A rapid screening method of mycotoxins in grains by liquid chromatograph tandem mass spectrometry

Significance of the topic

Mycotoxins produced by filamentous fungi contaminate cereal grains worldwide, posing acute and chronic health risks to humans and livestock. Regulatory authorities have established maximum permissible limits for individual mycotoxins in food and feed, driving the need for fast, accurate, and comprehensive screening methods in quality control laboratories.

Objectives and overview of the study

This work presents the development of a rapid, simple screening protocol for simultaneous analysis of eighteen key mycotoxins—including aflatoxins, fumonisins, ochratoxin A, trichothecenes, zearalenone, fusarenon-X, diacetoxyscirpenol, and patulin—in grain samples. The approach integrates a one-step spin-column clean-up with high-speed liquid chromatography–tandem mass spectrometry (LC-MS/MS) to achieve complete analysis within a 15-minute cycle.

Methodology and used instrumentation

Sample preparation employs the MycoSpin™400 multitoxin spin column for fast extraction and clean-up without evaporation steps. A 2.5 g grain sample is extracted with 50 % acetonitrile in water, vortexed, centrifuged, and passed through the spin column to yield purified extract in under five minutes.

• UHPLC system: Nexera™ X2 equipped with a 150 × 2.1 mm, 3 µm pentafluorophenyl (PFP) column.
• Mobile phases: 10 mM ammonium acetate/water (A) and methanol with 2 % acetic acid (B); gradient elution over 15 min at 0.4 mL/min, column at 40 °C.
• Mass spectrometer: Shimadzu LCMS-8050 triple quadrupole with ESI in positive/negative fast polarity-switching MRM mode.
• Autosampler rinse: multi-step internal and external needle washes to minimize carryover.

Main results and discussion

Baseline separation of all eighteen mycotoxins was achieved in a single 15-minute run. Recovery studies revealed that 50 % acetonitrile provided superior extraction of fumonisins compared to higher organic content. Significant matrix effects remained in wheat, corn, peanut, and almond extracts, so a standard addition calibration was employed.

• MRM transitions were optimized for each analyte, achieving method detection limits consistent with EC 1881/2006 requirements.
• Recoveries in wheat and corn ranged from 60 % to 149 % with RSDs below 20 %, and calibration curves showed R2 values above 0.997.

Benefits and practical applications

The combined spin-column cleanup and high-speed LC-MS/MS workflow offers:

• Rapid throughput with 15 min analysis time per sample.
• Minimal sample handling and solvent consumption.
• Simultaneous detection of diverse mycotoxin classes in a single run.
• Sufficient sensitivity and precision for regulatory compliance testing.

Future trends and potential applications

Further improvements may include evaluation of multifunctional and ion-exchange clean-up cartridges to reduce matrix interferences, incorporation of isotopically labeled internal standards for absolute quantification, and automation of sample preparation. Expansion to other commodities and integration with high-resolution mass spectrometry could broaden applicability.

Conclusion

A robust, high-throughput LC-MS/MS screening method for eighteen mycotoxins in grains has been established. The use of MycoSpin™400 spin-column clean-up combined with Shimadzu’s LCMS-8050 and Nexera X2 UHPLC provides effective separation, sensitivity, and accuracy suitable for routine QA/QC and regulatory monitoring.

References

• No external literature references were provided in the source document.