Extraction of Deoxynivalenol From Grain Using ISOLUTE® Myco prior to LC-MS/MS Analysis

Significance of the Topic

Deoxynivalenol (DON) is a prevalent mycotoxin in cereal grains, posing health risks and strict regulatory limits in food and feed. Reliable extraction and quantification are essential for compliance with European legislation and for ensuring consumer safety.

Objectives and Study Overview

This application note presents a solid phase extraction (SPE) workflow using ISOLUTE® Myco columns to isolate DON from wheat, maize and barley. The study aims to deliver a rapid, reproducible protocol compatible with LC-MS/MS analysis and meeting EU criteria for sensitivity, repeatability and recovery.

Methodology

1. Sample Pre-treatment
   • Grind 50 g of whole grain (wheat, maize or barley) and store at room temperature.
   • Weigh 5 g of ground sample and add 20 mL of water; shake for 30 min.
   • Centrifuge at 3000 g for 10 min, collect 8 mL supernatant, dilute with 32 mL water and re-centrifuge.
2. SPE Cleanup (flow rate 1 mL/min)
   • Condition ISOLUTE Myco column (60 mg/3 mL) with 2 mL acetonitrile, then equilibrate with 2 mL water.
   • Load 3 mL of diluted extract by gravity.
   • Wash with 3 mL water to remove interferents.
   • Elute DON with 3 mL of 10 % acetonitrile in water.
   • Evaporate eluate (TurboVap LV or vacuum concentrator) and reconstitute in 1 mL of 0.1 % acetic acid in 20 % acetonitrile/methanol (1:1 v/v). Filter through 0.2 µm PTFE prior to analysis.

Used Instrumentation

• UHPLC: Shimadzu Nexera system with Kinetex XB-C18 column (50×2.1 mm, 2.6 µm).
• Mobile Phase: A = 1 mM ammonium acetate/0.5 % acetic acid in water; B = same additives in 95 % methanol; 0.45 mL/min gradient over 10 min.
• MS/MS: AB Sciex Triple Quad 5500 with Turbo-V ESI source in negative ion mode; MRM transition 355.1→59.0 for primary quantifier; source at 500 °C and ‑4.5 kV.

Main Results and Discussion

The retention time of DON was approximately 0.7 min. Matrix-matched calibration (2–200 ng/mL on column) yielded excellent linearity (r² > 0.999). The method LOQ was 26.7 µg/kg, with intra-day RSDs below 11 % and recoveries ranging from 67 % to 85 % across wheat, maize and barley, all meeting EU performance criteria.

Benefits and Practical Applications

• Single SPE format simplifies sample preparation for multiple grain types.
• High throughput compatible with routine QA/QC laboratories.
• Robust performance ensures compliance with regulatory limits for DON.

Future Trends and Opportunities

Advancements may include multi-mycotoxin SPE sorbents for simultaneous cleanup, automated high-throughput platforms, and integration of high-resolution MS for broader contaminant screening.

Conclusion

The ISOLUTE Myco SPE procedure combined with LC-MS/MS provides a rapid, sensitive and reproducible method for deoxynivalenol quantification in cereal grains, fulfilling stringent regulatory and laboratory requirements.

References

• Biotage Application Note AN783, 2013.