Aflatoxin Testing: Detection Methods, How to Choose, and Regulatory Limits

A batch of peanuts is about to ship, and a single question stands between it and the market: does it carry more aflatoxin than the law allows? The answer can come from a ten-minute strip test at the warehouse or from a precise laboratory measurement to the part per billion, and choosing the right method for the situation is as important as running it well. This guide walks through how aflatoxins are detected, and, just as importantly, how to pick the method that fits the job.

Mycotoxins, the secondary metabolites of fungi, are toxic compounds to animals and humans (mycotoxicosis). A widely cited FAO estimate suggests that a significant share of the world's food crops, historically put at around a quarter, may be affected by mycotoxins, though more recent analyses vary. The ingestion of mycotoxins causes various acute and chronic biological effects, including carcinomas, mutations, genetic variation, and immunological effects.

Aflatoxins are a group of closely related mycotoxins, of which six matter most in food: B1, B2, G1, G2, M1, and M2. Aflatoxin B1 is the most important, because it is the most potent naturally occurring carcinogen known and is classified by the International Agency for Research on Cancer (IARC) as a Group 1 (proven) human carcinogen, strongly linked to liver cancer. The methods used to detect and quantify aflatoxins fall into a few families: chromatographic, immunochemical (immunoassay), immunosensor (biosensor), and spectroscopic techniques.

Aflatoxin (AF)

• Sources of aflatoxins: aflatoxins are produced mainly by the molds Aspergillus flavus and Aspergillus parasiticus (and the less common Aspergillus nomius). A. flavus produces B-type aflatoxins, while A. parasiticus produces both B and G types. These molds contaminate crops such as corn, peanuts, tree nuts, and cottonseed. Note that aflatoxins are essentially Aspergillus toxins; the various Penicillium species produce different mycotoxins (such as citrinin and patulin), not aflatoxins.

• Factors favoring the production: Temperature and grain moisture.

• Types of aflatoxins: B1, B2, G1, G2, M1, and M2 are the family of aflatoxins in food. B-type aflatoxins have a characteristic cyclopentanone (E) ring, and G-type have a xanthone ring in its place, which is the structural difference behind the "B" and "G" names. (Aflatoxin B1's significance as a carcinogen is covered above). AFM1 and AFM2 are the metabolites of AFB1 and AFB2. When a dairy animal eats feed contaminated with AFB1, it excretes AFM1 in its milk, which is why milk and dairy products are tested specifically for AFM1.

• Mechanism of action: the liver activates aflatoxin B1 using cytochrome P450 enzymes, converting it into a reactive form (an epoxide). This reactive form binds to guanine bases in DNA, forming DNA adducts that cause mutations, classically in the TP53 tumor suppressor gene. This damage is what drives liver cancer. Aflatoxins also inhibit protein and nucleic acid synthesis, and high acute exposure damages liver cells directly (aflatoxicosis). Hepatitis B infection strongly increases the cancer risk from aflatoxin.

• Toxicity: AF is more toxic in children than adults. Its susceptibility increases during nutrition deficiency.

• Clinical sign: It decreases the growth rate.The toxin reduces feed efficiency.Mild anemia. Increased susceptibility to infectious disease.

• Management in animal feed: binders such as hydrated sodium calcium aluminosilicate (HSCAS) are added to contaminated feed to reduce absorption in animals. There is no specific antidote for aflatoxicosis in humans; management is supportive, and prevention is the priority.

• Prevention:Inhibition of mold growth.Treatment of grain with anhydrous ammonia for 10-14 days.

Aflatoxin testing Methods at a Glance

Aflatoxin testing always follows the same three steps: extract the toxin from the food, clean up the extract, then detect and measure the toxin. The methods differ mainly in the final detection step, and each suits a different situation.

The rest of this article explains each method in detail, but the table above is the quick answer to "which test should I use?"

Which method should you use?

The right method depends on where you are testing, how precise you need to be, and what you are testing.

