Multi-residue analysis of 18 regulated mycotoxins by LC-MS/MS (2)

Significance of the Topic

The contamination of food and feed by Fusarium mycotoxins poses significant health risks to humans and animals. Regulatory bodies in the EU have established strict maximum levels for these toxins to ensure safety. Reliable, sensitive, and rapid multi-residue analytical methods are essential to monitor compliance and protect public health.

Objectives and Study Overview

This study aimed to develop and validate a single LC-MS/MS method capable of quantifying 18 regulated mycotoxins—including aflatoxins, fumonisins, ochratoxin A, and trichothecenes—at or below EU maximum levels. The method targets complete separation of critical regioisomer pairs and achieves a total analysis cycle time of 12.5 minutes.

Materials and Methods

Sample extracts were prepared following validated protocols by Concept Life Sciences. A pentafluorophenyl (PFP) column was evaluated against a C18 phase for chromatographic separation. Various mobile phase additives—including ammonium acetate, formate, fluoride, and acetic acid—were tested. Calibration curves were generated using 13C-labelled internal standards spiked into samples during extraction. All solvents were LC-MS grade.

Used Instrumentation

• UHPLC system: Shimadzu Nexera X2
• Mass spectrometer: Shimadzu LCMS-8060 triple quadrupole with positive/negative polarity switching
• Analytical column: Mastro PFP (100 mm × 2.1 mm, 3 µm)
• Column temperature: 40 °C; Flow rate: 0.4 mL/min
• Mobile phase A: 0.15 mM ammonium fluoride in water
• Mobile phase B: 0.15 mM ammonium fluoride in methanol + 2% acetic acid
• Gradient: 15% B at 0 min to 100% B at 7.5–10 min, return to 15% B by 10.1 min, stop at 12.5 min
• Source temperatures (interface/heat block/dry gas): 300/400/250 °C; Gas flows (nebuliser/heating/drying): 3/10/10 L/min

Main Results and Discussion

Ammonium fluoride significantly enhanced signal intensity in positive ion mode compared to formate and acetic acid additives. The PFP column achieved near-baseline resolution of the 3-AcDON/15-AcDON and FB2/FB3 regioisomer pairs, which could not be resolved on C18. Spiked mixed spice and pepper extracts yielded reproducible quantitation (RSD < 10%, n=12) for aflatoxins B1, B2, G1, G2 (2.5 µg/kg) and ochratoxin A (10 µg/kg).

Benefits and Practical Applications

• Enhanced positive-mode sensitivity for a broad panel of mycotoxins
• Rapid 12.5-minute cycle suitable for high-throughput laboratories
• Effective resolution of critical regioisomers using a PFP phase
• Robust routine quantitation with minimal carryover by adding 2% acetic acid

Future Trends and Potential Applications

Ongoing developments may include novel stationary phases for improved isomer separation, optimized additive combinations for further sensitivity gains, integration with high-resolution MS for non-target screening, and automating sample preparation workflows. Expanding the method to additional emerging mycotoxins will enhance comprehensive food safety monitoring.

Conclusion

This study presents a robust LC-MS/MS workflow that combines ammonium fluoride mobile phases with a PFP column to achieve high sensitivity and reliable separation of 18 regulated mycotoxins in food matrices within a 12.5-minute cycle. The approach meets regulatory requirements and is readily implementable in routine quality control and research laboratories.