Simultaneous Analysis of Mycotoxins in Dairy Products by Liquid Chromatography - Quadrupole Orbitrap Mass Spectrometry

Importance of the Topic

Mycotoxins are toxic metabolites produced by fungi that can contaminate dairy products, posing serious health risks to consumers and challenges for food safety monitoring. A robust, sensitive, and high-throughput method capable of simultaneously detecting multiple mycotoxins at trace levels is essential for quality control, regulatory compliance, and safeguarding public health.

Objectives and Study Overview

The study aimed to develop and validate a comprehensive liquid chromatography–quadrupole Orbitrap mass spectrometry (LC-Orbitrap MS) approach for the simultaneous analysis of 58 mycotoxins in various dairy matrices. Key goals included:

• Establishing a simple sample preparation workflow suitable for milk, milk beverages, and yogurt.
• Optimizing chromatographic separation and high-resolution mass detection parameters.
• Assessing method performance in terms of recovery, linearity, limits of detection, and precision.
• Demonstrating real-world applicability by analyzing commercial dairy samples.

Methodology and Instrumentation

Sample Preparation:
A modified QuEChERS protocol was employed. Dairy samples were extracted with acetonitrile–water containing 1% acetic acid, magnesium sulfate, and sodium acetate. Cleanup used dispersive solid-phase extraction (d-SPE) with magnesium sulfate, primary-secondary amine (PSA), and C18 sorbents.

Chromatography:
An ultra-high-performance liquid chromatography (UHPLC) system with a Thermo Scientific Accela 1250 pump and Accucore aQ column (100 × 2.1 mm, 2.6 μm) equipped with a guard column was used. Mobile phase A was water and B was methanol, both containing 4 mM ammonium formate and 0.1% formic acid. The flow rate was 300 μL/min with a 5 μL injection.

Mass Spectrometry:
A Thermo Scientific Q Exactive hybrid quadrupole-Orbitrap mass spectrometer with heated electrospray ionization (HESI) operated in positive and negative modes. Full MS scans (resolution 70,000 at m/z 200) were followed by data-dependent MS2 (resolution 17,500) when target precursor ions were detected. Key settings included 3.5 kV spray voltage (positive), 3.0 kV (negative), 320 °C capillary, 350 °C heater, sheath gas 18, auxiliary gas 3, and normalized collision energy of 35%.

Main Results and Discussion

The method achieved extraction recoveries between 86.6% and 113.7% and coefficients of variation below 6.2% across three concentration levels. Calibration was linear (R2 > 0.99) over 0.001–100 μg/kg, with limits of detection ranging from 0.001 to 0.92 μg/kg. Validation parameters such as decision limits (CCα) and detection capabilities (CCβ) were established for each analyte.
The approach was applied to 30 commercial dairy products. Four mycotoxins—aflatoxin M1, ochratoxin A, ochratoxin-α, and fumonisin B1—were detected at trace levels (0.002–0.11 μg/kg) with reproducibility (RSD) below 3.1%. These results demonstrate the method’s suitability for routine dairy safety screening.

Benefits and Practical Applications

• High-throughput multi-target screening for routine QA/QC in dairy laboratories.
• Regulatory compliance via detection at sub-ppb levels.
• Versatile platform adaptable to other food matrices and contaminant classes.
• Reduced solvent and sample consumption through UHPLC and miniaturized workflows.

Future Trends and Applications

Advancements may include integration with automated data-processing pipelines and suspect screening libraries to expand target lists. Coupling Orbitrap-based workflows with ion mobility or enhanced fragmentation techniques could improve isomer separation and structural elucidation. Application to broader food and environmental matrices will further strengthen contamination surveillance.

Conclusion

The validated UHPLC–Q Exactive Orbitrap MS method offers a reliable, sensitive, and efficient solution for simultaneous detection of 58 mycotoxins in dairy products. Its strong analytical performance and adaptability make it a powerful tool for ensuring food safety and supporting regulatory monitoring programs.

Reference

Jia W, Streaty RP, Park J-S, et al. Multi-Mycotoxin Analysis in Dairy Products by Liquid Chromatography Coupled to Quadrupole Orbitrap Mass Spectrometry. Journal of Chromatography A. 2014;1345:107–114.