Determination of aflatoxins in peanut samples - from extraction to high efficient detection

Importance of the Topic

Aflatoxin contamination in peanuts poses a significant health risk due to the potent carcinogenic and toxic nature of these fungal metabolites. Since peanuts are often cultivated and dried under warm, humid conditions or stored improperly, routine monitoring of aflatoxin levels is essential to protect consumer safety and meet stringent regulatory standards worldwide.

Objectives and Overview of the Study

This study presents an end-to-end workflow for extracting and detecting four major aflatoxins (B1, B2, G1, G2) from peanut samples using a combination of solvent extraction, defatting, SPE cleanup and high-sensitivity HPLC with non-toxic photochemical post-column derivatization. The goal is to achieve detection limits well below those mandated by the European Commission.

Methodology and Instrumentation

Sample preparation integrates three key steps to minimize matrix interferences:

• Solid-liquid extraction: grind 50 g of shelled peanuts and mix with methanol:water (17:3 v/v) for 30 min.
• Liquid-liquid defatting: remove lipids by partitioning with n-hexane (2×) and extract the aqueous phase twice with chloroform.
• SPE cleanup: concentrate the chloroform extracts to 3 mL, load onto silica sorbent cartridges, wash out fats with hexane, diethyl ether and chloroform, then elute aflatoxins in chloroform:acetone (9:1 v/v).

HPLC and detection setup:

• System: AZURA Analytical pump, autosampler, column thermostat and RF-20A fluorescence detector.
• Column: Eurospher II C18 (150 × 4.6 mm, 3 µm) at 60 °C.
• Mobile phase gradient: from 83% water/5% acetonitrile/12% methanol to 100% acetonitrile over 9 min, flow rate 2.4 mL/min.
• Post-column: UVE photochemical reactor (254 nm) enables hydroxylation of B1 and G1 for enhanced fluorescence.
• Detection wavelengths: excitation 365 nm, emission 460 nm, data rate 50 Hz.

Key Results and Discussion

The combined extraction/C18-SPE protocol reduced matrix fluorescence from 900 counts to below 25 counts in the aflatoxin elution window. Multi-matrix samples spiked at 20 ng/mL (B1/G1) and 6 ng/mL (B2/G2) demonstrated limits of detection of 0.05 ng/mL for B1/G1 and 0.015 ng/mL for B2/G2—3.4- to 11.3-fold below European limits. Triplicate extractions confirmed high reproducibility and robustness.

Benefits and Practical Applications

• Effective removal of co-extracted lipids and pigments delivers accurate, interference-free quantification across diverse food matrices.
• Non-toxic photochemical derivatization eliminates halogenated reagents and associated waste.
• Single-run HPLC analysis with sub-ppb sensitivity supports high-throughput quality control in food and feed laboratories.

Future Trends and Potential Applications

Future developments may include automated SPE workflows, integration with mass spectrometry for multiplexed mycotoxin panels, and miniaturized flow systems to reduce solvent use. Expanding the protocol to other commodities and portable screening devices could further enhance real-time monitoring capabilities.

Conclusion

The described workflow offers a streamlined, eco-friendly, and highly sensitive solution for routine aflatoxin analysis in peanuts and related products. With detection limits well below regulatory thresholds and scalable sample preparation, this method is ideally suited for industrial QA/QC and regulatory surveillance.

References

1. Hedayati MT, Pasqualotto AC, Bowyer P, Denning DW. Aspergillus flavus: human pathogen, allergen and mycotoxin producer. Microbiology. 2007;153(6):1677-1692.
2. Mupunga I, Lebelo SL, Mangwawa P, Rheeder JP, Katerere DR. Natural occurrence of aflatoxins in peanuts and peanut butter from Bulawayo, Zimbabwe. J Food Prot. 2014;77(10):1814-1818.
3. World Health Organization. Aflatoxins. Food Safety Digest. 2018.
4. U.S. FDA. Action levels for aflatoxins in animal food. CPG Sec. 683.100. 2019.
5. European Commission. Regulation (EC) No 401/2006 on sampling and analysis of mycotoxins. 2006.
6. European Commission. Regulation (EC) No 1881/2006 setting maximum contaminant levels in food. 2006.
7. Folmert K, Margraf M, Monks K. Quick determination of aflatoxins with photochemical post-column derivatization. KNAUER AppNote VFD0178. 2019.