Simultaneous analysis of major allergens in food matrices by high sensitive mass spectrometer

Importance of the topic

Food allergies affect millions worldwide and can provoke severe immune reactions ranging from mild itching to life-threatening anaphylaxis. Reliable detection of allergenic proteins in complex food products is essential for consumer safety and regulatory compliance. Traditional immunoassays and PCR methods face cross-reactivity and specificity limitations, highlighting the need for advanced analytical techniques.

Objectives and study overview

This study aimed to develop and validate a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for simultaneous detection of major allergens from eight regulated food groups (milk, eggs, fish, crustacean shellfish, tree nuts, peanuts, wheat, and soybeans). Using a high-sensitivity triple quadrupole mass spectrometer, the method targets 31 signature peptides derived from 13 allergenic proteins to achieve high selectivity and sensitivity in various food matrices.

Materials and methods

Sample preparation involved grinding commercial food products into fine powders, defatting with hexane, and extracting proteins in Tris-HCl buffer with urea and protease inhibitors. Protein extracts (100–250 µg) were denatured, alkylated, and enzymatically digested into peptides, which were desalted by solid-phase extraction and lyophilized prior to analysis.

Analytical methodology and instrumentation

Chromatographic separation was performed on a Shimadzu Nexera X2 UHPLC using a Shim-pack XR-ODS III column with a 0.1% formic acid/acetonitrile gradient at 0.5 mL/min. Peptide detection employed a Shimadzu LCMS-8050 triple quadrupole mass spectrometer in positive electrospray ionization mode. Multiple reaction monitoring (MRM) transitions were optimized using Skyline software. Key instrument parameters included:

• Nebulizing gas: 3 L/min
• Heating gas: 20 L/min; drying gas: 5 L/min
• Interface temperature: 250 °C; DL temperature: 150 °C; heat block: 200 °C

Results and discussion

The optimized LC-MS/MS method monitors 150 MRM transitions for 31 peptides from eight allergen groups. All peptides eluted within 6.5 minutes with excellent peak shape and linearity. Validation in various cooked and processed food matrices—including gluten-free bread spiked at 10–20 ppm wheat, beer-battered fish, shrimp dishes, and nut-containing products—demonstrated reliable detection of labeled allergens. Specificity was ensured by selecting peptide sequences absent in common cereal cross-reactants such as barley and rye.

Benefits and practical applications

The developed approach offers significant advantages over ELISA and PCR:

• High multiplexing capacity for simultaneous screening of multiple allergens
• Enhanced analytical specificity by targeting unique peptide markers
• Quantitative performance down to regulatory thresholds (10–20 ppm)
• Applicability to diverse food formats, including processed and heat-treated samples

Future trends and potential applications

Further developments may include expansion of the peptide panel to cover additional allergens, integration with high-resolution mass spectrometry for untargeted screening, and automation of sample preparation for high-throughput food safety testing. Data sharing and spectral libraries will enhance method transferability across laboratories.

Conclusion

This study presents a robust LC-MS/MS platform for simultaneous, sensitive, and specific detection of major food allergens in complex matrices. With regulatory relevance and broad applicability, the method supports improved food allergen surveillance and consumer protection.

Reference

Ogura T. Simultaneous analysis of major allergens in food matrices by high sensitive mass spectrometer. Shimadzu Scientific Instruments, Inc.; 2017. PO-CON1758E.