Evaluation of the Performance of a Simple Method for Regulated Mycotoxins in Cereals by LC-MS/MS Using an Interlaboratory Study

Importance of the Topic

Mycotoxins are toxic secondary metabolites produced by fungi that commonly contaminate cereal crops during cultivation, storage, and transport. Chronic exposure to regulated mycotoxins—such as aflatoxins, fumonisins, deoxynivalenol, zearalenone, and ochratoxin A—poses serious health risks to humans and animals and can lead to substantial economic losses. Reliable, high-throughput analytical methods are essential to ensure food safety, regulatory compliance, and the protection of public health.

Study Objectives and Overview

This interlaboratory study evaluated a simplified LC-MS/MS approach for simultaneous quantification of the 12 mycotoxins regulated by EU legislation in cereal matrices. Four laboratories across Europe and the USA analyzed two FAPAS maize flour quality control materials in triplicate. The aims were to assess method accuracy, repeatability, and reproducibility under routine testing conditions and to verify compliance with European Commission performance criteria.

Methodology and Instrumentation

• Sample preparation relied on a generic extraction solvent followed by dilution without additional cleanup steps to maintain simplicity and throughput.
• Quantification employed a bracketed, internally standardized calibration using 13C-labeled analogues.
• Each laboratory used the same UPLC column chemistry and gradient conditions to achieve consistent peak shape and retention patterns.

Applied Instrumentation

• ACQUITY UPLC BEH C18, 1.7 µm, 2.1 × 100 mm column
• Xevo TQ-XS Triple Quadrupole Mass Spectrometer
• MassLynx MS data acquisition and processing software
• 13C-labeled internal standards for each target mycotoxin

Main Results and Discussion

• Accuracy (trueness) ranged from 85 % to 113 % compared to assigned FAPAS values.
• Within-laboratory repeatability (RSDr) varied between 3.0 % and 13 %.
• Between-laboratory reproducibility (RSDRL) was between 3.1 % and 23 %, with fumonisins showing the greatest variance.
• No false positives were reported, and retention times and ion ratios met acceptance criteria (<±0.05 min deviation).
• All performance metrics satisfied the European Commission’s prescribed limits for official mycotoxin control.

Benefits and Practical Applications

• The streamlined extraction and dilution protocol reduces sample preparation time and solvent use.
• High sensitivity and selectivity of LC-MS/MS enable detection of multiple mycotoxins in a single run.
• Demonstrated consistency across laboratories supports method adoption for official control and food industry testing.
• Compliance with EU performance standards ensures data are fit for regulatory decision-making.

Future Trends and Potential Applications

• Expansion of the method to additional mycotoxin analogues and emerging fungal metabolites.
• Adaptation to diverse food matrices, including processed and complex cereal products.
• Integration with high-resolution mass spectrometry for non-targeted screening.
• Development of automated sample preparation and data evaluation workflows.
• Application of chemometric and machine-learning tools to enhance data interpretation and QC monitoring.

Conclusion

This interlaboratory study confirms that the simple LC-MS/MS method provides robust, accurate, and precise quantification of EU-regulated mycotoxins in cereal flour. Its ease of implementation and compliance with regulatory performance criteria make it suitable for routine monitoring by official laboratories and food business operators.

References

1. Commission Regulation (EC) No 1881/2006 of 19 December 2006 Setting Maximum Levels for Certain Contaminants in Foodstuffs. Official Journal of the European Union L364, 5–23 (2006).
2. Commission Recommendation 2013/165/EU of 27 March 2013 on the Presence of T-2 and HT-2 Toxin in Cereals and Cereal Products. Official Journal of the European Union L91, 12–15 (2013).
3. Dreolin N.; Stead S. LC-MS/MS Method Development and Validation for the Quantitative Determination of Regulated Mycotoxins in Cereal Grain Flours Using Simplified Sample Preparation Conditions on Xevo TQ-XS. Waters Application Note 720006685EN (2019).
4. European Commission, Document No. SANTE/12089/2016. Guidance Document on Identification of Mycotoxins in Food and Feed.
5. European Commission Regulation No. 519/2014 of 16 May 2014 Amending Regulation (EC) No. 401/2006 as Regards Methods of Sampling of Large Lots, Spice and Food Supplements, Performance Criteria for T-2, HT-2 Toxin and Citrinin and Screening Methods of Analysis. Official Journal of the European Union L147, 29–43 (2014).