For rapid, on-site screening (a warehouse, a grain depot, a border checkpoint): use a lateral flow strip test. It gives a yes/no or approximate answer in minutes with no laboratory.

For screening many samples in a laboratory: use ELISA. It handles up to 96 samples at once and is cost-effective for routine monitoring.

For a precise, legally defensible measurement: use HPLC (often HPLC with fluorescence detection, or HPLC-MS/MS for confirmation). This is the reference approach when an exact concentration is needed, for example to confirm a screening-positive result or for export certification.

For testing specific commodities:

• Peanuts, maize (corn), tree nuts, spices: any of the above; strip tests or ELISA for screening, HPLC to confirm.
• Milk and dairy: test specifically for AFM1, usually by ELISA or HPLC.
• Animal feed: strip tests and ELISA are common for routine checks.

A common workflow combines methods: screen with a rapid test or ELISA, then confirm any positive result with HPLC.

Regulatory limits

Because aflatoxins are so toxic, food and feed are subject to strict legal limits, usually expressed in micrograms per kilogram (μg/kg), which is the same as parts per billion (ppb).

The European Union sets some of the most widely referenced limits. As a general guide, the EU limit for total aflatoxins in many foods is around 4 μg/kg, with a lower limit (around 2 μg/kg) for aflatoxin B1 alone, and a much lower limit for AFM1 in milk (around 0.05 μg/kg). Limits vary by commodity and by country, so always check the specific regulation that applies.

The key point for testing is that the method must be sensitive enough to measure at or below these limits. This is why precise methods like HPLC are used for enforcement, while rapid tests are calibrated to flag samples near the legal threshold.

Materials for Aflatoxin Testing

Aflatoxin in food after consumption poses a significant health risk due to its severe toxicity. So, aflatoxin testing is very crucial in foods. Due to its low concentration, analytical methods like chromatographic, spectroscopic, electrochemical sensors, and immunochemical techniques are preferred. The sample is suspected food material.

The materials required for aflatoxin testing depend on the methods used for extraction, detection, and quantification. The materials needed are as follows:

For extraction and purification

• An organic solvent like methanol, acetone, or acetonitrile mixed or not mixed with water.
• Solid matrix and method/water and acetonitrile/water mixture
• Flask, centrifuge tube, or vials and ultrasonic bath
• Supercritical fluid (CO₂)
• Immunoaffinity column for purification

Chromatographic method

• Frits
• Columns
• Flow cells
• Pumps
• Detectors
• Collectors
• Cylinder with gas (gas chromatography)
• Silica gel, aluminum foil, solvent (thin layer chromatography)
• Solvent (high-performance liquid chromatography)

Spectroscopic analysis

• Spectrophotometer

Immunological detection

• Pipettes
• For ELISA (enzyme-linked immunosorbent assay): Coated plate with 96-wells, reader, conjugate, conjugates, and stop solution.
• For RIA (radioimmunoassay):
• Lateral flow kit

Immunosensor detection

• QCM (quartz crystal microbalance) sensor
• SPR (surface plasma resonance) sensor
• Electrochemical immunosensor

Methods of Aflatoxin Testing

Figure: Different methods of aflatoxin testing

The first step of testing aflatoxin is extraction. Then it is followed by purification and concentration. The final step is detection and quantification.

Extraction and Purification (Clean-up)

Extraction is key to better detection and quantification of aflatoxin in food samples. The extraction and purification of aflatoxin can occur in the following ways.

