Aflatoxins

Species	Formula	CAS Number	MW [g/mol]
B1	C17H12O6	1162-65-8	312.3
B2	C17H14O6	7220-81-7	314.3
G1	C17H12O7	1165-39-5	328.3
G2	C17H14O7	7241-98-7	330.3
M1	C17H12O7	6795-23-9	328.3
M2	C17H14O7	6885-57-0	330.3

Sampling

Sampling and sample preparation remain a considerable source of error in the analytical identification of aflatoxins. Therefore a systematic approach is necessary for the determination at low levels (i.e. in the (sub-)micrograms/kg range). Specific plans have been developed for commodities such as corn and peanuts and have been included in directives of the European Union (Directive 98/53/EC, 16 July, 1998). The distribution of aflatoxin M1 in liquid milk is reasonably homogeneous, resulting in less uncertainty.

A number of analytical methods have been developed for the determination of aflatoxins in food and feed. The species aflatoxin B₁, B₂, and G₁, G₂ can be determined simultaneously with some HPLC methods, whereas the species M₁ and M₂ for dairy products require a different protocol. All methods have in common that the extraction with organic solvents is followed by a clean-up step before separation can take place using a chromatographic method. Immunochemical methods (e.g. ELISA) have also been developed for screening purposes.

Rapid Methods

A large number of antibody-based tests are commercially available that can identify and measure aflatoxins in food within a very short time. These tests are based on the affinities of antibodies for aflatoxins.

1. Aflatoxin B1

2. Aflatoxin B2

3. Aflatoxin G1

4. Aflatoxin G2

5. Aflatoxin M1

6. Aflatoxin M2

Structural formula of aflatoxins:

Three types of immunochemical methods are available: Radio immunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and immunoaffinity column assay (ICA).

Thin layer chromatography (TLC), is a technique, which is easy to use and to identify and quantitate aflatoxins at levels as low as 1 ng/g. Other TLC methods involve 2 separation steps using a mixture of ether, methanol and water for the first step and chloroform and acetone for the second step after extraction and clean-up.

Extraction

Extraction usually involves conventional procedures using acetone, chloroform and methanol or mixtures thereof. Small amounts of water give higher extraction efficiencies. Dunne et al. have described a multi-mycotoxin method, which uses hydrochloric acid and dichloromethane for extraction.

Clean-up

The use of solid-phase extraction (SPE) with C₁₈ cartridges, or immunoaffinity columns (IAC) is now well established in aflatoxin determination. Mycosep™ columns, which remove matrix components efficiently and can produce a purified extract within a very short time, are also available. Conventional clean-up with silica columns has also been reported.

Liquid Chromatography (HPLC)

Liquid chromatography methods for the determination of aflatoxins in foods include Normal-Phase and Reversed-Phase HPLC. Pre- or post-column derivatisation is usually performed for low-level detection, because aflatoxins are rather weak emitter of fluorescent light. Derivatisation can take place using Br₂, I₂ (for B and G aflatoxins) or trifluoro-acetic acid (TFA for aflatoxin M₁). The emitted light is detected at 435 nm after excitation at 365 nm. With derivatisation detection is possible in the ng/g range. Stationary phases for HPLC usually include C₁₈ material with mobile phases being mixtures of water, methanol or acetonitrile.

Identification with Mass Spectroscopy (LC/MS) is becoming increasingly popular. Methods feature high specificity, good reliability and possibility of automation. They are sensitive enough to operate in concentration ranges, where legal limits were established for commodities like peanuts, figs and even spices. Nevertheless the application of these methods is expensive and requires expert knowledge.

Standardisation/Reference Materials

For the purpose of quality assurance a number of certified reference materials have been produced for different matrices that allow the user to check the method’s performance. These materials include aflatoxin M₁ in chloroform and milk powder and aflatoxin B₁ in peanut meal and compound feed. Reference materials are for sale at the IRMM (Institute for Reference Materials and Measurements; http://www.irmm.jrc.be/).

A number of methods (TLC, HPLC) for various types of matrices have been considered AOAC methods (e.g. AOAC Method II, aflatoxin in peanuts and peanut products no. 26.030). The AOAC-IUPAC method 991.31 using post-column derivatisation with iodine was adopted as an official method.

Four methods are currently investigated by CEN to become standardised methods:

• Foodstuffs - Determination of aflatoxin M₁ in milk and milk powder - TLC method (ISO/NP 14674), under development
• Milk and milk products - Guidelines for a standardized description of competitive enzyme immunoassays - Determination of aflatoxin M₁ content (ISO/DIS 14675:2001), under approval
• Animal feeding stuffs - Determination of aflatoxin B₁ content of mixed feeding stuffs - Method using high-performance liquid chromatography (ISO 14718:1998), under approval
• Foodstuffs - Determination of aflatoxin B_1/ and the sum of aflatoxin B₁, B₂, G₁ and G₂ in peanut butter, pistachio paste, fig paste, and paprika powder - High performance liquid chromatographic method with post column derivatisation and immunoaffinity column, prEN 14123, under approval

Safety Issues

Aflatoxins, especially aflatoxin B₁ are carcinogenic and genotoxic compounds. Therefore safety is an important issue for scientists working with aflatoxin-contaminated samples. Exposure to the toxins and to the producing microorganisms must be minimised.

References

[1] USDA, Grain Fungal Disease and Mycotoxin Reference, GIPSA Technical Services Division, Kansas City, MO (1999)

[2] C. Dunne, M. Meaney and M. Smyth, Multimycotoxin Detection and Clean-up for Aflatoxins, Ochratoxin and Zearalenone in Animal Feed Ingredients Using High Performance Liquid Chromatography and Gel Permeation Chromatography, Journal of Chromatography, 629, 229-235 (1993)

[3] K.A. Scudamore, S. Nawaz and M.T. Hetmanski, Mycotoxins in Ingredients of Animal Feeding Stuffs: II: Determination of Mycotoxins in Maize and Maize Products, Food Additives and Contaminants, 15, 1, 30-55 (1998)

[4] CEN standardisation work programme: http://www.cenorm.be

[5] K. Reif, W. Metzger, Determination of aflatoxins in medicinal herbs and plant extracts; Journal of Chromatography A, 692, 131-136 (1995)

[6] J. Jaimez, C.A. Fente, B.I. Vazquez, C.M. Franco, A. Cepeda, G. Mahuzier, P. Prognon, Review: Application of the Assay of Aflatoxins by Liquid Chromatography with Fluorescence Detection in Food Analysis, Journal of Chromatography A, 882, 1-10 (2000)