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

Importance of the Topic

The simultaneous determination of multiple mycotoxins across diverse food matrices addresses critical needs in food safety monitoring and regulatory compliance. A unified analytical workflow reduces time and cost while ensuring accurate detection of regulated and emerging toxins produced by fungi such as Aspergillus, Fusarium and Alternaria.

Goals and Study Overview

This study aimed to develop and validate a streamlined sample preparation protocol combined with a fast 11-minute LC-MS/MS method for the quantitative analysis of 37 mycotoxins, including:

• Five Alternaria toxins
• Six major ergot alkaloids and their epimers
• Other regulated mycotoxins (e.g., aflatoxins, fumonisins, ochratoxin A, trichothecenes)

Four representative food matrices were selected for validation: baby wheat cereal, raw peanut, tomato puree and a blended gluten-free flour.

Methodology

Sample Preparation:

• Weigh 2 g of homogenized matrix into a 50 mL polypropylene tube.
• Fortify at 5, 50 and 200 µg/kg for recovery studies.
• Add 16 mL of extraction solvent (80:20 acetonitrile:water with 0.5% formic acid; omit formic acid for tomato puree).
• Shake at 800 rpm for 20 minutes, centrifuge at 4000 rpm for 5 minutes.
• Evaporate 1 mL aliquot to dryness at 45 °C under nitrogen, reconstitute in 1 mL of 50:50 water:methanol, filter through a 0.2 µm PTFE vial.

Calibration:
Matrix-matched calibration standards covering 0.4–400 µg/kg were prepared from extracted blank matrices, using quadratic (1/x) regression.

Instrumental Setup

System Configuration:

• Waters Xevo TQ-S triple quadrupole mass spectrometer with Acquity UPLC.
• Analytical column: Raptor Biphenyl 2.7 µm, 100 × 2.1 mm i.d., or inert Biphenyl equivalent.
• Guard column: Biphenyl EXP 5 mm × 2.1 mm i.d.
• Mobile phase A: 0.05% formic acid in water; B: 0.05% formic acid in methanol.
• Gradient: 25% B to 100% B over 9 minutes, total cycle 11 minutes.
• Flow: 0.4 mL/min, column temperature 60 °C, injection volume 5 µL.
• Ionization: Electrospray positive mode, scheduled MRM transitions for each analyte.

Main Results and Discussion

Chromatography and Sensitivity:

• Complete separation of ergot alkaloid epimers within an 11-minute cycle.
• Use of an inert-coated Biphenyl column mitigated non-specific binding (NSB) on metal surfaces, improving peak symmetry and sensitivity by over 50% for metal-sensitive analytes (e.g., fumonisins, tenuazonic acid).

Linearity and Detection Limits:

• Calibration curves for all compounds exhibited r²>0.997 over 0.4–400 µg/kg.
• Most analytes were quantifiable across the full range; some matrix-specific limitations observed (e.g., citrinin in solid matrices, nivalenol in baby cereal).

Accuracy and Precision:

• Recovery ranged from 70–120% for all mycotoxins except citrinin in solid samples.
• Relative standard deviations were below 12% across three fortification levels and nine replicates.

Benefits and Practical Applications

This workflow offers:

• Rapid 11-minute analysis for broad mycotoxin screening.
• Simple sample extraction without complex cleanup steps.
• Robust quantification using matrix-matched calibration.
• Enhanced performance for metal-sensitive compounds via inert column hardware.

It can be applied in quality control, regulatory testing laboratories and research settings for comprehensive food safety evaluation.

Future Trends and Possibilities

Future developments may include:

• Extension to additional food matrices and complex feed formulations.
• Automation of sample extraction and online cleanup to increase throughput.
• Integration with high-resolution mass spectrometry for non-target screening.
• Further optimization of column coatings and mobile phases to minimize NSB and expand analyte coverage.

Conclusion

A fast, reliable LC-MS/MS method has been established for the simultaneous analysis of 37 regulated and emerging mycotoxins across diverse food matrices. The simplified preparation, short run time and use of inert column hardware deliver accurate, precise and sensitive quantification suitable for modern food safety laboratories.