A rapid and simple LC-MS/MS method for the quantification of the EU regulated mycotoxins in cereal-based products

Importance of the Topic

The presence of regulated mycotoxins in cereal-based products poses a serious health risk and is strictly controlled by EU legislation. A rapid, reliable method capable of simultaneous quantification of multiple mycotoxins supports food safety monitoring, streamlines laboratory workflows and ensures compliance with maximum permitted levels.

Objectives and Study Overview

This work describes the development and validation of a multi-mycotoxin LC-MS/MS method for wheat flour, extended to various cereal matrices. Key goals included achieving high sensitivity, broad analyte coverage, simplified sample preparation and robust quantification through isotopically labelled internal standards. Method performance was evaluated according to EC Regulation 401/2006 and SANTE guidelines, including an interlaboratory study.

Methodology and Instrumentation

Sample Preparation:

• Weigh 5 g homogenized flour, extract with 20 mL MeCN:H2O (80:20) containing 0.75% acetic acid and 0.2% formic acid.
• Shake or vortex 5 min, centrifuge >5000 g, transfer 50 µL supernatant into vial, dilute with 450 µL water (40× dilution).
• Add 10 µL mixed 13C-labelled internal standard to each vial; filter cloudy solutions through 0.2 µm glass fibre.

Caryover Mitigation:

• Use acidic needle washes (pH <3) with citric acid or EDTA to chelate metal-binding fumonisins.
• Replace stainless-steel injector components with MP35N or Kalrez to reduce adsorption.

Instrumentation Used

• UPLC: ACQUITY UPLC I-Class-FL system
• MS/MS: Xevo TQ-XS
• Column: ACQUITY UPLC BEH C18, 1.7 µm, 2.1×100 mm
• Mobile phases: A) 1 mM ammonium acetate in H2O + 0.5% acetic acid + 0.1% formic acid; B) MeOH + 0.5% acetic acid + 0.1% formic acid
• Column temp: 40 °C; Sample temp: 15 °C; Injection volume: 15 µL
• Needle wash solutions optimized for carryover control

Key Results and Discussion

Linearity and Sensitivity:

• Calibration linear from LOQ to MPLs with R2 = 0.994–1.000; residuals < 20%.
• Method LOQs defined at lowest calibration level; LOD/LOQ confirmed by Eurachem guidelines.

Accuracy and Precision:

• Recoveries 90–115% across three spiking levels; intra-day RSD < 10% (n = 7).

Matrix Effects:

• Signal suppression up to 30% for some analytes; enhancement > 1000% for ochratoxin A.
• Use of isotopic standards effectively compensates for matrix variability.

Interlaboratory Study:

• Four labs analyzed two cereal QC materials in triplicate.
• Trueness 85–113%; within-lab RSD 3–13%; between-lab reproducibility 3.1–23%.

Benefits and Practical Applications

This method leverages ultra-sensitive MS/MS detection to simplify extraction and dilution steps, reducing analysis time and solvent consumption. The incorporation of 13C-labelled internal standards ensures accurate quantitation across different cereal matrices. Laboratories gain a turnkey solution for compliance testing, QA/QC and high-throughput screening of mycotoxins.

Future Trends and Opportunities

Advances may include expanding analyte panels to emerging mycotoxins, integrating automation and robotics for sample prep, employing high-resolution instruments for non-target screening, and applying AI-driven data processing for faster decision-making. Miniaturized flow-injection approaches and ambient ionization may further accelerate routine monitoring.

Conclusion

The validated LC-MS/MS approach provides robust, sensitive, and high-throughput determination of EU-regulated mycotoxins in cereals. Its simplicity, accuracy and interlaboratory consistency make it a valuable tool for food safety laboratories.

Reference

EC Regulation No. 1881/2006
1. B. Magnusson and U. Ornemark (eds.), Eurachem Guide, 2nd ed. (2014). ISBN 978-91-87461-59-0.