Multi-Mycotoxin Analysis CrossTOX® Sample Clean-up and Matrix Removal

Significance of the Topic

Multi-mycotoxin contamination in food and feed has become increasingly prevalent, with over 90% of samples containing multiple toxins. Maintaining regulatory compliance and ensuring food safety requires robust analytical workflows capable of detecting diverse mycotoxins in complex matrices such as cereals, nuts, dried fruits and spices. Traditional clean-up methods often struggle with high fat or pigment content, leading to instrument fouling, extended downtime and compromised data quality.

Objectives and Study Overview

This study aimed to develop a unified sample clean-up protocol for the simultaneous determination of at least 16 mycotoxins using a single extraction and clean-up step. The objectives included:

• Evaluating the performance of the CrossTOX® SPE column across various food matrices
• Assessing recoveries and matrix effects for cereals, nuts, dried fruits and spices
• Demonstrating instrument robustness and reduced maintenance through efficient matrix removal

Methodology and Instrumentation

Samples (10–20 g) were homogenized and extracted with acetonitrile/water/acetic acid (84/15/1, v/v/v). An additional n-hexane partition step was applied for high-lipid spices. The supernatant (0.25–3 mL) was passed through the CrossTOX® SPE column, which selectively retained matrix constituents. Cleaned extracts were directly injected into an LC-MS/MS system.

• Extraction solvent: acetonitrile/water/acetic acid (84/15/1)
• Optional n-hexane for spice lipid removal
• CrossTOX® SPE column (P/N 17900)
• LC: UPLC Accucore Biphenyl column (100 × 2.1 mm, 2.6 µm) with guard
• Mobile phase A: water/methanol (98/2) + 1% acetic acid + 5 mM ammonium acetate
• Mobile phase B: water/methanol (2/98) + 1% acetic acid + 5 mM ammonium acetate
• Gradient: 95% A to 5% A (0–5 min), hold, then re-equilibration (total 16 min)
• MS detection: heated ESI, positive and negative modes, two qualifier ions per analyte

Main Results and Discussion

Recovery studies across cereals (spelt, rice, wheat, oats, corn) yielded 73–127% for 16 mycotoxins, demonstrating consistent performance. Nut matrices (peanut, hazelnut, walnut, pistachio, almond) showed recoveries of 65–118%, while raisins achieved 84–109%. Spice tests (bell pepper, ginger, nutmeg, turmeric) indicated acceptable recoveries (73–146%) despite high pigment loads. Visual and LC-MS/MS data confirmed effective removal of dyes, fats and other interferences, with negligible backpressure buildup and stable signal sensitivity after 150 injections.

Benefits and Practical Applications

The integrated CrossTOX® approach offers:

• A single clean-up protocol for diverse matrices
• Reduced sample preparation time and solvent use
• Enhanced LC-MS/MS robustness and extended column lifetime
• Improved throughput by minimizing instrument downtime

Future Trends and Possibilities

Advances may include automation of CrossTOX® clean-up on online SPE platforms, expansion to emerging mycotoxins and integration with high-resolution mass spectrometry. Broader adoption could streamline regulatory testing and support rapid screening in food safety laboratories.

Conclusion

The CrossTOX® SPE column enables efficient removal of matrix interferences while delivering reliable recoveries for multi-mycotoxin analysis across a wide range of food and feed matrices. Implementation of this protocol enhances analytical throughput, data quality and instrument longevity.

References

1. LCTech GmbH (2020) Application Note AN0042: Multi-Mycotoxin Analysis CrossTOX® Sample Clean-up and Matrix Removal