Aflatoxin - HPLC analysis and photochemical post column derivatization

Importance of the Topic

Aflatoxins are potent fungal toxins with significant health risks due to their strong carcinogenicity, particularly in the liver. They commonly contaminate agricultural products such as nuts, cereals, spices and milk. Stringent regulatory limits necessitate highly sensitive and reliable analytical methods for routine monitoring.

Study Objectives and Overview

This application note describes an HPLC method combined with photochemical postcolumn derivatization to enhance fluorescence detection of aflatoxins B1 and G1. The goal is to achieve low parts per trillion detection limits, minimize laborious sample preparation steps and ensure compliance with European regulation EC 1881 2006.

Methodology and Instrumentation

Samples are extracted with methanol or acetonitrile and cleaned up using immunoaffinity columns. An isocratic HPLC separation on a 150 x 4.6 mm C18 column with guard at 36 °C uses a water acetonitrile methanol mobile phase at 1.2 mL/min. Postcolumn derivatization is performed by passing the eluent through a 1 mL photochemical reactor at 254 nm. Fluorescence detection is carried out at excitation 365 nm and emission 460 nm.

Used Instrumentation

• HPLC system equipped with fluorescence detector
• Photochemical reactor UVE with 1 mL coil and 254 nm UV lamp
• C18 analytical column 150 x 4.6 mm with guard cartridge
• Immunoaffinity columns AflaCLEAN and Afla-OtaCLEAN

Main Results and Discussion

Photochemical derivatization increased the fluorescence response of aflatoxins B1 and G1 approximately 30 fold. Chromatograms demonstrated clear baseline separation of G2 G1 B2 and B1 within 8 minutes. Achievable detection limits reach the low ppt range depending on sample preparation and detector sensitivity. The UVE approach matches chemical bromination performance while eliminating reagent use and simplifying operation.

Benefits and Practical Applications

• Streamlined workflow by avoiding evaporation and reconstitution steps
• Enhanced sensitivity to meet strict regulatory thresholds
• Reduced matrix loading for cleaner chromatograms
• Reagent-free derivatization with minimal maintenance

Future Trends and Applications

Future developments may integrate online multidimensional separation, coupling with high resolution mass spectrometry for confirmation, and further automation of immunoaffinity cleanup. Miniaturized photochemical reactors and expanded detection of other mycotoxins offer promising directions.

Conclusion

The described HPLC method with photochemical postcolumn derivatization provides a robust sensitive and efficient solution for aflatoxin analysis in food matrices. It simplifies preparation achieves low ppt detection limits and complies with regulatory requirements without chemical reagents.

Reference

1. Papadopoulou Bouraoui A Stroka J Anklam E Journal of AOAC International 85 2 2002 411 416
2. Sobolev V Dorner JW Journal of AOAC International 85 3 2002 642 645
3. AOAC Official Method 2005 08 Aflatoxins in Corn Raw Peanuts and Peanut Butter Liquid Chromatography with Post Column Photochemical Derivatization