Development and application of an exact mass LC-MS/MS library for the screening of mycotoxins and fungal metabolites in food and feed

Importance of the topic

Mycotoxins are toxic secondary metabolites produced by various fungi, including Fusarium, Aspergillus and Penicillium species. They pose significant health risks such as hepatotoxicity, mutagenicity and carcinogenicity when contaminating food and feed. The chemical diversity and emergence of masked or modified mycotoxins create analytical challenges, making accurate mass screening essential for food safety and regulatory compliance.

Objectives and study overview

This work aimed to develop an exact-mass high-resolution MS/MS library for over 400 mycotoxins and fungal metabolites, and to establish robust screening workflows for food and feed commodities. Two complementary approaches were evaluated: a classical targeted MS/MS screening and an All-Ions fragmentation strategy for broad, untargeted confirmation.

Methodology and instrumentation

Sample preparation followed a generic extraction without cleanup to avoid bias. Ground and homogenized samples were extracted with acetonitrile:water:acetic acid (79:20:1, v/v/v) for 90 minutes on a rotary shaker. A Zorbax SB-C18 RRHD column (150×2.1 mm, 1.8 μm) was used on an Agilent 1290 Infinity UHPLC coupled to a 6550 iFunnel QTOF. Data were acquired in positive and negative ionization modes, correcting mass axis throughout the run.

Creation of the HR-MS/MS library

A spectral library of ~150 standards was built by flow injection of individual analyte solutions, applying collision energies of 10, 20 and 40 eV. Narrow isolation windows (1.3 amu) and acquisition rates of 5 spectra/s enabled high-quality fragmentation. Fragments were curated via in silico annotation (MassHunter Molecular Structure Correlator) and only explainable ions with exact m/z values were retained.

Screening approaches and data processing

• Classical screening: full-scan MS followed by targeted MS/MS on selected precursors; library matching confirmed analyte identity based on retention time and fragment spectra.
• All-Ions MS/MS: simultaneous acquisition of low- and high-energy channels without precursor isolation. Data processing involved “Find by Formula” database search on the low-energy channel, extraction of fragment ion chromatograms from the high-energy channel, and coelution scoring for confirmation.

Main results and discussion

Validation included 45 representative mycotoxins spiked into maize, hazelnut and wine across three concentration levels. Both approaches achieved high library match scores and eliminated false positives effectively. At spiking levels of 100 µg/kg, most analytes were detected and confirmed. While regulated aflatoxins at lower levels remained challenging, the All-Ions method reduced analysis time (single injection) and supported retrospective data evaluation.

Benefits and practical applications

• Broad analyte coverage—over 400 database entries with ~150 confirmed by MS/MS.
• High confidence in screening and confirmation, minimizing false positives.
• Time-efficient workflow using All-Ions acquisition for one-shot analysis.
• Post-acquisition reprocessing allows detection of emerging or masked mycotoxins without reanalysis.

Future trends and potential applications

Expansion of the library to include emerging and masked mycotoxins will enhance screening scope. Integration with machine-learning algorithms may improve fragment annotation and coelution scoring. Development of quantitative HR-MS/MS methods and automation of data workflows will support routine QA/QC in food and feed safety laboratories.

Conclusion

The combination of UHPLC-QTOF high-resolution MS/MS and an exact mass spectral library provides a powerful tool for the comprehensive screening and confirmation of mycotoxins in complex matrices. The All-Ions approach streamlines analysis and enables retrospective data mining, while the growing library underpins future method development.

References

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