Determination of Aflatoxins

Summary

This video outlines a laboratory procedure for detecting aflatoxin B1 in feedstuff materials using high-performance liquid chromatography (HPLC). The process includes sample preparation, immunoaffinity column cleanup, and HPLC analysis with post-column bromination. Emphasis is placed on safety precautions, particularly when handling mycotoxins, and on ensuring accurate results through precise calibration and sample handling. The procedure is validated by the European Commission's Joint Research Centre and adheres to international safety standards for food and animal feed analysis.

Takeaways

• 😀 Ensure laboratory safety when handling mycotoxins: wear gloves, glasses, and follow proper waste disposal protocols.
• 😀 Aflatoxins, toxic metabolites of *Aspergillus flavus*, can contaminate feedstuff and affect both animal and human health.
• 😀 Aflatoxin B1 levels in feedstuff are regulated in over 50 countries, requiring accurate testing methods for enforcement.
• 😀 The method described involves extracting aflatoxin B1 from feedstuff followed by immunoaffinity column cleanup and HPLC analysis.
• 😀 Feedstuff must be ground or milled appropriately before extraction if it consists of compound material or grain.
• 😀 Proper sample preparation includes weighing 50 grams of feedstuff, diluting it in an acetone-water mixture, and shaking it for 30 minutes.
• 😀 A 5 mL aliquot from the filtered extract is diluted to 100 mL for immunoaffinity cleanup using phosphate-buffered saline or water.
• 😀 The immunoaffinity column uses antibodies specific to aflatoxin B1, selectively binding and isolating it from the sample.
• 😀 The elution of aflatoxin B1 from the immunoaffinity column is done in two stages using methanol, followed by dilution with water.
• 😀 The HPLC method requires post-column bromination of aflatoxin B1 before fluorescence detection for accurate measurement.
• 😀 A calibration curve made with known aflatoxin B1 standards is essential for determining the concentration in the sample.
• 😀 Automated systems like ASP EC can be used for this analysis, but the specified protocol must be strictly followed for consistent results.

Q & A

What is the main purpose of the method described in the video?

The main purpose of the method is to determine aflatoxin B1 levels in feedstuff, which can be contaminated by mycotoxins. This is crucial for ensuring food and animal feed safety.

What safety precautions are necessary when handling mycotoxins?

Laboratory safety precautions must be followed, including wearing gloves, safety glasses, and handling spillages properly. Special care must also be taken in disposing of contaminated waste.

Why is aflatoxin B1 a concern in feedstuff and food?

Aflatoxin B1 is a toxic metabolite of Aspergillus fungi and poses a health risk to both humans and animals, especially when it contaminates animal feed and finds its way into dairy products.

What are the primary steps in the sample preparation process?

The sample preparation involves weighing 50 grams of feedstuff, diluting it in an acetone-water mixture, shaking for homogeneity, mechanically shaking for 30 minutes, filtering the mixture, and then diluting the filtrate for further analysis.

How does the immunoaffinity column work in the process?

The immunoaffinity column contains antibodies specific to aflatoxin B1. These antibodies bind to the toxin, allowing other impurities to be washed away. The aflatoxin B1 is then eluted from the column using methanol.

What role does high-performance liquid chromatography (HPLC) play in the analysis?

HPLC is used to separate and detect aflatoxin B1 in the sample. After the sample is processed through the immunoaffinity column, it is injected into the HPLC system for further analysis, with post-column bromination aiding in detection.

What is the purpose of post-column bromination in the HPLC process?

Post-column bromination enhances the fluorescence detection of aflatoxin B1, allowing for more accurate quantification of the toxin.

How is the concentration of aflatoxin B1 determined after HPLC analysis?

The concentration of aflatoxin B1 is determined by comparing the sample’s chromatographic peak to a calibration curve created using known concentrations of aflatoxin B1 standards.

What should the chromatogram look like when aflatoxin B1 is successfully detected?

The chromatogram should show a well-resolved peak corresponding to aflatoxin B1, free from interference from other extracted components.

Can automated systems be used for this method?

Yes, fully automated systems, such as the ASP EC system, can be used, as long as they follow the specified analytical protocol, including proper volumes of reagents and flow rates.