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How to Detect Aflatoxins in Food: Step-by-Step Lab Methods

Aflatoxins are toxic secondary metabolites produced mainly by Aspergillus flavus and Aspergillus parasiticus.

They frequently contaminate grains, nuts, spices, and dairy products, especially in warm and humid regions.

To protect consumers and meet international food safety standards, accurate laboratory detection of aflatoxins is essential.

In this guide, we’ll walk through the step-by-step methods used by laboratories worldwide to detect and quantify aflatoxins from sample preparation to result interpretation and show you how rapid aflatoxin test kits can simplify routine screening.

Light microscopy of Aspergillus flavus with lactophenol cotton blue Photo

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General characteristics of Aspergillus Flavus

Aspergillus flavus is one of the most frequently isolated mold species in agriculture and medicine.

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Step 1: Sample Collection and Preparation

Effective aflatoxin detection starts with representative sampling.

Since contamination is often unevenly distributed, it’s crucial to collect small portions from multiple areas of the batch (for example, maize, peanuts, or dried fruits).

Important steps:

  1. Collect a composite sample (about 1–2 kg).
  2. Grind it finely to obtain a homogeneous powder.
  3. Mix thoroughly and store in a clean, dry, opaque container.

Tip: Avoid exposure to light and moisture, which may degrade aflatoxins and affect test results.

Step 2: Extraction of Aflatoxins

The goal of this step is to separate aflatoxins from the food matrix into a measurable solution.

Common extraction solvents:

  • Methanol–water (70:30 v/v)
  • Acetonitrile–water (80:20 v/v)

Procedure:

  1. Weigh ~25 g of ground sample.
  2. Add 100 mL of solvent mixture.
  3. Shake or blend for 2–3 minutes.
  4. Filter or centrifuge the extract.

This filtered solution is now ready for clean-up or direct analysis, depending on the chosen method.

Step 3: Clean-Up Techniques

Clean-up reduces matrix interference to improve accuracy and sensitivity.

Common approaches:

  • Immunoaffinity Columns (IAC): Highly specific antibodies bind to aflatoxins, allowing pure elution for quantification.Learn more




  • Solid Phase Extraction (SPE): Useful for high-throughput screening.Learn more

Clean-up ensures the extract is compatible with sensitive instruments like HPLC or LC-MS.

Step 4: Detection and Quantification Methods

Laboratories use several analytical techniques to identify and quantify aflatoxins. Each has its own level of precision, cost, and time requirement.

1. ELISA (Enzyme-Linked Immunosorbent Assay)

Best for: Routine, rapid, and cost-effective screening.

Principle: Specific antibodies bind to aflatoxin molecules; color intensity is inversely proportional to toxin concentration.

Advantages:

  • Fast (under 30 min)
  • Requires minimal instrumentation
  • High sensitivity (ppb level)

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2. HPLC (High-Performance Liquid Chromatography)

Best for: Quantitative confirmation and regulatory compliance.

Principle: Aflatoxins are separated on a column and detected by fluorescence after derivatization.

Advantages:

  • Excellent accuracy and reproducibility
  • Simultaneous detection of multiple aflatoxins (B1, B2, G1, G2)


3. LC-MS/MS (Liquid Chromatography–Mass Spectrometry)

Best for: Advanced research and reference laboratories.

Principle: Combines chromatographic separation with mass analysis for exact identification.

Advantages:

  • Extremely high specificity
  • Detects multiple mycotoxins in one run

4. Rapid Test Strips (Lateral Flow Devices)

Best for: On-site testing in farms, factories, or border control.

Principle: Visual or instrumental reading of color lines indicating presence or concentration.

Advantages:

  • Results within minutes
  • No lab expertise required

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Step 5: Data Interpretation and Validation

After detection, results must be interpreted according to international standards such as:

  • EU Regulation (EC) No 1881/2006
  • FDA maximum levels (20 ppb for total aflatoxins)
  • Codex Alimentarius limits

Always include calibration curves, quality control samples, and replicate measurements to validate accuracy.

Step 6: Reporting and Decision-Making

Once results are validated, laboratories classify food batches as:

  • Compliant: Below regulatory limits
  • Non-compliant: Above permissible levels → requires further action (e.g., segregation or disposal)

Conclusion

Aflatoxin detection is a critical step in ensuring global food safety and trade compliance.

From ELISA and HPLC to on-site rapid testing, each method offers advantages depending on the need for speed, precision, and resources.

To simplify your workflow, explore our complete range of Aflatoxin Detection Kits and Accessories:

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Top 5 Methods to Detect Aflatoxins in Food