DETERMINATION OF AFLATOXIN M1 IN MILK

Significance of the topic

Aflatoxin M1 is a regulated mycotoxin derived from animal metabolism of aflatoxins B1 and B2. Its presence in milk poses a serious public health concern due to carcinogenicity and strict regulatory limits (0.5 µg/L). Rapid, reliable determination of aflatoxin M1 ensures food safety and compliance with international standards.

Objectives and Study Overview

The aim of this study was to develop a fast, isocratic HPLC method combined with photochemical post-column derivatization for sensitive quantification of aflatoxin M1 in raw and processed milk. A straightforward solid phase extraction protocol was integrated to concentrate and clean up the analyte prior to analysis.

Methodology

Sample pretreatment involved online SPE with a CHROMABOND C18 ec column: 20 mL spiked milk was diluted, loaded, washed with water and n-hexane, dried, and eluted with acetonitrile. Chromatographic separation used an isocratic mobile phase of water/methanol/acetonitrile (60:25:15, v/v) at 0.8 mL/min, 30 °C, and a 10 min run time. Detection was by fluorescence at excitation 365 nm and emission 455 nm, with high sensitivity gain.

Used Instrumentation

• AZURA P 6.1L LPG pump (10 mL)
• Autosampler 3950 analytical version
• AZURA CT 2.1 column thermostat
• UVE photochemical reactor for post-column derivatization
• RF-20Axs fluorescence detector
• KNAUER Eurospher II 100-3 C18 column (150 × 3 mm ID)
• OpenLAB CDS EZChrom Edition software

Main Results and Discussion

Fluorescence chromatograms of a 1 µg/mL aflatoxin M1 standard showed clear, sharp peaks after photochemical derivatization. Spiked milk samples at the regulatory limit of 0.5 µg/L were successfully quantified despite matrix effects from SPE. Additional tests demonstrated detection down to 0.001 µg/mL. The method delivered reproducible retention times and peak shapes within a 10 min analysis.

Benefits and Practical Applications of the Method

• High sensitivity to meet or exceed regulatory requirements.
• Short analysis time supports high sample throughput.
• Isocratic conditions simplify method transfer and maintenance.
• Robust SPE cleanup reduces matrix interferences.
• Photochemical derivatization avoids chemical reagents and simplifies system setup.

Future Trends and Applications

Advancements may include coupling this approach with mass spectrometry for confirmatory analysis, automating SPE steps for full online workflows, and adapting the method to processed dairy matrices. Miniaturized or multiplexed systems could further increase throughput and reduce solvent consumption.

Conclusion

The combination of SPE, isocratic HPLC, UVE photochemical derivatization, and fluorescence detection provides a fast, sensitive, and reliable method for quantifying aflatoxin M1 in milk at or below regulatory limits. Its simplicity and robustness make it well suited for routine quality control in dairy industries.

Reference

1. FDA Mycotoxin Regulatory Guidance, National Grain and Feed Association, Washington, D.C., USA, August 2011.
2. Commission Regulation (EC) No 1881/2006, Official Journal of the European Union L 364, 19 December 2006.
3. Additional application note data, MN-net, date unspecified.