REGULATED MYCOTOXINS IN CEREAL GRAIN FLOURS: A SIMPLIFIED SAMPLE PREPARATION AND LC-MS/MS METHOD

Importance of the Topic

Contamination of cereal grains by regulated mycotoxins poses significant health risks for consumers and can lead to trade restrictions. Reliable quantitation is essential to ensure compliance with EU regulations and safeguard public health.

Objectives and Study Overview

This work aims to establish a simplified sample preparation and LC-MS/MS method for simultaneous determination of EU-regulated mycotoxins in various cereal flours. The method was validated according to Commission Regulation (EU) 519/2014 and SANTE/12089/2016 using wheat, oats, maize, rice, buckwheat, potato and tapioca flours.

Methodology and Instrumentation

A dilute-and-shoot protocol was developed to reduce sample handling:

• Spike 200 mg powdered flour with isotopic internal standard mix
• Extract with acidified acetonitrile:water (MeCN:H₂O)
• Centrifuge at ~5300 g and collect the supernatant
• Dilute 1:10 with acidified water and inject

Used Instrumentation

• Waters ACQUITY I-Class FL UPLC system
• Waters Xevo TQ-XS triple quadrupole mass spectrometer

Main Results and Discussion

Calibration curves for all analytes exhibited excellent linearity (R² ≥ 0.994) with residuals < 20%. Instrumental limits of detection ranged from 11 to 14 fg on column for aflatoxins and up to 22.5 pg for other mycotoxins. Method LOD and LOQ were confirmed per Eurachem guidelines. Recoveries at three spiking levels ranged from 90 to 115% with RSDr < 10% (n = 3). Mixed flour matrices spiked at LOQ met EU recovery criteria (81–114%). Significant matrix effects (up to >1000% enhancement for ochratoxin A and >30% suppression for nivalenol) validated the use of 13C-labelled internal standards to ensure accurate quantitation and repeatability without matrix-matched calibration.

Benefits and Practical Applications

• Minimized sample preparation time and solvent consumption
• High sensitivity and selectivity enabling compliance monitoring below regulatory limits
• Robust quantitation across diverse cereal flours using solvent calibration curves

Future Trends and Opportunities

Future developments may include extension to masked mycotoxins, integration of automated high-throughput extraction, and application of high-resolution mass spectrometry for non-targeted screening. Advances in isotope-labelling and miniaturized workflows could further enhance analytical efficiency.

Conclusion

The simplified dilute-and-shoot LC-MS/MS method is fit-for-purpose for quantitative analysis of EU-regulated mycotoxins in cereal‐based flours. It delivers excellent sensitivity, reproducibility and operational efficiency suitable for routine QA/QC laboratories.

References

• Commission Regulation (EU) 519/2014 on sampling and performance criteria for T2, HT2 and citrinin, 2014.
• Guidance on identification of mycotoxins in food and feed, SANTE/12089/2016, 2017.
• Eurachem. The Fitness for Purpose of Analytical Methods: A Laboratory Guide to Method Validation and Related Topics, 2014.