Quantification of Mycotoxins in Food Matrices

Significance of the topic

The control of mycotoxins in food is critical for public health and regulatory compliance. Aflatoxins (B1, B2, G1, G2) and ochratoxin A are potent contaminants in cereals, spices, and processed foodstuffs; they require sensitive, robust analytical methods able to meet low limits of quantitation imposed by EU regulations. Two-dimensional liquid chromatography coupled to triple quadrupole mass spectrometry (2D-LC/TQ) offers an analytical strategy to reduce matrix effects and enable direct analysis of minimally prepared extracts, potentially removing the need for time‑consuming cleanup such as solid-phase extraction or immunoaffinity purification.

Objectives and study overview

This application note demonstrates a 2D-LC/TQ method for quantitative analysis of five mycotoxins (aflatoxins B1, B2, G1, G2, and ochratoxin A) in three food matrices (pasta, brown rice, and cayenne pepper). Key goals were to: validate that direct analysis of dilute extracts after simple solvent extraction meets the EU required LOQ; achieve a short total runtime; and provide a single‑software solution for method control and data acquisition.

Methodology

Sample preparation:

• 5 g of homogenized sample extracted with acetonitrile/water, shaken and centrifuged.
• Aliquot diluted to a final solvent composition of 26/74 (organic/water, v/v) for measurement.
• Matrix-matched calibration prepared from a 50 ng/mL standard in 20/80 acetonitrile/water and diluted to produce ten levels covering 0.01–5 ng/mL.
• Internal standards: stable-isotope labeled analogs (13C7-AFB1, 13C7-AFB2, 13C17-AFG1, 13C17-AFG2, 13C20-OTA) were delivered by injector program prior to injection.

2D-LC strategy:

• Multiple heart-cutting (MHC) approach: time-based cuts from the first-dimension separation are trapped in 40 µL loops and transferred sequentially to a second-dimension column for further separation to minimize coeluting matrix interferences.
• First-dimension column: Agilent Poroshell Phenyl Hexyl, 2.1 × 50 mm, 2.7 µm; gradient elution produced analyte elution within ~8.1 min.
• Second-dimension column: Agilent ZORBAX Eclipse Plus C18, 2.1 × 50 mm, 1.8 µm; analytical gradient per cut with runtime including backwash/equilibration of 1.75 min per cut.
• Total injection cycle time across both dimensions: 14 minutes.
• Active Solvent Modulation (ASM) used to optimize transfer between dimensions.

Mass spectrometry and acquisition:

• Agilent 6495D Triple Quadrupole LC/TQ with Agilent Jet Stream ESI in positive ion mode.
• MRM acquisition with compound-specific transitions, iFunnel optimization and dwell time of 30 ms per transition; fragmentor set to 166 V.
• Data acquisition and 2D control via Agilent MassHunter Acquisition (rev. 12.1) and 2D-LC software; quantitation with MassHunter Quantitative Analysis for QQQ.

Instrumentation used

The 2D-LC/TQ configuration employed for the method:

• Agilent 1290 Infinity II 2D-LC Solution: two high-speed pumps, multisampler with cooler, multicolumn thermostats, valve drives and ASM valve, and two multiple heart-cutting valves with 40 µL loops.
• Columns: Agilent Poroshell Phenyl Hexyl (1D) and Agilent ZORBAX Eclipse Plus C18 (2D).
• Mass spectrometer: Agilent 6495D Triple Quadrupole with Jet Stream ESI.
• Software: Agilent MassHunter Acquisition and 2D-LC software, MassHunter Quantitative Analysis for QQQ.

Main results and discussion

Analytical performance:

• Linearity: Aflatoxins B1, B2, G1 linear from 0.01 to 5 ng/mL; AFG2 and OTA linear from 0.03 to 5 ng/mL. Correlation coefficients (R2) exceeded 0.998 for all analytes.
• Sensitivity and LOQ: The low-level matrix spike of 0.08 ng/mL (equivalent to 0.96 µg/kg) met the LOQ requirements specified by Commission Implementing Regulation (EU) 2023/2782 for all tested matrices and analytes.
• Precision: Seven replicate injections of the 0.08 ng/mL calibration level produced peak area RSDs between 1.89 and 4.53% and retention time RSD ≤ 0.07%, demonstrating method robustness. Low-level spike triplicate injections yielded concentration RSDs of 0.71–8.18%.
• Recovery: Matrix-spike recoveries, with internal standard correction, ranged from 70–120% across all mycotoxins and matrices at both low (0.08 ng/mL) and high (2 ng/mL; 24.01 µg/kg) spike levels.

Practical implications of 2D-LC:

• Multiple heart-cutting reduced matrix interference sufficiently to allow direct injection of diluted extracts, obviating SPE or IAC for the tested matrices.
• The use of ASM and short transfer loops enabled rapid second-dimension separations and a compact overall cycle time (14 min), supporting higher throughput.

Benefits and practical applications

• Regulatory compliance: method sensitivity and validated LOQs align with EU mycotoxin monitoring requirements, enabling routine control in food testing laboratories.
• Operational efficiency: reduced sample preparation lowers consumable costs and labor, while the short runtime increases sample throughput.
• Flexibility: 2D-LC/MHC can be adapted to additional mycotoxins or challenging food matrices by adjusting cut windows and second-dimension chemistry.

Future trends and potential applications

• Broader multiclass screening: expanding 2D-LC/TQ panels to include emerging mycotoxins and other low-level contaminants will enhance food safety surveillance.
• Automation and software integration: improved 2D-LC workflows and intelligent cut selection algorithms will simplify method transfer and reduce operator input.
• Miniaturization and speed: advances in column technology, faster second-dimension separations, and improved interface designs (e.g., optimized ASM) will further reduce cycle times and solvent consumption.
• Hybrid workflows: combining limited cleanup with 2D-LC may offer the best balance for extremely complex matrices or ultra-trace analyte requirements.

Conclusion

The presented 2D-LC/TQ method reliably quantifies aflatoxins B1, B2, G1, G2 and ochratoxin A in pasta, brown rice, and cayenne pepper using direct analysis of simple solvent extracts. The approach meets regulatory LOQs, delivers robust precision and acceptable recoveries (70–120%), and reduces or eliminates extensive cleanup steps while maintaining a short total runtime (14 min). Integrated software control streamlines method setup and acquisition, supporting practical implementation in routine food testing laboratories.

References

1. European Commission. Commission Regulation (EU) 2023/915 of 25 April 2023 on maximum levels of certain contaminants in food. Official Journal of the European Union.
2. Sobieski, T.; et al. Analysis Of Mycotoxins in Food Matrices Using the Agilent Ultivo Triple Quadrupole LC/MS. Agilent Technologies application note, publication 5991-8962EN, 2019.
3. Zhao, L.; Zhao, H. Determination of Multiclass Multiresidue Mycotoxins in Pet Food. Agilent Technologies application note, publication 5994-7471EN, 2024.
4. Carr, P. W.; Stoll, D. R. Two-Dimensional Liquid Chromatography–Principles, Practical Implementation and Applications. Agilent Technologies primer, publication 5991-2359EN, 2015.
5. Agilent InfinityLab 2D-LC Solutions. Two Dimensions for Ultimate Separation Power. Agilent Technologies brochure, publication 5994-4876EN, 2022.
6. European Commission. Commission Implementing Regulation (EU) 2023/2782 of 14 December 2023 laying down methods of sampling and analysis for the control of mycotoxins in food. Official Journal of the European Union.