Gluten contamination of naturally gluten-free flours and starches used by Canadians with celiac disease.

A large national investigation into the extent of gluten cross-contamination of naturally gluten-free ingredients (flours and starches) sold in Canada was performed. Samples (n = 640) were purchased from eight Canadian cities and via the internet during the period 2010-2012 and analysed for gluten contamination. The results showed that 61 of the 640 (9.5%) samples were contaminated above the Codex-recommended maximum level for gluten-free products (20 mg kg⁻¹) with a range of 5-7995 mg kg⁻¹. For the ingredients that were labelled gluten-free the contamination range (5-141 mg kg⁻¹) and number of samples were lower (3 of 268). This picture was consistent over time, with approximately the same percentage of samples above 20 mg kg⁻¹ in both the initial set and the subsequent lot. Looking at the total mean (composite) contamination for specific ingredients the largest and most consistent contaminations come from higher fibre ingredients such as soy (902 mg kg⁻¹), millet (272 mg kg⁻¹) and buckwheat (153 mg kg⁻¹). Of the naturally gluten-free flours and starches tested that do not contain a gluten-free label, the higher fibre ingredients would constitute the greatest probability of being contaminated with gluten above 20 mg kg⁻¹.

Analysis of glabrous canary seeds by ELISA, mass spectrometry, and Western blotting for the absence of cross-reactivity with major plant food allergens.

Glabrous (hairless) canary seed belongs to the Poaceae (Gramineae) family and could serve as an alternative source of gluten-free cereal grain. In this study, allergenic cross-reactivities between hairless, dehulled canary seeds (Phalaris canariensis) and major allergenic proteins from gluten, soy, peanuts, tree nuts, sesame, and mustard were studied using commercial enzyme-linked immune sorbent assay (ELISA) kits specific for these target allergens. Mass spectrometry (MS) and immunoblotting were further used to assess for the presence of gluten-specific protein fragments. MS results revealed the likely presence of proteins homologous with rice, oat, corn, carrot, tomato, radish, beet, and chickpea. However, no presence of celiac-related gluten fragments from wheat, rye, barley, or their derivatives was found. Immunoblotting studies yielded negative results, further confirming the absence of gluten in the canary seed samples tested. No cross-reactivities were detected between canary seeds and almond, hazelnut, mustard, peanut, sesame, soy, walnut, and gluten using ELISA.

Emerging analytical methods to determine gluten markers in processed foods--method development in support of standard setting.

The availability of analytical methods to detect and determine levels of markers of priority allergens in foods is of the utmost importance to support standard setting initiatives, the development of compliance and enforcement activities, as well as to provide guidance to industry on implementation of quality control practices, ensuring the effectiveness of allergen-related sanitation techniques. This paper describes the development and implementation of a mass-spectrometry-based technique to determine markers for individual sources of gluten in beer products. This methodology was shown to answer the requirements of Health Canada's proposed labeling standard for individual gluten source declaration, in order to achieve its policy objectives (i.e., protection of sensitive consumers, while promoting choice). Minimal sample work-up was required and the results obtained by ELISA were further complemented using the LC-MS/MS method. This paper aims to demonstrate the feasibility of alternative techniques to ELISA-based methodologies to determine allergen and gluten markers in food.

Generation and characterization of polyclonal antibodies against microcystins-Application to immunoassays and immunoaffinity sample preparation prior to analysis by liquid chromatography and UV detection.

