Sensitive multi-mycotoxins analysis with a single sample preparation by LC-MS/MS (RAFA)

Importance of the topic

Mycotoxins are naturally occurring toxins produced by fungi that can contaminate agricultural commodities, posing serious health risks such as liver damage, nephritis and pulmonary disorders. Regulatory agencies worldwide have established maximum limits for various mycotoxins in food and feed, creating demand for sensitive and reliable analytical methods. A robust multi-mycotoxin assay supports food safety, regulatory compliance and public health protection.

Objectives and overview

The study aimed to develop a single-preparation liquid chromatography–tandem mass spectrometry (LC-MS/MS) method capable of simultaneously quantifying more than a dozen regulated mycotoxins in rice matrix. Key goals included achieving high sensitivity, wide linear range, minimal carryover and compatibility with global regulatory limits.

Methods and sample preparation

The workflow combined a simple solvent extraction with an integrated spin purification step:

• 50 g ground rice flour extracted with 100 mL water/acetonitrile (15/85) and shaken for 30 minutes.
• Filtration through glass-fiber paper, dilution with aqueous acetic acid (Solution A).
• Loading of 1 mL Solution A onto a MycoSpinTM400 purification column, vortex mixing and centrifugation at 10 000 rpm for 2 minutes.
• Collection of purified supernatant for LC-MS/MS analysis.

Used instrumentation

The analysis employed a Shimadzu Nexera X2 UHPLC system coupled to a LCMS-8060 triple quadrupole mass spectrometer. Chromatographic separation was achieved on a Mastro PFP2 column (2.1 mm×150 mm, 3 μm) with a gradient of ammonium acetate and acetic acid in methanol. Electrospray ionization in positive and negative modes was optimized by adjusting drying gas temperature, flow rates and nebulizing gas. A metal-free column and an automated multi-rinse cleaning protocol reduced analyte carryover.

Main results and discussion

The developed method covered 13 mycotoxins including aflatoxins B1, B2, G1, G2; deoxynivalenol, 3- and 15-acetyl-deoxynivalenol; nivalenol; fusarenon-X; fumonisins B2, B3; ochratoxin A; T-2 and HT-2 toxins; zearalenone. Key performance metrics in spiked rice matrix were:

• Linearity coefficients (R2) ≥ 0.998 across broad concentration ranges aligned with global limits.
• Limits of quantification from 0.0125 to 50 µg/kg, below typical regulatory thresholds.
• Sharp, well-resolved MRM chromatograms with retention times reproducible within ±0.05 min.
• Minimal carryover due to metal-free flow path and multi-rinse modes.

Benefits and practical applications

This single-prep LC-MS/MS assay streamlines multi-mycotoxin testing, reducing sample handling and analysis time while maintaining high accuracy and sensitivity. It supports laboratories engaged in quality control, regulatory compliance and routine surveillance of agricultural products.

Future trends and applications

Advancements may include automated sample preparation, integration of high-resolution mass spectrometry for non-target screening and expansion to other complex matrices. Coupling miniaturized extraction devices and enhanced ion sources can further improve throughput and lower detection limits.

Conclusion

The study demonstrates a robust, efficient LC-MS/MS approach for comprehensive mycotoxin profiling in rice. The method achieves sensitive quantification, broad dynamic range and low carryover, aligning with international regulatory requirements and offering a practical tool for food safety laboratories.

References

1. Masayoshi Tamura, Keiko Matsumoto, Jun Watanabe, Naoki Mochizuki, et al. Journal of Separation Science. 2014;37:1552–1560.