• Liquid-liquid extraction (LLE): Aflatoxin is highly soluble in polar-protic solutions like acetone, chloroform, methanol, and acetonitrile. These can either be mixed with water or used in their pure state. It does not produce clean analytes.
• Liquid-solid extraction (LSE): A fixed amount of solid food particles is mixed using centrifugal force (mixer or vortex) with an extraction reagent, either acetonitrile/water or methanol/water, in varying ratios. It is the most common method, followed by the purification method like IAC (immunoaffinity column).
• Ultrasound extraction: The mixed sample from LSE containing flask, centrifuge tube, or vial) is dipped into the ultrasonic bath with water. The ultrasound helps quick transfer the toxin from the sample to the extraction reagent.
• Supercritical fluid extraction (SFE): The use of supercritical fluid CO₂ to extract apolar aflatoxin has shown promising results. However, it is not ideal for polar aflatoxins because it has a low recovery of the toxin and a higher concentration of co-extracts.
• Solid phase extraction (SPE): It is a popular method of clean-up or purification. It uses a C-18 (octadecylsilane) column. Its application is in the immunoaffinity clean-up column (IAC) and SPME (solid-phase microextraction). SPME is compatible with chromatographic techniques, and IAC has columns with antibodies specific to the toxins. SPME has high specificity in foods like nuts, spices, dried fruits, and cereals. In contrast, IAC is useful in pasteurized milk. During IAC, the crude extracted sample is applied to the column. The aflatoxin binds to a specific antibody immobilized in the column. A washing step is required after IAC.

Detection and Quantification

Various methods have been applied to detect and quantify extracted and purified aflatoxins. They are chromatographic techniques (TLC, HPLC, and GC), Spectroscopic techniques (fluorescence and infrared spectroscopy), immunological techniques (RIA, ELISA, and lateral flow immunoassay), and immunosensors (electrochemical, optical, and QCM).

Chromatographic Techniques

Chromatographic techniques involve interaction between a mobile and a stationary phase. The mobile phase is usually a liquid, gas, or supercritical fluid that moves around the stationary phase (solid or liquid). The mixture with desired components gets distributed between the two phases (mobile and stationary). The general practice is dissolving the mixture into the mobile phase and applying it as a spot in the stationary phase. The mobile phase carries the mixture along the stationary phase, and the speed of different components of the mixture varies, which is the basis of separation.

The chromatographic techniques used commonly are thin-layer chromatography (TLC), gas chromatography (GC), and high-performance liquid chromatography (HPLC).

Thin layer chromatography (TLC)

TLC has been widely used in detecting aflatoxins in food and has found aflatoxins as low as 1-20 ppb (parts per billion). Its stationary phase comprises silica, alumina, or cellulose immobilized in glass or plastic. The mobile phase used for separating aflatoxins is a mixture of the sample with acetonitrile, methanol, and water. The difference in solubility of aflatoxins determines the interaction time between the mobile and stationary phases. Depending on the interaction time and molecular weight of the toxin, it either adheres to the stationary or mobile phase. The longer it adheres to the stationary phase, the later it separates from the mixture and vice versa. TLC helps to detect different types of toxins in a single test. Although it is sensitive, it requires highly trained professionals and lacks precision due to applying samples, plate development, and interpretation errors.

HPTLC (high-performance thin layer chromatography) has been developed in order to improve the limitation of TLC. The HPTLC is an automated process where sample application, plate development, and interpretation are automatic. HPTLC is currently the most precise and efficient technique for determining mycotoxins.

High-performance liquid chromatography (HPLC)

HPLC is the most common technique used for the separation of organic components. It has an absorbent inside a glass or plastic tube (column) as a stationary phase and an aqueous/organic solvent as a mobile phase. The sample is injected into the stationary phase and flows through it with the help of the mobile phase. A pump provides high pressure for fast movement of the mobile phase through the column (stationary phase). The separation occurs due to the different affinities of the components toward the stationary and mobile phases. The higher the affinity, the longer analyte reacts with the particular phase. If an analyte interacts longer with the mobile phase, it separates faster.

The time taken for the elution (separation followed by removal from the setup) of analytes (mixture) is called retention time. The retention time is detected using UV, fluorescent, or diode array detectors. HPLC is used widely in the determination of aflatoxins. However, it requires rigorous sample purification using immunoaffinity columns and tiresome pre and post-column derivatization for detecting AFB1 and AFG1 due to the use of UV for detection. Hence to overcome the derivatization, HPLC is combined with mass spectroscopy limiting the sample size for generating structural information. But HPLC-MS/MS is bulky and suitable only in laboratory environments.

