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Enhanced Automated Online Immunoaffinity Liquid Chromatography-Fluorescence Method for the Determination of Aflatoxin
Jackie E Wood1, Brendon D Gill1, Iain J McGrail1
1Fonterra Co-operative Group Ltd, PO Box 7, Waitoa 3341, New Zealand.
Background:
Aflatoxin M1 (AFM1) is found in the milk of cows exposed to feed spoiled by Aspergillus fungi species. These fungi may produce the secondary metabolite aflatoxin B1, which is converted in the cow liver by hydroxylation to AFM1 and is then expressed in milk. AFM1 is regulated in milk and other dairy products because it can cause serious health issues, such as liver and kidney cancers, in humans and is an immunosuppressant.
Objective:
To optimize the chromatographic protocol and to extend the matrix scope to include a wider range of dairy products: whey powder, whey protein concentrate, whey protein isolate, liquid milk, skim milk powder, whole milk powder, adult nutritional products, and yogurt.
Methods:
AFM1 is extracted using 1% acetic acid in acetonitrile incorporating ionic salts. The AFM1 in the resulting extract is concentrated using an automated RIDA®CREST IMMUNOPREP® online cartridge coupled to quantification by HPLC-fluorescence.
Results:
The method was shown to be accurate, with acceptable recovery (81.2-97.1%) from spiked samples. Acceptable precision was confirmed, with a relative standard deviation (RSD) for repeatability of 6.6-11.2% and an RSD for intermediate precision of 7.5-16.7%. Method LOD and robustness experiments further demonstrated the suitability of this method for routine compliance testing. Analysis of an international proficiency trial sample generated results that were comparable with the value assigned from alternative independent methods.
Conclusion:
A method with improved chromatography for high-throughput, routine testing of AFM1 in an extended range of dairy products is described. The method was subjected to single-laboratory validation and was found to be accurate, precise, and fit for purpose.
Highlights:
Single-laboratory validation of an automated online immunoaffinity cleanup fluorescence HPLC method for AFM1 in whey proteins, milk powders, nutritional products, liquid milk, and yogurt. Allows for high-throughput analysis of AFM1 with enhanced chromatographic performance. Method applicable to the analysis of AFM1 in an extended range of milk and milk-based products.
Insights
A new method accurately detects Aflatoxin M1 (AFM1) in various dairy products using automated HPLC-fluorescence. This validated approach ensures reliable, high-throughput testing for regulatory compliance and consumer safety.
Area of Science:
- Food Safety Analysis
- Analytical Chemistry
- Mycotoxin Detection
Background:
- Aflatoxin M1 (AFM1) is a toxic metabolite found in milk from cows consuming contaminated feed.
- AFM1 poses health risks, including carcinogenicity and immunosuppression in humans.
- Regulation of AFM1 in dairy products is crucial for public health.
Purpose of the Study:
- To optimize a chromatographic protocol for AFM1 detection.
- To extend the method's applicability to a wider range of dairy products.
- To validate a high-throughput analytical method for routine testing.
Main Methods:
- AFM1 extraction using acetic acid in acetonitrile with ionic salts.
- Automated online immunoaffinity cleanup (RIDA®CREST IMMUNOPREP®).
- Quantification by High-Performance Liquid Chromatography with fluorescence detection (HPLC-fluorescence).
Main Results:
- The method demonstrated high accuracy with recovery rates of 81.2-97.1%.
- Precision was acceptable, with relative standard deviations (RSD) for repeatability (6.6-11.2%) and intermediate precision (7.5-16.7%).
- Method validation confirmed suitability for routine compliance testing, with comparable results to independent methods.
Conclusions:
- An optimized, automated HPLC-fluorescence method for AFM1 detection in diverse dairy products was successfully validated.
- The method offers enhanced chromatographic performance for high-throughput analysis.
- This validated method is suitable for routine compliance testing of AFM1 in milk and milk-based products.
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