allergen and grain-specific ultra-HPLC coupled to MS/MS method allowing the identification and the discrimination of gluten from seven cereals, simultaneously with the detection and identification of 10 allergens in only one analysis, is thus described here. Conclusions: This method can be used for the analysis of a broad range of foodstuff matrices containing wheat and/or its derivatives, including cereals, flours, heat-treated and foodstuffs, but also more complex samples having undergone fermentation processes (such as beers).