Difficulties in detecting and quantifying allergens identified

By Joseph James Whitworth contact

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Difficulties for labs in detecting and quantifying allergens have been looked at by researchers using ultra-high performance liquid chromatography coupled to tandem MS (UHPLC-MS/MS).

They demonstrated the importance of determining method performance in different matrixes under conditions similar to industrial manufacturing to guarantee detection of allergens in food products.

Food allergen detection methods are mostly characterized by sensitivity thresholds in spiked materials and it is hard to relate this method sensitivity to method performances in industry processes.

Problems such as the absence of conversion factors between reporting units and lack of correspondence between analytical methods such as PCR (DNA copies) and ELISAs or MS (ingredients, soluble and total proteins) complicate comparing the performances of methods for allergen detection.

Bottlenecks to developing efficient methods include the lack of regulatory thresholds, delays in emergence of reference materials and guidelines and the need to detect processed allergens.

Ultra-HPLC coupled to tandem MS

Ultra-HPLC coupled to tandem MS was used to analyze highly processed and spiked materials.

The impacts of processing and matrix effects were evaluated and compared for detection of egg, milk, soy and peanut allergens in ice cream (fat), cookie (processed) and tomato sauce (acid) matrixes.

In UHPLC-MS/MS, the sensitivity (LOQ) is defined as the lowest concentration of analyte corresponding to an S/N higher than 10.

They looked at three matrixes (tomato sauce, ice cream and cookie) spiked with 0.5 mg/kg total milk proteins. Sensitivity of the method depended greatly on the matrix used: the SN was 3.2 times as high in ice cream and 8.8 times as high in tomato sauce as in cookie.

“These results show that the matrix effect can be considerable and that foodstuff composition must be taken into account when assessing method performance.”

Researchers said despite efforts of industry, it is hard to achieve complete elimination of cross-contact with allergens during food manufacturing, transport and storage.

To limit the risk of allergy, industry uses precautionary labeling (i.e., “may contain…”), but there are still recalls due to unlabeled allergens.

The lack of correlation between precautionary labeling and presence of allergens leads allergenic people to ignore the labelling and absence of regulatory thresholds does not help producers establish trustworthy labeling.

For global harmonization, reference materials should be tested and used by different food allergen control labs but some such materials are unavailable (cashew, pistachio, Brazil nut and others).

Thresholds have been set by the Allergen Bureau’s Voluntary Incidental Trace Allergen Labeling (VITAL) Program but despite manufacturers’ improvements and emergence of allergen thresholds, it remains necessary to check for possible contaminations using reliable analytical methods.

Gaps between real and quantified allergens

ELISA is most widely used in routine laboratories to detect allergens in food products. However, detecting highly processed allergens at VITAL thresholds is very difficult because of protein modifications and interfering compounds (polyphenols, high fat content, etc).

This limitation has led to development of methods based on ultra-HPLC (UHPLC) coupled to tandem MS (MS/MS) for detection of allergens in products processed at high temperature.

In the ELISA technique, quantification of allergens is based mainly on a single calibration curve done in a solvent or extracted matrix, regardless of food matrix to be analyzed.

This means that matrix effects and thermal processes are usually not taken into account and leads to gaps between real and quantified amounts of allergens, said the study.

Researchers found that for main egg, milk, soy and peanut peptides, the use of labeled peptides does not correct for matrix effects linked to the extraction and digestion steps, but it does correct for matrix effects, purification and UHPLC-MS/MS analysis.

Results supported the use of labeled peptides as internal standards, but correcting for effects during extraction/digestion seemed crucial to allowing the use of a single calibration curve to quantify an allergen in various foodstuffs.

Source: Journal of AOAC International​, Volume 100, Number 4, July-August 2017, pp. 1126-1130(5)

“Highlight on Bottlenecks in Food Allergen Analysis: Detection and Quantification by Mass Spectrometry”

Authors: Mélanie Planque; Thierry Arnould; Patricia Renard; Philippe Delahaut; Marc Dieu; Nathlie Gillard

Related topics: HACCP, R&D

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