Validation of a confirmatory analytical method for the determination of aflatoxins B₁, B₂, G₁ and G₂ in foods and feed materials by HPLC with on-line photochemical derivatization and fluorescence detection

Abstract

A sensitive and selective analytical method for the simultaneous separation and quantitative determination of aflatoxins B₁, B₂, G₁ and G₂ in foodstuffs and materials for feed has been validated. The method is based on high performance liquid chromatography with on-line post-column photochemical derivatization and fluorescence detection. The chromatographic separation of aflatoxins was accomplished using a C₁₈ column eluted with an isocratic mobile phase consisting of water, methanol and acetonitrile. The sample preparation required a simple extraction of aflatoxins with MeOH/H₂O (80:20, v/v) and a purification step by immunoaffinity column cleanup. The total analysis time, including sample preparation and chromatographic separation, did not exceed 40 min with a run time of 10 min. The on-line photochemical derivatization ensures better results in terms of simplicity, sensitivity and reproducibility with respect to chemical derivatization techniques, and provides an increase of the peak resolution and an extent of automation in comparison with the electrochemical ones. The procedure for the determination of aflatoxins in food samples and cereals for animal consumption was extensively validated following Regulation (EC) No. 882/2004. Detection limits in wheat bran samples of 0.08 µg kg⁻¹ for AFB₁, 0.02 µg kg⁻¹ for AFB₂, 0.16 µg kg⁻¹ for AFG₁ and 0.04 µg kg⁻¹ for AFG₂ were attained. The method allows high recovery with mean values ranging from 72 to 94% and it satisfies the necessary requirements for sensitivity, linearity, selectivity, precision and ruggedness, demonstrating the conformity of the method with provisions of Regulation (EC) No. 401/2006.

Keywords:

• cleanup, affinity columns
• HPLC
• method validation
• aflatoxins
• cereals

Acknowledgements

Ministero della Salute (Rome, Italy) is thanked for providing financial support. P. D’Antini and G. Battafarano from Istituto Zooprofilattico Sperimentale di Puglia e Basilicata-Foggia are gratefully acknowledged for technical assistance.