Determination of Aflatoxin M1 via FREESTYLE ThermELUTE™ with Online HPLC-Measurement

Importance of the topic

Aflatoxin M1 poses significant health risks when present in dairy products due to its carcinogenic and hepatotoxic effects. Strict regulatory limits require sensitive and reliable methods to monitor its levels in milk, dairy products, and related matrices.

Aims and study overview

This study presents an automated workflow combining the FREESTYLE ThermELUTE system with online HPLC-fluorescence detection to quantify Aflatoxin M1 in various dairy matrices. The goal is to achieve rapid, high-throughput analysis with detection limits in the low ppt range, meeting European Commission requirements.

Methodology and instrumentation

Samples are extracted according to official protocols for liquid milk, raw milk, and milk powder, followed by dilution and filtration. A 16 mL aliquot is loaded into the FREESTYLE ThermELUTE system, where immunoaffinity columns (AflaCLEAN M1 SMART) perform automated cleanup. Eluted analytes are directly injected online into the HPLC system equipped with a fluorescence detector. The total process time per sample is approximately 30 minutes, enabling over 70 samples per day in continuous operation.

Instrumentation used

• FREESTYLE ThermELUTE system (P/N 13691) with supporting racks and adapter frames
• Immunoaffinity columns AflaCLEAN M1 SMART (P/N 14246/14248)
• HPLC system with RP C18 column (LCTech P/N 10522) and fluorescence detector
• Solvents: methanol, acetonitrile, water (HPLC grade)

Main results and discussion

Recovery studies at 80 ppt spiking level yielded 96% in milk, 90% in raw milk, and 98% in milk powder after correction for native toxin levels. Chromatographic separation used an acetonitrile/water/methanol (15/60/30 v/v/v) mobile phase at 1.2 mL/min, excitation at 365 nm and emission at 435 nm. The method demonstrated precise quantification with low background interference and high reproducibility across matrices.

Benefits and practical applications

• Fully automated sample cleanup reduces manual labor and variability
• High sample throughput supports routine quality control and monitoring
• Sensitive detection in the low ppt range ensures compliance with EC regulations
• Applicability to multiple dairy matrices enhances laboratory flexibility

Future trends and potential applications

Advances may include multiplexed immunoaffinity cleanup for simultaneous analysis of multiple mycotoxins, coupling with mass spectrometric detectors for enhanced specificity, miniaturization of online systems for point-of-need testing, and integration with digital lab management platforms for real-time data processing.

Conclusion

The automated FREESTYLE ThermELUTE coupled with online HPLC-fluorescence detection provides a fast, reliable, and sensitive method for Aflatoxin M1 analysis in dairy products. Its high throughput and robust performance make it suitable for regulatory compliance and routine laboratory operations.

References

1. AOAC Official Method 2000.08. Aflatoxin M1 in Liquid Milk by Immunoaffinity Column Liquid Chromatography.
2. DIN EN ISO 14501:2008-01. Extraction and Analysis of Aflatoxin M1 in Milk and Milk Powder.
3. Commission Regulation (EU) No 165/2010 of 26 February 2010 amending Regulation (EC) No 1881/2006 on maximum levels for aflatoxins in foodstuffs.
4. Marina Martins H. (2007). Aflatoxin M1 Determination in Cheese by Immunoaffinity Column Clean-up Coupled to HPLC. RPCV, 102(563-564), 321-325.