Gas chromatography (GC)

In GC, the mobile phase is carrier gas, and the stationary phase is liquid coated on inert solid particles confined to a long stainless steel or glass tube (column). The stationary phase needs to maintain an appropriate temperature for separation. The sample is vaporized into its gaseous state and carried by the carrier gas along the stationary phase. Like all chromatographic techniques, the basis of the separation of components is their distribution among the two phases. The sample’s movement depends on the components’ affinity with the stationary phase. The higher the affinity, the slower the movement and the longer it takes to separate and vice versa. Once the separation completes, the separated particles are detected using FID (flame ionization detector), or electron capture detector (ECD), and a mass spectrometer. Since aflatoxins are non-volatile, these may need derivation for detection and are less commonly used.

Spectroscopic Techniques

Fluorescence spectroscopy

Aflatoxins are fluorescing compounds that emit light of various wavelengths after absorption. The fluorometric method help quantify aflatoxins in the range of 5-5000 ppb. However, derivatization is required for better analysis, and the limit for detection is slightly higher than the limit of 4 μg/kg set by European standards.

Infrared spectroscopy

Infrared spectroscopy depends on the difference in molecular vibrations after irradiation with infrared. Atomic size, bond length, and strength vary from one molecule to another. The absorption rate of infrared radiation by a particular bond depends on the bond type and mode of vibrations. So, different frequency ranges of infrared radiation are passed through the sample during the analysis of compounds using an infrared spectrometer. The absorbed radiant energy by every bond of the sample molecule is then measured. The measurement helps construct the spectrum graph, and no two organic molecules have the same spectrum, helping identify the organic compound. It has been used in determining aflatoxins in peanuts, peanut cakes, and corn kernels.

Immunological Techniques

Immunological techniques involve the specific binding of antibodies and antigens. Various immunological methods have been developed based on the specificity and high-affinity interaction between antibodies and antigens and are expanded to receptor-ligand bonding. These interactions can also be quantified using the absorbance of photons of light energy by spectrophotometer. The other ways of detection and quantification are labeling with radioisotopes, fluorophores, and enzymes. The immunochemical methods used commonly are radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunosensors, and ICA (immunocolumn assay).

Radioimmunoassay (RIA)

RIA relies on the competitive binding of the radiolabeled antigens and non-radiolabeled antigens with available antibodies. The unlabeled antigen competes with the radioactive antigen for binding with a fixed number of antibodies. The amount of radiolabeled antigen and antibody is known, whereas the quantity of non-labeled antigens is unknown, but the amount is inversely proportional to the amount of radiolabeled antigens. RIA is used in the qualitative and quantitative determination of aflatoxins. Although the RIA can analyze multiple samples at once, these require antigens in the pure state, and the radioactive isotope is hazardous and hard to dispose of and store.

ELISA (enzyme-linked immunosorbent assay)

Another immunochemical method that overcomes the hazardous limitation of RIA is ELISA, where an antigen or antibody is bound with enzymes. The principle of ELISA is similar to that of other immunochemical methods that rely on specific antigen-antibody binding and increased sensitivity due to antigen or antibody linked with an enzyme that combines with a particular substrate.

ELISA is available in a kit and can easily detect aflatoxins in agricultural products. Although it requires many washing steps, it is cost-effective, can analyze 96 samples simultaneously, and carries no health hazards.

Lateral flow devices (kit)

Lateral flow devices are immunochromatographic assays. The principle is based on the antigen-antibody interaction. These consist of a porous membrane for flowing samples, an absorbent pad for increasing the volume of flowing liquid, and a sample pad to ensure contact between the liquid sample and the membrane. The devices use colloidal gold or gold-coated antibodies to detect the red-colored binding zones. The sample moves through the sample pad due to capillary action. When the sample with aflatoxins reaches the gold particles, these bind with the particle giving the red colored line. A device made by Delmulle et al., can detect 5 μg/Kg AFB1 in pig feed within 10 minutes. These devices are easy to use, cost less, and provide rapid on-site detection.

