Development of a Multi-Toxin UPLC™ MS/MS Method for 50 Mycotoxins and Tropane Alkaloids in Cereal Commodities

Significance of the Topic

The reliable detection of mycotoxins and tropane alkaloids in cereal-based commodities is critical for food safety, regulatory compliance, and public health. Advances in ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) allow simultaneous quantification of multiple toxins at trace levels, streamlining testing workflows and improving analytical throughput.

Objectives and Study Overview

This study extends a previous method to develop a single UPLC-MS/MS assay for over fifty mycotoxins and plant-derived toxins in cereals. Key aims included achieving ultra-low detection limits, stable quantitation in complex grain matrices, and efficient data processing using modern software tools.

Methodology and Instrumentation

Sample Preparation:

• Homogenize 5 g of cereal flour (wheat, barley, rice, maize).
• Extract with 20 mL of acetonitrile:water:acetic acid (79:20:1, v/v/v) using automated vortex mixing for 10 min.
• Centrifuge at >5000 g for 6 min and dilute 150 µL supernatant with 1350 µL water (1:10), yielding a 40× overall dilution.
• Filter through glass fiber syringe filter prior to analysis.

Solvent and Matrix-Matched Standards:

• Prepare a stock mix of toxins and perform serial dilutions in water:acetonitrile (95:5, v/v).
• Matrix-matched calibrants generated by spiking blank cereal extract.

Chromatography and Detection:

• UPLC separation based on established gradient conditions; void volume ~0.55 min.
• Detection using Xevo TQ-XS triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode with two transitions per analyte.
• Data processing via MS Quan and waters_connect™ quantitation software.

Used Instrumentation:

• Waters UPLC system
• Xevo TQ-XS Mass Spectrometer
• Oasis PRiME HLB SPE cartridges (optional cleanup)

Main Results and Discussion

Sensitivity and Quantification:

• Instrument limits of detection (i-LOD) reached as low as 0.0003 ng/mL for aflatoxins B1, B2, G1, G2, and M1.
• Method limits of quantification (m-LOQ) achieved 0.1 µg/kg for aflatoxins; ≤1 µg/kg for ochratoxin A, sterigmatocystin, beauvericin, enniatins, atropine, scopolamine, and roquefortine C; ≤10 µg/kg for most others, except nivalenol, zearalenone, and penitrem A.

Chromatographic Performance:

• All analytes (except highly polar moniliformin) exhibited stable retention times (±0.03 min) and clear baseline separation.
• Calibration curves covered three orders of magnitude with coefficients of determination (R2) >0.99 and residuals <20%.

Moniliformin requires a HILIC approach for confirmation due to its strong polarity and early elution.

Benefits and Practical Applications

This multi-toxin UPLC-MS/MS method offers:

• Comprehensive screening of regulated, emerging, and masked mycotoxins plus tropane alkaloids in cereals.
• High sensitivity allowing significant extract dilution and reduction of matrix effects.
• Streamlined “dilute-and-shoot” sample prep, minimizing labor and consumables.
• Rapid data review and quantitation via advanced software integration.

Future Trends and Potential Uses

Emerging directions include:

• Integration of 13C-labelled internal standards to further enhance quantitation accuracy.
• Automation of sample preparation steps and SPE clean-up to boost throughput.
• Expansion of the toxin panel to cover additional food matrices and international regulatory portfolios.
• Implementation of high-resolution MS for non-target screening of unknown mycotoxin metabolites.

Conclusion

The developed UPLC-MS/MS assay on the Xevo TQ-XS platform provides a robust, sensitive, and efficient solution for multi-toxin monitoring in cereal commodities. Its simple sample preparation, low detection limits, and broad analyte scope support routine QA/QC, regulatory compliance, and surveillance of mycotoxin contamination.

Reference

SANTE 12089/2016 guidelines on method validation in the context of pesticide and contaminant analysis.