Quick and easy determination of aflatoxins in food matrices with photochemical post column derivatization

Significance of the topic

Aflatoxins are among the most toxic natural contaminants in food and feed, with aflatoxin B1 classified as a group 1 carcinogen by the World Health Organization. Produced by Aspergillus species under warm, humid conditions, these compounds persist through processing and storage, posing serious health risks to humans and animals. Regulatory agencies worldwide enforce strict limits, particularly for infant and medical diets, creating a demand for highly sensitive and reliable detection methods.

Objectives and study overview

This work aimed to develop a quick, robust, and sensitive HPLC method incorporating photochemical post-column derivatization for simultaneous determination of aflatoxins B1, B2, G1, and G2 in diverse food matrices. The method was validated using a composite extract from nuts, dried fruits, and baby cereal products to simulate routine monitoring conditions.

Methodology and instrumentation

Sample preparation combined liquid-liquid and solid-phase extraction as described in KNAUER application notes. Chromatographic separation was achieved on a Eurospher II C18 column using a 9-minute gradient at 2.4 mL/min, followed by a 6-minute wash and re-equilibration. A photochemical reactor irradiating at 254 nm converted aflatoxins B1 and G1 into strongly fluorescent derivatives, detected at excitation 365 nm and emission 460 nm. The total cycle time of 15 minutes supports high throughput.

Instrumentation used

• AZURA P 6.1L low-pressure gradient pump
• AZURA AS 6.1L autosampler
• AZURA CT 2.1 column thermostat
• RF-20A fluorescence detector with UVE photochemical reactor
• Eurospher II C18 100-3 column (150 × 4.6 mm)
• ClarityChrom 8.1 chromatography software

Main results and discussion

The optimized method delivered baseline separation of all four aflatoxins and matrix interferences within 9 minutes. Empirical limits of detection were 0.05 ng/mL for B1/G1 and 0.015 ng/mL for B2/G2, surpassing EU requirements by factors of 3.4 and 11.3. Recoveries at LOQ, 2× LOQ, and 20 ng/mL ranged from 78 % to 87 % with RSD < 6 %. Repeatability tests (n = 8) showed RSD < 0.1 % for retention times and < 0.5 % for peak areas. Robustness trials confirmed consistent performance under ±2 °C temperature, ±0.2 mL/min flow rate, and ±2 % mobile phase composition variations.

Benefits and practical applications

The non-toxic photochemical derivatization replaces hazardous halogen reagents and reduces solvent waste. Rapid analysis and high sensitivity make this approach ideal for routine QA/QC in food and feed industries, ensuring compliance with stringent regulatory limits, especially for infant and special medical diets.

Future trends and applications

Future developments may involve ultra-high-performance liquid chromatography to shorten run times and coupling with mass spectrometry for confirmatory analysis. Advances in automated sample preparation and integrated derivatization modules will further increase throughput and reliability in large-scale monitoring.

Conclusion

This study demonstrates a fast, sensitive, and environmentally friendly HPLC method with photochemical post-column derivatization for simultaneous quantification of aflatoxins B1, B2, G1, and G2. Its excellent robustness, precision, and compliance with regulatory requirements make it a valuable tool for safeguarding consumer health.

References

• Hedayati MT, Pasqualotto AC, Bowyer P, Denning DW. Microbiology. 2007;153(6):1677-1692. doi:10.1099/mic.0.2007/007641-0
• World Health Organization. Food Safety Digest - Aflatoxins. Department of Food Safety and Zoonoses. 2018. Ref No WH O/NHM/FOS/RAM/18.1
• U.S. Department of Health and Human Services. Food and Drug Administration. Section 683.100 Action Levels for Aflatoxins in Animal Food. 2019
• European Commission. Commission Regulation (EC) No 401/2006 on sampling and analysis methods for official control of mycotoxins in foodstuffs. 2016
• European Commission. Commission Regulation (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Official Journal of the European Union. 2006;L364:5-24