Immunosensor Techniques for Detection

Immunosensors are biosensors that use antigens or antibodies as recognition components combined with signal transducers like graphite, gold, or carbon. These help to detect binding between complementary species. Based on the signal transduction used, immunosensors are of three types; piezoelectric, optical, and electrochemical.

Electrochemical immunosensors

These devices use antibodies added in a biorecognition layer for producing electroactive signals that are detectable by transducers resulting in the generation of measurable signals. Aflatoxin analysis can be done using various types of electrochemical immunosensors. The most common type consists of antibodies immobilized to the surface of the glassy carbon electrode, and an enzyme is a biological component used. However, there has also been the construction of non-enzymatic electrochemical immunosensors using chitosan, gold particles, anti-aflatoxin B1 and Fe3O4 as electrodes.

Optical immunosensors

Surface plasmon resonance (SPR) is the optical immunosensors used for detecting aflatoxins. It depends on measuring the refractive index produced after binding of the analyte with its biospecific partner (specific antibodies) that is immobilized in the sensor surface. It is used in detected AFB1 using monoclonal or polyclonal antibodies of AFB1. OWLS (optical waveguide light-mode spectroscopy) is another label-free biosensor that relies on fluorescence excitation for measuring the binding events at the surface of the waveguide. OWLS detects aflatoxins and ochratoxins; the detection range is 0.5-10 ng/ml in wheat flour and barley.

Piezoelectric QCMs (quartz crystal microbalances)

These are label-free devices used in detecting antigens. T depends on the change in mass of the electrode when antigens react with the immobilized antibody in the quartz crystal surface. The difference in mass is proportional to the concentration of the antibody-antigen complex. QCM sensor is coated with gold for detecting aflatoxin B1 in the range of 0.5-10 ppb.

How to Remember

Extract, clean up, detect. Every aflatoxin test follows these three steps. The methods differ mainly in the last one.

Field, lab-screen, lab-confirm. Strip test in the field, ELISA to screen many samples, HPLC to confirm precisely.

B1 is the worst. Aflatoxin B1 is the most potent natural carcinogen, a Group 1 human carcinogen, and it targets the liver.

M is for milk. AFM1 is the milk metabolite of B1, and it is what dairy is tested for.

Aspergillus, not Penicillium. Aflatoxins come from Aspergillus flavus and A. parasiticus. A. flavus makes B only; A. parasiticus makes B and G.

Key exam facts

Where Students Get Confused

"Aflatoxins come from Penicillium and Aspergillus." No. Aflatoxins are produced by Aspergillus species, mainly A. flavus and A. parasiticus. Penicillium species produce different mycotoxins, such as citrinin and patulin.

"A positive strip test proves the food is illegal." Not by itself. Strip tests and ELISA are screening methods. A positive screen is confirmed with a precise method like HPLC before a regulatory decision.

"All aflatoxins are equally dangerous." No. Aflatoxin B1 is the most potent and the most carcinogenic, which is why it is regulated most strictly and often measured on its own.

"Milk is tested for aflatoxin B1." Milk is tested for AFM1, the metabolite of B1 that appears in milk after an animal eats contaminated feed.

"HPLC is always the best choice." It is the most precise, but it is slow, costly, and needs a lab. For rapid field screening, a lateral flow strip is the better tool. The best method depends on the situation.

References and further reading

• Wacoo, A. P., Wendiro, D., Vuzi, P. C., and Hawumba, J. F. (2014). Methods for detection of aflatoxins in agricultural food crops. Journal of Applied Chemistry, 2014, 706291.
• Miklós, G., et al. (2020). Detection of aflatoxins in different matrices and food-chain positions. Frontiers in Microbiology, 11, 1916.
• International Agency for Research on Cancer. (2012). Aflatoxins. In IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Vol. 100F.
• Food and Agriculture Organization / World Health Organization (Codex Alimentarius). Standards for mycotoxins in food.