Targeted Peptide Quantitation of Seven Food Allergens in Dark Chocolate Using Triple Quadrupole LC/MS

Importance of the Topic

A precise and sensitive method for detecting trace levels of multiple food allergens in dark chocolate addresses a critical need in public health.
Undeclared allergens can pose severe risks to allergic consumers, while inconsistent precautionary labeling practices worldwide cause confusion.
Robust analytical techniques support evidence-based risk assessment and enforcement of labeling regulations, enhancing consumer safety and industry compliance.

Objectives and Study Overview

This study aimed to develop and validate a rapid sample preparation workflow coupled with triple quadrupole LC/MS for targeted quantitation of seven major food allergens (egg, milk casein, milk whey, soy, peanut, almond, hazelnut, walnut) in dark chocolate.
The goal was to achieve sensitivity levels that meet or exceed recommendations from VITAL 3.0 and AOAC SMPR 2016.002, while demonstrating broad analytical range, high recovery, and good precision.

Methodology and Instrumentation

Sample preparation included:

• Weighing 0.1 g defatted dark chocolate and adding 1 mL 40 mM Tris‐HCl buffer (pH 8).
• Centrifugation at 14,000 × g for 30 min and transferring 500 µL supernatant to a 3 kDa MWCO spin filter.
• Rinsing the filter twice with buffer and reverse spinning to concentrate proteins.
• Reduction with 240 mM DTT at 75 °C for 30 min, followed by alkylation with 500 mM IAA at 25 °C for 30 min.
• Rapid digestion by adding 200 µg trypsin and incubating at 37 °C for 1 h.
• Spiking with isotope-labeled peptide internal standards before LC/MS analysis.

Chromatography and mass spectrometry conditions:

• UHPLC: Agilent 1290 Infinity II with Poroshell 120 EC-C18 column (2.1 × 100 mm, 2.7 µm), 0.4 mL/min flow, 40 °C, gradient from 2% to 98% acetonitrile over 14.6 min.
• MS: Agilent 6495 Triple Quadrupole LC/MS, positive AJS ESI, dynamic MRM, gas temperature 150 °C, drying gas 16 L/min, sheath gas 11 L/min, capillary voltage 3500 V.

Key Results and Discussion

Peptide Marker Selection:

• Two unique peptides and two MRM transitions per allergen were selected from peptide mapping to ensure specificity in the chocolate matrix.
• Optimal separation achieved in a short 10-min LC gradient.

Method Sensitivity and LOQ:

• LOQ values ranged from 2.5 mg/kg (almond) to 10 mg/kg (milk proteins, peanut), meeting or exceeding VITAL 3.0 and AOAC SMPR guidelines.
• Signal-to-noise ratios above 10 at LOQ demonstrated reliable detection in dark chocolate.

Analytical Range and Linearity:

• All allergens showed 3–4 orders of magnitude linear range (R² > 0.99) across typical spiking levels.

Recovery and Precision:

• Recoveries of 75–102% at QC levels of 40 and 200 mg/kg, within AOAC recommended 60–120%.
• Precision (RSD) between 1.5–10.4% across replicates.

Benefits and Practical Applications

This method provides a fast, standardized workflow for simultaneous quantitation of multiple allergens in dark chocolate.
It supports manufacturers and regulators in validating precautionary labeling, ensuring consumer protection and compliance.
The approach can be extended to related confectionery and bakery products.

Future Trends and Potential Applications

The protocol may be adapted for high-throughput automation and expanded allergen panels.
Emerging high-resolution MS and data processing tools will further enhance sensitivity and multiplexing capacity.
Integration with risk assessment models and real-time monitoring can improve food safety management.

Conclusion

A comprehensive sample preparation coupled with triple quadrupole LC/MS enables sensitive, accurate quantitation of seven key food allergens in dark chocolate.
The method meets regulatory performance criteria and offers a versatile platform for routine allergen surveillance in food production.

Instrumentation Used

• Agilent 1290 Infinity II UHPLC system
• Agilent Poroshell 120 EC-C18 column (2.1 × 100 mm, 2.7 µm)
• Agilent 6495 Triple Quadrupole LC/MS (AJS ESI source)

References

1. The Allergen Bureau Limited. Food Industry Guide to the Voluntary Incidental Trace Allergen Labelling (VITAL) Program Version 3.0, 2019.
2. Paez V, et al. AOAC SMPR 2016.002 Standard Method Performance Requirements (SMPRs) for Detection and Quantitation of Selected Food Allergens. J AOAC Int. 2016, 99(4), 1122–1124.