Determination of Multiclass Multiresidue Mycotoxins in Pet Food

Importance of the Topic

Mycotoxins are toxic fungal metabolites that can contaminate raw ingredients used in dry pet food, posing health risks to companion animals. Routine monitoring of multiple mycotoxin classes is critical for ensuring pet health and meeting regulatory requirements. The complexity of pet food matrices demands robust cleanup and sensitive detection strategies to deliver reliable results.

Study Objectives and Overview

This work aimed to develop and validate a streamlined analytical method for simultaneous quantitation of 21 multiclass mycotoxins in dog and cat dry food. Key goals included:

• Implementing QuEChERS extraction followed by Agilent Captiva EMR Mycotoxins mixed-mode passthrough cleanup
• Comparing method performance against a stable isotope dilution assay (SIDA) and a competitor’s SPE/dSPE workflow
• Assessing recovery, precision, calibration linearity, and matrix cleanup efficiency for routine use in pet food quality control

Methodology and Instrumentation

Sample Preparation:

• Weigh 2 g of finely ground pet food into a 50 mL tube, spike with standards or ISTDs, and vortex
• Add 7.5 mL water with 1% formic acid and 10 mL acetonitrile with 2% formic acid plus QuEChERS EN salts
• Shake on Geno/Grinder (1,500 rpm, 5 min), centrifuge (5,000 rpm, 5 min)
• Transfer 4.5 mL supernatant, dilute with 0.5 mL water, and load onto Captiva EMR Mycotoxins 6 mL cartridge
• Elute by gravity or low pressure, dry sorbent, collect eluent, dilute with water and ISTDs for LC/MS/MS analysis

Instrumentation:

• Agilent 1290 Infinity II LC system with ZORBAX RRHD Eclipse Plus C18 columns (2.1 × 100 mm, 1.8 µm)
• Agilent 6475 triple quadrupole LC/MS/MS with MassHunter Workstation 12.0

Results and Discussion

Matrix Cleanup Efficiency:

• EMR cleanup removed over 90% of fats, pigments, carbohydrates, and other co-extractives compared with SIDA and competitor methods
• LC/Q-TOF background scans showed significantly lower noise in EMR-treated samples

Recovery and Precision:

• Recoveries in cat and dog food ranged 79–134% across three spiking levels, with RSDs ≤15%
• Isotopic dilution using five labeled ISTDs achieved linear calibration (R² >0.99) over 100–500× dynamic range for most mycotoxins
• Higher LOQs and reporting limits were noted for fumonisins and a few compounds due to native background levels

Comparison with Other Methods:

• Procedure time and complexity were reduced versus SIDA (solvent extraction + filtration) and competitor SPE/dSPE workflows
• EMR method produced cleaner extracts and improved sensitivity for low-abundance mycotoxins

Benefits and Practical Applications

Key advantages of the EMR-based workflow include:

• Rapid and reproducible cleanup compatible with high-throughput testing
• Enhanced sensitivity and reduced matrix effects in LC/MS/MS detection
• Simplified sample preparation steps, lowering solvent consumption and labor
• Applicability to a broad range of complex dry feed and food matrices

Future Trends and Potential Applications

Emerging directions to further improve mycotoxin surveillance:

• Integration of automated sample preparation platforms for higher throughput
• Extension to wet and semi-moist pet food matrices and other animal feeds
• Adoption of high-resolution MS and multiplexed assays for expanded mycotoxin panels
• Implementation of real-time monitoring and data analytics for rapid QC decision support

Conclusion

The QuEChERS extraction followed by Agilent Captiva EMR Mycotoxins passthrough cleanup and LC/MS/MS yields a robust, sensitive, and efficient method for multiclass multiresidue mycotoxin analysis in dry pet food. Validation demonstrated excellent recovery, precision, and matrix cleanup, supporting its use in routine pet food safety testing.

References

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