Polyclonal antibodies against microcystin-LR (MC-LR), a cyclic heptapeptide toxin, were generated in rabbits using MC-LR-BSA. An enzyme-linked immunosorbent assay (ELISA) was developed for the characterization of the antibodies and their potential use for analytical purposes. The concentration of MC-LR that inhibits 50% of antibody-antigen binding (IC(50)) was 0.5mugL(-1) for the indirect ELISA format and 0.9mugL(-1) for the direct ELISA, using MC-LR-horseradish peroxidase conjugate. The limit of detection corresponding to IC(80) was found to be 0.06mugL(-1), well below the Word Health Organization level for drinking water of 1mugL(-1). The direct competitive ELISA was applied to water samples and was shown useful for screening purposes. The developed anti-microcystin antibodies were immobilized on solid supports for use in selective solid phase extraction (SPE) systems, prior to liquid chromatography (LC) quantification. An immunoaffinity cartridge (IAC), a Sepharose((R))-based cartridge incorporating 2mg of antibodies allowed the selective and quantitative recovery of a mixture of 0.2mug of MCs showing potential use in sample preparation of real matrices. When applied to water and green algae samples, average recoveries from Sepharose((R))-based cartridges were in the range of 86-113% for water samples and 85-92% for blue-green algae samples. Selectivity of the IAC clean-up was proven by comparison with non-specific solid phase extraction using octadecylsilica (ODS) sorbent. Results obtained using LC/UV after IAC clean-up agreed well with results obtained using liquid chromatography and mass spectrometry detection (LC/MS and LC/MS/MS) after SPE-C18 clean-up, allowing therefore to validate the resulting technique.

Regulatory and compliance activities to protect food-allergic consumers in Canada: research in support of standard setting and consumer protection.

An overview is presented of the activities of Health Canada and the Canadian Food Inspection Agency (CFIA) in the area of food allergens. Since the 1990s, changes were made in the Food and Drug Regulations in order to better protect allergic consumers by imposing labeling requirements to clearly identify sources of priority food allergens in prepackaged foods. Policies of application as well as risk management strategies are discussed with some statistics on allergen-related food recalls in Canada for the years 1997--2001. Health Canada's allergen method development program is a pioneering research initiative that was developed in the early 1990s in support of the changing Canadian regulatory environment. The objectives and some of the accomplishments of this program are presented. The development of the Canadian Compendium of Allergen Methodologies under a Web-based application to compile data on evaluated allergen detection methods will provide further support to compliance activities nationally, as well as to the international analytical community in both government and the food industry. Some emerging techniques for the confirmation of results generated by enzyme-linked immunosorbent assays are also discussed.

Liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry of the Alternaria mycotoxins alternariol and alternariol monomethyl ether in fruit juices and beverages.

Alternariol (AOH) and alternariol monomethyl ether (AME) are among the main mycotoxins formed in apples and other fruits infected by Alternaria alternata. For determination of AOH and AME by LC, apple juice and other fruit beverages were cleaned up on C18 and aminopropyl solid-phase extraction columns. Positive and negative ion mass spectra of AOH and AME under electrospray (ESI) and atmospheric pressure chemical ionization (APCI) conditions were obtained. Collision-induced dissociation of the [M+H]+ and [M-H]- ions for both compounds were also studied. The phenolic anions of both compounds are more stable with less fragmentation. In quantitative analysis, negative ion detection also offers lower background and better sensitivity. Sensitive LC-MS and LC-MS-MS confirmatory procedures based on APCI with negative ion detection were applied to confirm the natural occurrence of AOH in nine samples of apple juice and in single samples of some other clear fruit beverages--grape juice, cranberry nectar, raspberry juice, red wine, and prune nectar (which also contained 1.4 ng AME/ml)--at levels of up to 6 ng AOH/ml. Electrospray LC-MS-MS with negative ion detection and in multiple reaction monitoring mode offers higher sensitivity and specificity. Absolute detection was better than 4 pg per injection for both compounds.

Immunochemical-based method for detection of hazelnut proteins in processed foods.

A competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect hazelnut by using polyclonal antibodies generated against a protein extract of roasted hazelnut. No cross-reactivity was observed in tests against 39 commodities, including many common allergens, tree nuts, and legumes. Hazelnut protein standard solutions at 0.45 ng/mL [inhibition concentration (IC80) of the competitive test] were clearly identified by the ELISA. An extraction and quantification method was developed and optimized for chocolate, cookies, breakfast cereals, and ice cream, major food commodities likely to be cross-contaminated with undeclared hazelnut during food processing. No sample cleanup was required when extracts were diluted 10-fold. Recovery results were generated with blank matrixes spiked at 4 levels from 1 to 10 microg/g hazelnut protein. With the developed extraction and sample handling procedure, hazelnut proteins were recovered at 64-83% from chocolate and at 78-97% from other matrixes. A confirmatory technique was developed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western transfer. The developed methods were applied to a small market survey of chocolate products and allowed the identification of undeclared hazelnut in these products.