Simultaneous determination of 16 mycotoxins in cereals using an Agilent Triple Quadrupole LC/MS system and e-Method

Simultaneous Determination of 16 Mycotoxins in Cereals Using LC-MS/MS

Importance of the Topic

Mycotoxins are diverse toxic metabolites produced by fungal contamination of cereal grains. Chronic exposure can cause carcinogenic, teratogenic, and mutagenic effects in humans and animals. Monitoring multiple mycotoxins in staple crops like wheat and maize is essential for food safety, regulatory compliance, and protecting public health. Rapid, accurate multi-residue testing supports high-throughput screening in quality control laboratories.

Study Objectives and Overview

This work presents the development and validation of a streamlined liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of 16 common mycotoxins in cereal matrices. The method leverages a triple quadrupole LC/MS system and isotope-labeled internal standards to achieve high sensitivity, accuracy, and throughput. Key aims included:

• Establishing a single extraction and cleanup procedure without SPE or immunoaffinity steps.
• Achieving baseline separation and accurate detection of isomeric toxins such as 3-Acetyldeoxynivalenol (3-AcDON) and 15-Acetyldeoxynivalenol (15-AcDON).
• Validating linearity, recovery, precision, and applicability to proficiency test samples.

Methodology and Sample Preparation

Crushed wheat and maize (5 g) were extracted with 20 mL acetonitrile–water–acetic acid (70:29:1, v/v/v), shaken for 30 min, and centrifuged. The supernatant was diluted 1:1 with water, centrifuged again, and filtered through a 0.2 µm PTFE membrane. An aliquot was spiked with a mixture of 14 13C-labeled internal standards before injection. Standard calibration solutions spanned low to high µg/L levels to cover regulatory limits.

Chromatographic Conditions

A C18 column (2.1 × 100 mm, 1.8 µm) operated at 35 °C with a gradient of aqueous 1% acetic acid/ammonium acetate and methanol at 0.3 mL/min. Total run time was 22 min, with solvent diverted to waste during initial and final periods to protect the source.

Mass Spectrometry Conditions

An electrospray triple quadrupole LC/MS in MRM mode and rapid polarity switching detected 12 compounds in positive mode and 4 in negative mode. Key parameters (fragmentor voltage, collision energy, retention-time segments) were optimized for each analyte to maximize sensitivity and selectivity.

Instrumentation

The following instrumentation was used:

• Agilent 1290 Infinity II LC system (binary or high-speed pump, autosampler, column compartment).
• Agilent 6460/6470/6490/6495 Triple Quadrupole LC/MS with Jet Stream electrospray ionization.
• Agilent MassHunter software for acquisition and quantitative processing.

Key Results and Discussion

Calibration curves for all 16 mycotoxins exhibited excellent linearity (R2 > 0.996). Spike-recovery tests in wheat and maize at three concentration levels yielded recoveries of 85–120% and RSDs below 8.2%. Six consecutive injections in maize showed stable retention times and peak areas. Analysis of a FAPAS proficiency test sample demonstrated agreement within established uncertainty ranges, confirming method accuracy.

Benefits and Practical Applications

The developed protocol offers:

• Simple sample preparation without time-consuming cleanup steps.
• Rapid analysis within a 22 min run time for 16 analytes.
• High sensitivity and precision using isotope dilution quantitation.
• Plug-and-play e-Method files enabling quick method deployment across laboratories and instrument models.

This approach suits routine monitoring programs in food safety laboratories, grain inspection agencies, and research facilities.

Future Trends and Applications

Advances in ultra-high-performance LC and high-resolution MS may further reduce analysis time and expand target analyte lists. Integration of automated sample preparation and online cleanup can enhance throughput. Emerging mycotoxins and modified forms will require ongoing method adaptation. Wider adoption of standardized e-Methods will facilitate inter-laboratory harmonization and data comparability.

Conclusion

A robust LC-MS/MS method for simultaneous quantification of 16 mycotoxins in cereals has been established and validated. It demonstrates excellent linearity, recovery, precision, and suitability for proficiency testing. The use of isotope-labeled standards and e-Method templates streamlines setup and ensures reproducible results across laboratories, supporting reliable food safety monitoring.

References

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5. Advances in LC-MS for mycotoxin assay in foods. Sci Tech Food Ind. 2016.