Simultaneous Determination of Alternaria Toxins, Ergot Alkaloid Epimers, and Other Major Mycotoxins in Various Food Matrices by LC-MS/MS

Importance of the Topic

Food commodities can be contaminated by a diverse range of mycotoxins produced by fungal pathogens, posing health risks and regulatory challenges. A generic multi-mycotoxin analysis enables efficient monitoring across various matrices, supporting food safety and compliance. The simultaneous detection of Alternaria toxins, ergot alkaloid epimers, and other major mycotoxins addresses demand for high-throughput, robust screening methods.

Objectives and Study Overview

This study aimed to develop and validate a simplified and rapid LC-MS/MS workflow for the quantification of 37 regulated and emerging mycotoxins across four representative food matrices: baby wheat cereal, peanut, tomato puree, and blended gluten-free flour. Key goals included achieving full chromatographic separation of ergot alkaloid epimers, maintaining high sensitivity, and mitigating non-specific binding issues.

Methodology and Sample Preparation

Sample extraction involved weighing 2 g of each food sample into polypropylene tubes, fortifying at 5, 50, and 200 µg/kg, and extracting with an 80:20 acetonitrile:water solution acidified with 0.5 % formic acid (excluding tomato puree). After shaking for 20 minutes and centrifugation, extracts were dried under nitrogen at 45 °C, reconstituted in 50:50 water:methanol, and filtered through a 0.2 µm PTFE vial filter. Matrix-matched calibration standards were prepared by spiking blank extracts in the 0.05–50 ng/mL range.

Instrumentation Used

• LC-MS/MS system: Waters Xevo TQ-S with Acquity UPLC
• Analytical columns: Raptor Biphenyl and inert Biphenyl (2.7 µm, 100 mm × 2.1 mm)
• Guard column: Raptor Biphenyl EXP Guard Cartridge
• Mobile phases: 0.05 % formic acid in water (A) and methanol (B)
• Gradient cycle: 11-minute total run, flow rate 0.4 mL/min, column temperature 60 °C
• Detection: ESI+ with scheduled MRM transitions for 38 analytes

Main Results and Discussion

• Chromatographic performance: A fast 11-minute cycle achieved full separation of all target analytes and clear resolution of ergot alkaloid epimer pairs.
• Linearity: Quadratic regression with 1/x weighting provided r2 > 0.997 across 0.4–400 µg/kg, with deviations below 30 %.
• Accuracy and precision: Recoveries of 72–112 % for most mycotoxins (excluding citrinin in solids), RSDs below 12 % over nine replicates. Tomato puree recoveries improved without formic acid for key analytes.
• Non-specific binding: Stainless-steel hardware caused signal loss and peak tailing for metal-sensitive mycotoxins. An inert Biphenyl column coating mitigated NSB, boosting response by over 50 % and ensuring consistent peak shapes.

Benefits and Practical Applications

• Comprehensive coverage: Single method quantifies a broad spectrum of regulated and emerging mycotoxins.
• High throughput: 11-minute run time suitable for routine food safety laboratories and QA/QC operations.
• Improved sensitivity: Inert column hardware reduces NSB, enhancing detection of adsorptive analytes like fumonisins and tenuazonic acid.
• Matrix versatility: Validated across diverse commodities with minimal adjustments.

Future Trends and Opportunities

The integration of inert column technologies with ultra-fast LC-MS/MS will gain traction for metal-sensitive analytes. Further expansion to include additional emerging toxins and implementation of automated sample preparation will elevate laboratory throughput. Coupling with high-resolution mass spectrometry could enhance unknown screening and confirmatory analyses.

Conclusion

This study presents a robust, rapid, and reliable LC-MS/MS workflow for simultaneous determination of 37 mycotoxins across multiple food matrices. The adoption of inert column hardware effectively addresses NSB challenges, delivering improved sensitivity and reproducibility. The method’s simplicity and versatility make it well suited for food safety monitoring and regulatory compliance.