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

Significance of the Topic

Mycotoxins are harmful fungal metabolites frequently contaminating staple grains such as wheat, maize and barley. Regulatory bodies worldwide, particularly in Europe, enforce strict limits on mycotoxin levels to protect public health. Reliable sample preparation and sensitive detection methods are essential to ensure food safety and compliance.

Objectives and Study Overview

This application note describes a streamlined solid phase extraction (SPE) workflow using ISOLUTE® Myco columns to isolate multiple mycotoxins from grain matrices prior to LC-MS/MS analysis. The method aims to achieve high recoveries, low limits of quantitation (LOQs) and reproducible performance across wheat flour, whole grain wheat, maize and barley, meeting current European regulatory criteria.

Methodology and Instrumentation

Sample Preparation Procedure:

• Grind 50 g of grain; weigh 5 g for extraction.
• Extract with 20 mL 50% acetonitrile for 30 min; centrifuge and collect 8 mL supernatant.
• Dilute to 40 mL with water; centrifuge again.

SPE Protocol (1 mL/min):

• Condition column (ISOLUTE Myco 60 mg/3 mL) with 2 mL acetonitrile, then 2 mL water.
• Load 3 mL diluted extract by gravity.
• Wash with 3 mL water and 3 mL 10% acetonitrile.
• Dry under vacuum; elute with 2 mL 0.1% formic acid in acetonitrile, then 2 mL methanol.
• Evaporate to dryness at 35 °C; reconstitute in 1 mL 0.1% acetic acid in 20% acetonitrile/methanol, filter (0.2 µm PTFE).

LC-MS/MS Conditions:

• UHPLC: Kinetex XB-C18 (50×2.1 mm, 2.6 µm); gradient 20–100% organic in 10 min; 0.45 mL/min; 40 °C column.
• MS: AB Sciex Triple Quad 5500 with Turbo-V ESI; dual-polarity MRM transitions optimized per analyte; desolvation at 500 °C.

Main Results and Discussion

The method delivered excellent linearity (r² > 0.998) and LOQs down to 0.67 µg/kg for aflatoxins and 13.3 µg/kg for other toxins. Recoveries ranged from 73% to 110% across matrices, with %RSD ≤ 6% for most analytes. Representative extracted ion chromatograms demonstrated clear separation and sensitivity at spiking levels of 5 µg/kg (aflatoxins, ochratoxin A) and 50 µg/kg (others). Performance met or exceeded European Commission performance criteria for precision and recovery.

Benefits and Practical Applications

This single-product SPE protocol simplifies multi-mycotoxin analysis, reducing preparation time and variability. Its robust performance supports routine QA/QC laboratories in regulatory compliance testing of grains and grain-derived products.

Future Trends and Applications

Advances may include automation of SPE workflows, miniaturized sorbent formats and expansion to additional food matrices. Coupling with high-resolution mass spectrometry could further enhance detection of emerging mycotoxins. Development of rapid, on-site screening devices remains an area of active research.

Conclusion

The ISOLUTE® Myco SPE method coupled with LC-MS/MS provides a reliable, efficient and fully validated approach for multi-mycotoxin extraction from grains, fulfilling stringent regulatory requirements and supporting food safety monitoring programs.