Immunoaffinity Solid-Phase Extraction with HPLC-FLD Detection for the Determination of Aflatoxins B2, B1, G2, AND G1 in Ground Hazelnut

Significance of the Topic

Aflatoxins are highly toxic secondary metabolites produced by Aspergillus fungi and pose serious health risks when present in food commodities. Ground hazelnuts, being high‐fat matrices, can harbor trace levels of aflatoxins B2, B1, G2 and G1 that must be accurately monitored to comply with stringent European Commission limits. The development of rapid, sensitive and selective analytical workflows is essential to ensure food safety and regulatory compliance.

Study Objectives and Overview

This study aimed to establish a robust method for the quantification of four key aflatoxins in ground hazelnut using immunoaffinity solid‐phase extraction (SPE) combined with high‐performance liquid chromatography and fluorescence detection (HPLC‐FLD) without chemical derivatization. The goals included achieving low detection limits well below EU maximum levels, demonstrating linearity and recovery in the nut matrix, and confirming that the procedure supports high sample throughput.

Methodology

Standard addition calibration was employed to correct for matrix effects in hazelnut extracts that inherently contain aflatoxins. Calibration involved six fortified levels ranging from 0.2 to 2.1 µg/kg for G2 and 0.7 to 7.1 µg/kg for G1, B2 and B1. Sample preparation steps included:

• Mixing 20 g ground hazelnut with 2 g NaCl, 100 mL 80% methanol and 50 mL hexane, followed by 15 min stirring.
• Phase separation, dilution of 14 mL extract with phosphate‐buffered saline (pH 7.2).
• Loading 50 mL diluted extract onto an AflaClean Select immunoaffinity SPE cartridge.
• Washing with water, elution with methanol, dilution with acetic acid, and 0.2 µm membrane filtration.
• HPLC‐FLD analysis under isocratic conditions, excitation at 365 nm and emission at 450 nm.

Instrumentation

• Thermo Scientific Vanquish Flex UHPLC system with Quaternary Pump F, Split Sampler FT and Column Compartment.
• Acclaim C18 column (100 × 3 mm, 3 µm) at 30 °C with forced air.
• Mobile phase: 50% water, 30% methanol, 20% acetonitrile at 0.5 mL/min; run time 4 min; injection volume 20 µL.
• Thermo Scientific Fluorescence Detector F (Bio Flow Cell) set to high‐power lamp mode, sensitivity 8.

Main Results and Discussion

• Baseline separation of all four aflatoxins achieved within 4 min, with no matrix interference in the target retention window.
• Linearity coefficients (R2) ranged from 0.9920 to 0.9974; retention time RSD < 0.2%.
• Limits of detection between 0.075 and 1.056 µg/kg; limits of quantification between 0.185 and 1.329 µg/kg.
• Recovery rates from spiked hazelnut samples varied from 72% (G1) to 100% (G2, B2); B1 recovery was 95%.
• Immunoaffinity SPE effectively removed impurities present in standard solutions and nut matrix, enhancing selectivity without derivatization.

Benefits and Practical Applications

• The method delivers trace‐level detection far below EU regulatory limits, ensuring consumer safety.
• Omission of chemical derivatization simplifies workflow and reduces analysis time.
• Short run time (< 4 min) and reliable quantification metrics support high laboratory throughput.
• Suitable for routine quality control in food industry and regulatory labs monitoring aflatoxins in nuts and other commodities.

Future Trends and Potential Applications

Continued advances may include integration of online SPE automation, coupling to mass spectrometry for confirmatory analysis, and expansion to multi‐mycotoxin panels. Emerging immunoaffinity materials and microfluidic platforms could further reduce solvent usage and sample volume, enabling on‐site screening of aflatoxins in diverse food matrices.

Conclusion

The developed immunoaffinity SPE–HPLC‐FLD method offers a rapid, sensitive and selective approach for quantifying aflatoxins B2, B1, G2 and G1 in ground hazelnut. It meets European regulatory requirements, delivers excellent linearity, low detection limits and high recoveries, and supports high sample throughput for routine food safety monitoring.

References

1. Commission Regulation (EC) No 1881/2006 of 19 December 2006: setting maximum levels for certain contaminants in foodstuffs.
2. Thermo Fisher Scientific, Application Note 72686: Determination of underivatized aflatoxins B2, B1, G2, and G1 in ground hazelnut by immunoaffinity SPE with HPLC-FLD detection.