Quantitative Analysis of Aflatoxin in Edible Nuts by Using High-Performance Liquid Chromatography Coupled with Florescence Detector

Importance of the Topic

Aflatoxins are potent fungal metabolites that frequently contaminate edible nuts under warm and humid conditions. Even at trace levels, these toxins pose significant health hazards and economic losses. Regulatory agencies such as the European Union enforce strict maximum limits (e.g., 2 µg/kg for aflatoxin B₁ and 4 µg/kg for total aflatoxins in nuts) to ensure consumer safety. Reliable, sensitive, and rapid analytical methods are therefore essential for routine monitoring and quality assurance in the food industry.

Objectives and Study Overview

This work aimed to develop and validate a high-performance liquid chromatography method with fluorescence detection (HPLC-FLD) combined with post-column photochemical derivatization and immunoaffinity cleanup (AflaCLEAN) for quantifying aflatoxins B₁, B₂, G₁, and G₂ in five commercially available edible nuts (pine nuts, peanuts, cashews, almonds, and walnuts). The study assessed method linearity, sensitivity, accuracy, precision, recovery, and compliance of actual samples with EU regulatory limits.

Materials and Methods

Sample preparation involved grinding 20 g of nuts and extracting aflatoxins with methanol–water (80:20, v/v) and n-hexane, followed by centrifugation and phase separation. The methanolic phase was diluted with phosphate-buffered saline and passed through immunoaffinity cartridges for selective toxin cleanup. Bound aflatoxins were eluted with methanol and directly injected into the HPLC system. Calibration standards ranged from 0.05 µg/kg to 1 µg/kg, and spiked recovery experiments at 0.1–0.4 µg/kg were conducted.

Used Instrumentation

• Shimadzu i-Series LC-2060C 3D HPLC system
• RF-20Axs fluorescence detector (Shimadzu)
• LC Tech photochemical derivatization chamber
• AflaCLEAN immunoaffinity cartridges (LC-Tech)

Analytical Conditions

Isocratic elution on a Shim-pack GIST C18 column (150 mm × 4.6 mm, 5 µm) using water/methanol/acetonitrile (60:30:15) at 1.2 mL/min and 35 °C. Injection volume: 10 µL. Fluorescence excitation/emission: 365/460 nm. Total run time: 10 min.

Results and Discussion

Calibration curves exhibited excellent linearity (R² ≥ 0.998) across all aflatoxin standards. Limits of detection and quantification ranged from 0.002 to 0.020 µg/kg, with signal-to-noise ratios above 10. Recovery rates for spiked nut samples fell between 80% and 100%, demonstrating high accuracy and minimal matrix interference.
The naturally contaminated samples revealed:

• Pine nuts: B₂ at 0.066 µg/kg and B₁ at 0.346 µg/kg.
• Peanuts: B₂ at 0.015 µg/kg and B₁ at 0.057 µg/kg.
• Almonds, cashews, and walnuts: aflatoxins below quantification limits.

All results were well below EU maximum levels, confirming product safety and method reliability for regulatory compliance.

Benefits and Practical Applications

The combination of immunoaffinity cleanup and post-column photochemical derivatization enhances selectivity and sensitivity without chemical reagents. The rapid 10-minute run time, low detection limits, and robust recoveries make this HPLC-FLD method ideal for routine quality control in food safety laboratories, ensuring consumer protection and regulatory adherence.

Future Trends and Potential Applications

Emerging directions include:

• Integration of immunoaffinity cleanup with automated online systems to further increase throughput.
• Expansion to multi-mycotoxin screening using LC-MS/MS for broader contaminant profiling.
• Development of portable fluorescence-based detectors for field testing.
• Data-driven predictive tools (AI/ML) to forecast aflatoxin risk based on environmental parameters.

Conclusion

The validated HPLC-FLD method with immunoaffinity cleanup and photochemical derivatization offers a sensitive, accurate, and efficient approach for aflatoxin analysis in edible nuts. It meets stringent regulatory requirements and supports routine monitoring to ensure food safety.

References

1. European Commission. Commission Regulation (EC) No. 1881/2006 setting maximum levels for certain contaminants in foodstuffs, as amended by Regulation (EU) No. 165/2010. 2006.
2. LCTech GmbH. Application Note AN0038: Aflatoxin Analysis of Almond and Sesame Products. 2023.