Determination of underivatized aflatoxins B2, B1, G2, and G1 in ground hazelnuts by immunoaffinity solid-phase extraction with HPLC-FLD detection

Significance of the Topic

Aflatoxins are potent fungal toxins with high stability that can persist through food processing and pose significant health risks. Monitoring aflatoxin B2, B1, G2 and G1 in nut products is critical to ensure consumer safety and regulatory compliance.

Objectives and Study Overview

The study aimed to develop a rapid and sensitive chromatographic method for quantification of four underivatized aflatoxins in ground hazelnuts. Key goals included selective sample purification using immunoaffinity solid phase extraction and HPLC with fluorescence detection without derivatization.

Methodology and Instrumentation

Sample preparation combined methanol extraction, hexane defatting and phosphate buffer dilution followed by immunoaffinity SPE cleanup. Purified extracts were directly analyzed by HPLC-FLD under isocratic conditions.
Used instrumentations:

• Thermo Scientific Vanquish Flex UHPLC system
• Vanquish Flex Fluorescence Detector
• Acclaim C18 analytical column (100 × 3 mm, 3 µm)
• AflaCLEAN Select immunoaffinity SPE cartridges

Main Results and Discussion

Calibration via standard addition was linear over 0.2–2.1 µg/kg for G2 and B2 and 0.7–7.1 µg/kg for G1 and B1 (R2 0.992–0.997). Limits of detection were 0.075–1.056 µg/kg and limits of quantification ranged from 0.185 to 1.329 µg/kg. Recovery rates were 100% for G2 and B2, 95% for B1 and 72% for G1, attributed to cartridge shelf life. Selective cleanup eliminated matrix interferences and no carryover was observed.
Quantitative analysis of a commercial hazelnut sample yielded 0.4 µg/kg G2, 0.3 µg/kg B2, 2.2 µg/kg G1 and 3.4 µg/kg B1. These values comply with European Commission limits for aflatoxins in nuts (B1 up to 2–12 µg/kg; sum of four toxins 4–15 µg/kg).

Benefits and Practical Applications of the Method

• Direct FLD analysis without derivatization accelerates workflow
• Immunoaffinity SPE provides high selectivity and low detection limits in complex matrices
• Short analysis time (<4 minutes) increases sample throughput
• Robust quantification with good linearity and reproducibility

Future Trends and Applications

Further improvements may include integration with mass spectrometry for enhanced confirmatory analysis, broader mycotoxin panels and miniaturized SPE formats. Advances in immunoaffinity materials and UHPLC technology could enable faster, multiplexed testing in food safety laboratories.

Conclusion

The combination of immunoaffinity SPE cleanup with direct HPLC-FLD detection on the Vanquish Flex platform delivers a reliable, sensitive and high-throughput method for underivatized aflatoxin analysis in hazelnuts. The protocol meets regulatory requirements and supports routine quality control in food safety testing.

Reference

1. COMMISSION REGULATION (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs
2. Karsten M et al Fast and effective determination of aflatoxins in grains using accelerated solvent extraction followed by HPLC HPLC 2008 presentation