A quantitative LC-MS/MS method for 15 mycotoxins in corn-based animal feed

Importance of the Topic

Mycotoxins are toxic secondary metabolites produced by fungi that commonly contaminate corn and other feed grains. Ingestion of contaminated feed can lead to severe health issues in livestock, economic losses in animal production, and potential residues in animal-derived products. Routine and accurate quantitation of multiple mycotoxins in corn-based animal feed is therefore critical for veterinary diagnostics, food safety, and quality control in the agricultural industry.

Study Objectives and Overview

This study aimed to develop and validate a rapid, multi-class LC-MS/MS method for the simultaneous quantitation of 15 major mycotoxins in corn-based animal feed. The targeted analytes encompass trichothecenes, aflatoxins, fumonisins, zearalenones, and ochratoxin A. A simple dilute-and-shoot sample preparation was combined with liquid-liquid extraction to streamline analysis time while maintaining high accuracy and precision.

Methodology and Instrumentation

Sample Preparation and Extraction:

• Ground corn matrix was spiked with internal standards and analyte standards across an eight-point calibration range and quality control (QC) levels.
• Liquid-liquid extraction with 70:30 methanol/water (0.1% formic acid) was performed, followed by centrifugation and a 1:5 dilution of the supernatant.
• Extracts were transferred to amber LC vials for direct injection.

Chromatography and Mass Spectrometry:

• LC: Thermo Scientific Vanquish Flex binary system with Hypersil GOLD aQ column (100 x 2.1 mm, 1.9 µm) at 40 °C.
• Gradient from 0% to 100% organic mobile phase over 8 minutes at 0.3 mL/min, total run time 12 minutes.
• MS: Thermo Scientific TSQ Altis triple quadrupole operated in timed-SRM mode with both positive and negative electrospray ionization.
• Source parameters: positive spray voltage 3500 V, negative spray 2000 V, sheath gas 42, auxiliary gas 17, vaporizer 300 °C.
• Resolution: Q1/Q3 at 0.7 FWHM, cycle time 0.4 s.

Key Results and Discussion

Calibration and Sensitivity:

• All analytes exhibited excellent linearity (R² ≥ 0.983) over their respective ranges, with limits of quantitation from 0.5 to 75 ng/g.
• Representative calibration curves (aflatoxin B1, T-2 toxin, fumonisin B1, deoxynivalenol) showed consistent weighting (1/x) and minimal bias at low levels.

Recovery and Matrix Effects:

• Average analyte recoveries ranged from 86% to 100% across matrices.
• Signal suppression was observed for most analytes (matrix effects 31–66%), while fumonisins showed enhancement (up to 173%).

Accuracy and Precision:

• Intra- and inter-day bias for QC levels remained below 15% for all compounds, except minor deviations at the LLOQ for certain analytes, still within ±20%.
• Precision (CV) was ≤15% for all analytes at low, medium, and high QC levels, demonstrating method robustness (N=15 replicates).

Benefits and Practical Applications

This validated LC-MS/MS workflow offers:

• High throughput with a 12-minute run time per sample and minimal sample preparation steps.
• Simultaneous quantitation of 15 veterinary-relevant mycotoxins.
• Reliable performance in complex feed matrices, supporting veterinary diagnostics, feed quality assurance, and regulatory compliance.

Future Trends and Potential Applications

Advancements may include:

• Integration of high-resolution mass spectrometry for broader screening of unknown or emerging mycotoxins.
• Automation of sample preparation to further increase throughput.
• Expansion to multi-matrix methods covering other feedstuffs and finished animal diets.
• Use of isotopically labeled standards to improve quantitation in highly variable matrices.

Conclusion

A streamlined LC-MS/MS method was successfully developed and validated for rapid, accurate quantitation of 15 mycotoxins in corn-based animal feed. The approach demonstrates excellent sensitivity, precision, and robustness, meeting the demands of veterinary diagnostic laboratories and feed quality control programs.

Reference

1. Cheeke, P. R.; Natural toxicants in feeds, forages, and poisonous plants, 2nd ed.; Interstate Publishers: Danville, IL, 1998; pp 87–136.
2. Osweiler, G. D.; Mycotoxins: Contemporary Issues of Food Animal Health and Productivity. Veterinary Clinics of North America: Food Animal Practice 2000, 16(3), 511–530, DOI 10.1016/S0749-0720(15)30084-0.
3. Klaassen, C. D.; Casarett and Doull’s Toxicology: The Basic Science of Poisons, 8th ed.; McGraw-Hill: New York, 2013; pp 1139.