Multi-mycotoxin Screening and Quantitation Using UHPLC, High Resolution and Accurate Mass

Significance of the Topic

Beer consumption is widespread and cereals used in malting barley can be contaminated by mycotoxins produced by fungi such as Fusarium, Aspergillus, Penicillium and others. Monitoring multiple mycotoxins in beer is essential due to health risks from toxins like deoxynivalenol (DON), its glucoside, aflatoxins, ochratoxins and ergot alkaloids.

Objectives and Study Overview

This study aimed to develop a rapid, robust multi-mycotoxin screening and quantitation method for 32 mycotoxins in beer. It focused on sample preparation simplification, ultra-high performance liquid chromatography (UHPLC) separation, and Orbitrap high-resolution mass spectrometry (HRMS) detection, operating in full-scan mode without internal mass correction.

Methodology

• Sample Preparation:
  4 mL beer degassed ultrasonically, extracted with 16 mL acetonitrile, centrifuged, evaporated and reconstituted in 1 mL MeOH:water (50:50). Filtration with 0.2 µm PVDF microfilters. 13C-labelled DON and zearalenone added as surrogates.
• UHPLC Conditions:
  Column: Hypersil GOLD aQ 100 × 2.1 mm, 1.9 µm; mobile phases: 5 mM ammonium formate in water (A) and methanol (B); flow 0.5 mL/min; gradient 5–95% B over 15 min; temp 40 °C; injection 5 µL.
• MS Conditions (APCI Full Scan):
  Thermo Exactive Orbitrap; capillary ±60 V; vaporizer 250 °C; sheath gas 35; resolution settings 10k–100k FWHM; scan range m/z 100–1000.

Instrumention Used

• Thermo Scientific Accela UHPLC system
• Thermo Scientific Exactive single-stage Orbitrap MS
• Ultrasonic bath, centrifuge, PVDF microfilters

Main Results and Discussion

• APCI vs ESI: APCI improved detection limits for most Fusarium toxins by up to 1200% compared to ESI, except ochratoxin A which ionized better in ESI.
• High resolution (≥50,000 FWHM) allowed narrow mass extraction windows (±5 ppm), reducing matrix interferences and improving mass accuracy (<5 ppm).
• Method validation showed:
  
  • Limits of quantification 0.5–60 µg/L in matrix.
  • Linearity R² > 0.996 across 0.5–250 µg/L.
  • Recoveries 92–124% at spiking levels; RSDs 4–15% mid-level, 11–28% at LCL.
  • Matrix effects 63–112%; matrix-matched calibration applied.

Benefits and Practical Applications

• Simultaneous screening of 32 mycotoxins in beer with minimal preparation supports high-throughput QA/QC laboratories.
• High-resolution full-scan data enable retrospective review for emerging toxins.
• Simple acetonitrile precipitation eliminates need for solid-phase or immunoaffinity cleanup.

Future Trends and Potential Applications

• Integration of HRMS databases and non-targeted screening for new mycotoxin metabolites.
• Automation of sample prep and data processing to boost throughput.
• Extension to other complex food and beverage matrices such as wine, juice, and cereal-based products.

Conclusion

The developed UHPLC-APCI-Orbitrap method offers a reliable, high-throughput approach to quantify multiple mycotoxins in beer with excellent sensitivity and selectivity. High resolving power and narrow extraction windows minimize matrix effects and enable accurate retrospective analysis without internal calibration.

References

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