Development and Validation of on-line SFE-SFC-MS/MS Method for Screening of Aflatoxins B1, B2, G1 and G2 in Grain Matrices

Significance of the topic

Aflatoxins B1, B2, G1 and G2 are toxic metabolites produced by Aspergillus fungi and pose serious carcinogenic risks in food supplies. Rapid and reliable screening methods are essential for food safety compliance and public health. Conventional analysis often relies on manual extraction with large volumes of organic solvents and immunoaffinity clean-up, leading to increased cost, time and environmental impact. Integrating supercritical fluid extraction (SFE) with supercritical fluid chromatography (SFC) on a single platform offers a green, automated alternative for high-throughput aflatoxin screening.

Objectives and study overview

This work aimed to develop and validate an on-line SFE-SFC-MS/MS method for simultaneous quantitation of aflatoxins B1, B2, G1 and G2 in grain matrices such as maize and peanut flour. The study evaluated method linearity, accuracy, precision, detection limits and applicability for regulatory compliance.

Methodology

A 1 g food sample was mixed with water, acetonitrile, absorbent and 13C-labeled internal standards and loaded into a 5 mL extraction vessel. SFE was performed using supercritical CO2 with static and dynamic steps. Split flow was applied to reduce matrix interference. Following extraction, analytes were separated on a COSMOSIL 5PBr column using a gradient of CO2 and ammonium acetate modifier in methanol, with a formic acid make-up flow to enhance ESI ionization. Detection employed a Shimadzu LCMS-8050 with ESI positive mode and multiple reaction monitoring, using two transitions per aflatoxin.

Instrumental setup

Shimadzu Nexera UC system coupling SFE and SFC with an LCMS-8050 detector. Key components include CO2 pump, modifier pump, make-up pump, COSMOSIL 5PBr column (4.6 mm x 250 mm, 5 µm), ESI source at 300°C, block temperature 400°C, desolvation at 250°C, nitrogen nebulizer and drying gases.

Main results and discussion

Calibration showed linear response over 0.5–20 ng for B1 and G1 and 0.15–6 ng for B2 and G2 with R2>0.991 over three days. Recovery in cereal and peanut certified reference materials ranged from 87.5% to 134% with inter-day RSD <15%. Detection limits were 0.05 ng/g in maize and 0.15–0.2 ng/g in peanut. The technique matched conventional HPLC methods in maize and provided regulatory-level sensitivity.

Benefits and practical applications

• Fully automated on-line extraction and analysis reduces manual steps.
• Reduced organic solvent consumption and environmental impact due to supercritical CO2.
• High throughput with a 21.5 min cycle time per sample.
• Accurate quantitation with 13C internal standards compensates matrix effects.
• Fit for routine food safety screening and regulatory compliance.

Future trends and potential applications

• Extension to other mycotoxins and contaminants using tailored SFC methods.
• Integration with high resolution mass spectrometry for untargeted screening.
• Miniaturization and field-portable SFE-SFC platforms.
• Automation enhancements for real-time monitoring and process control in food production.
• Adoption of greener modifiers and solvent-free ionization techniques.

Conclusion

The developed on-line SFE-SFC-MS/MS method provides a green, automated, and robust approach for aflatoxin screening in grain products. It achieves regulatory-level sensitivity, high accuracy and precision, while minimizing solvent use and manual workflow. This platform is suitable for routine food safety analysis and can be adapted for broader contaminant monitoring.

References

• F Soleimany et al, Food Control 25 (2012) 647–653
• MC Spanjer et al, Food Addit Contam A 25 (2008) 472–489
• Shimadzu Application News LAAN-A-LC-E273 (2015)
• J Xing et al, ASMS 2016 Poster Session MP 283
• Commission Regulation (EC) No 1881/2006