Targeted Screening of Fungal and Plant Metabolites in Wheat, Corn, and Animal Feed Using Automated Online Sample Preparation Coupled to Orbitrap LC-MS

Importance of the Topic

Mycotoxin contamination of cereals and animal feed poses significant risks to food safety and public health. Beyond regulated toxins, more than 400 fungal and plant secondary metabolites may be present, including biosynthetic precursors and masked forms. High-resolution mass spectrometry (HRMS) combined with automated online sample preparation enables comprehensive targeted screening of a broad panel of compounds in complex matrices.

Study Objectives and Overview

This work extends an established Orbitrap LC-MS method for Fusarium toxins to a targeted screening of 21 fungal and plant metabolites in wheat, corn, and animal feed. The aim was to integrate automated online cleanup, high-resolution full scan and HCD fragmentation, and database-driven data processing to achieve fast, reliable detection and identification in compliance with current legislation.

Methodology and Instrumentation

Sample Preparation and Clean-Up:

• Weigh 5 g homogenized sample into 50 mL vial
• Extract with 20 mL water (0.1% formic acid)/acetonitrile (43:57) for 45 min
• Filter through 0.2 µm nylon membrane
• Inject 10 µL into automated Transcend TLX-1 TurboFlow system

Chromatography and MS Conditions:

• Online cleanup on TurboFlow Cyclone MCX column coupled to Hypersil GOLD C18 (50×4.6 mm, 5 µm)
• Total run time 18 min with dual-pump gradient (water/0.1% formic acid and methanol/0.1% formic acid)
• Orbitrap Exactive operated in positive/negative full scan (m/z 100–900) at 100 000 resolution and HCD MS2 (m/z 50–500, 35 eV)

Data Processing:

• ExactFinder software with custom database (.cdb) of >600 metabolites
• Identification criteria: accurate mass deviation <5 ppm, retention time tolerance ±2.5%, minimum one fragment ion

Used Instrumentation

• Thermo Scientific Transcend TLX-1 online sample prep system
• Thermo Scientific Exactive Orbitrap HRMS
• TurboFlow Cyclone MCX column, Hypersil GOLD analytical column
• Autoinjector, temperature-controlled tray, precision balances, ultrasonic bath, vortex

Main Results and Discussion

Spiked samples at 250 and 500 µg/kg in corn, wheat, and feed were analyzed in six replicates. Overall identification and confirmation rates were 99%, with an average of 98% confirmed in corn, 97% in wheat, and 100% in feed. Only 1% of targets were not detected, mainly due to matrix interferences or peak shape issues. False positives were absent. Quality control materials further validated compound confirmation and identification across various matrices.

Benefits and Practical Applications of the Method

• High throughput screening of multiple toxins in a single run
• Automated online sample cleanup reduces manual handling and solvent use
• HRMS ensures excellent selectivity and low false positive rate
• Adaptable database approach allows rapid inclusion of new metabolites
• Compliance with regulatory guidelines for screening methods

Future Trends and Potential Applications

Screening scope can expand to emerging mycotoxins and plant metabolites. Integration with advanced data analysis platforms and non-targeted workflows will enhance discovery of novel contaminants. Further automation and miniaturization of sample preparation, along with hybrid MS/MS strategies, may improve sensitivity and throughput for routine QA/QC in food and feed laboratories.

Conclusion

This targeted screening workflow combining automated online cleanup and Orbitrap HRMS delivers rapid, reliable detection of a broad range of fungal and plant metabolites in wheat, corn, and animal feed. The method achieves high identification rates, minimal false positives, and compliance with legislative requirements, offering a versatile tool for routine food safety monitoring.

References

1. European Commission Regulation 1881/2006 setting maximum levels for contaminants in foodstuffs
2. European Commission Regulation 1126/2007 amending limits for Fusarium toxins in maize
3. Commission Recommendation 576/2006 on mycotoxin presence in animal feed
4. Ates E, Mittendorf K, Stroka J, Senyuva H. Quantification of Fusarium mycotoxins in cereals using online cleanup and HRMS. Food Addit Contam A 2012;doi:10.1080/19440049.2012.729162
5. Commission Decision 657/2002 on analytical methods performance and interpretation
6. Guidelines for validation of veterinary medicine screening methods (2010)