Determination of Aflatoxins and Ochratoxin A in a Range of Food Commodities Using VICAM Immunoaffinity Chromatography Clean-up with UPLC-MS/MS

Significance of the Topic

Natural contamination of food commodities with aflatoxins and ochratoxin A poses serious health risks and is strictly regulated worldwide. Reliable, sensitive, and streamlined analytical methods are essential for ensuring compliance with regulatory limits and safeguarding public health. Immunoaffinity chromatography (IAC) coupled with UPLC-MS/MS offers highly selective cleanup, minimizes matrix effects, and simplifies quantitation workflows without the need for matrix-matched standards or isotopically labeled internal standards.

Objectives and Study Overview

This study presents three validated methods for quantifying aflatoxins and ochratoxin A in diverse food matrices:

• Method 1: Aflatoxins in groundnuts, pistachios, and hazelnuts using AflaTest WB IAC cleanup.
• Method 2: Ochratoxin A in roasted coffee beans and cocoa powder using OchraTest WB IAC cleanup.
• Method 3: Simultaneous determination of aflatoxins and ochratoxin A in black pepper using AflaOchra IAC cleanup.

Each method involves solid-liquid extraction, selective IAC trap-and-release cleanup, and UPLC-MS/MS detection, achieving method LOQs as low as 0.050 µg/kg for aflatoxins and 0.4 µg/kg for ochratoxin A.

Methodology and Instrumentation

Sample preparation protocols vary by commodity but share common steps:

• Weigh and homogenize sample; extract with aqueous methanol or acetonitrile mixtures.
• Centrifuge and dilute supernatant; load onto IAC column at controlled flow rate.
• Wash column to remove matrix interferences; elute mycotoxins with methanol.
• Evaporate or dilute eluate; reconstitute for UPLC-MS/MS analysis.

Instrumentation Used

• UPLC System: ACQUITY UPLC I-Class with BEH C18 (2.1 × 100 mm, 1.7 µm) column at 40 °C.
• Mobile Phases: 1 mM ammonium acetate in water with 0.5% acetic acid and 0.1% formic acid; methanol with 0.5% acetic acid and 0.1% formic acid.
• MS System: Xevo TQ-S cronos, ESI+ mode, MRM acquisition, capillary voltage +0.75 kV, desolvation at 600 °C.
• Software: MassLynx v4.2 with TargetLynx XS.

Results and Discussion

All three methods demonstrate excellent linearity across regulatory ranges, with instrument LOQs of 0.013–0.08 ng/mL and corresponding method LOQs of 0.050 µg/kg for aflatoxins and 0.4 µg/kg for ochratoxin A. Recovery studies in spiked samples and certified reference materials yielded 71–108% (mean ~90%) with repeatability (RSDr) typically below 5%. Matrix effect factors ranged from –4% to +13%, confirming minimal ion suppression or enhancement. The IAC cleanup effectively removed co-extractives, as shown by clean chromatograms and negligible background signals.

Benefits and Practical Applications

• High selectivity for targeted mycotoxins in complex food matrices.
• No requirement for matrix-matched calibration or isotopically labeled internal standards.
• Low method LOQs meet stringent regulatory limits, including those for infant foods.
• Robust repeatability (RSDr <5%) and minimal matrix effects (<±13%).
• Streamlined workflow suitable for routine compliance and QA/QC laboratories.

Future Trends and Potential Applications

Advances in antibody development and multiplexed IAC formats may enable simultaneous cleanup of a broader range of mycotoxins. Integration with high-throughput UPLC-MS/MS platforms and miniaturized extraction approaches could further reduce solvent use and analysis time. Application of these methods to emerging commodities and infant formula will support evolving regulatory requirements and food safety monitoring.

Conclusion

The described IAC-UPLC-MS/MS methods for aflatoxins and ochratoxin A in nuts, coffee, cocoa, and black pepper fulfill European performance criteria for trueness, precision, and sensitivity. The approach offers a robust, user-friendly solution for routine mycotoxin surveillance without complex calibration strategies.

Reference

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