Overview of analytical methods for mycotoxin contamination in maize and peanuts

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There are two main types of assay which have been developed for detection and/or determination of mycotoxins, viz biological and chemical. Bioassay techniques, are only qualitative or semi-quantitative and are often non-specific. They are useful for indicating the presence of unknown toxins, and are useful in studies to isolate new mycotoxins. Once a new toxin has been identified, it is then usually possible to develop a suitable chemical assay for its detection and quantification. The chemical assay is almost invariably quicker, cheaper, more specific, more reproducible, and more sensitive than the corresponding bioassay. Chemical assays are suitable for routine analyses, as required for quality control and survey work. Immunoassays, are a combination of a chemical and a biological assay. They are very sensitive and can be specific, making them suitable for screening body fluids etc. for traces of toxin.

CHEMICAL ANALYSES

All methods of chemical analysis for mycotoxins incorporate a combination of procedures, as indicated in the flow-chart in Figure 1.

Sampling and Sample Preparation

This aspect of the analysis is discussed in detail in the previous section. However, it must be emphasised here that this is a most important step. If the sample taken for analysis is not representative of the bulk, then the analytical results are meaningless. Because of the very uneven distribution of mycotoxins that are usually found in naturally contaminated commodities, it is essential to follow a suitable sampling plan. Sample preparation involves grinding and sample division to obtain a representative analytical sample. The use of water slurries enables larger, and therefore more representative analytical samples to be taken. Typically, 1 kg of ground material is blended at high speed with an appropriate amount of water to give an homogeneous slurry (paste) from which 100g aliquots are taken for analysis.

Extraction

The organic solvents most commonly used for extraction of mycotoxins are:chioroform, acetonitrile, methanol and acetone. These solvents are mixed with a given ratio of a more polar solvent (water, dilute acid, aqueous solution of salts) to aid the breaking of weak electrostatic bonds which bind some mycotoxins to other substrate molecules (eg proteins).

Figure 1. Flow diagram for Mycotoxin Analysis

The ground sample, or preferably an aqueous slurry, is either shaken with the extraction solvent for 30-45 minutes or blended at high speed for about 3 minutes. It should be noted that an explosion proof blender is recommended for use with inflammable solvents such as acetone and methanol.

Clean-Up

Mycotoxins are such a diverse group of chemical compounds that it is difficult to find a simple procedure which specifically removes non- mycotoxin "interfering" compounds whilst leaving the mycotoxins in the extract. For this reason it is difficult to find a good method for screening a wide-range of mycotoxins simultaneously. It is possible, however, to devise procedures which remove interfering non-mycotoxin compounds from the extract of a particular commodity and leave a particular mycotoxin or group of mycotoxins in the extract.

Common clean-up techniques which have been used are:

1. Defatting-this is carried out prior to the toxin extraction step and uses petroleum ether or hexane to extract lipids from the sample using a Soxhlet extractor. This step is only required when the subsequent clean-up step is not capable of removing the lipids.
2. Column-chromatography-this technique has very wide application, and is used in a number of regulatory or officially approved methods. A glass column is packed with one or more adsorbent materials and the crude extract is added to the top of the column. The column is then eluted with a series of solvents or solvent mixtures which are designed to first wash off interfering compounds and then elute the desired mycotoxins, whilst other interfering compounds remain strongly bound on the column.
   A miniature column, called a mini-column is used in many rapid aflatoxin assay methods to remove interfering compounds and to qualitatively detect aflatoxin down to a few parts per billion (ppb)
3. Precipitation-this is an extremely useful technique whereby certain chemicals, sometimes in colloidal form, are added to the crude extract and these absorb certain pigments, proteins and other interfering compounds onto their surface. The complex so formed precipitates out of solution and can be filtered off, leaving a "cleaned-up" solution. Useful precipitating agents include:cupric carbonate, ammonium sulphate, lead acetate, and ferric gel.
4. Liquid-liquid partition-is commonly used, often in conjunction with one of the other clean-up procedures, to provide additional clean-up and also to transfer toxins from one solvent system to another whilst at the same time effecting a considerable increase in concentration of the toxins. The partition is carried out in a separating funnel which contains the two immiscible solvents. The funnel is shaken for a few minutes to allow the dissolved compounds, including the mycotoxins, to partition between the two phases. The solvents are selected so that the mycotoxins are preferentially partitioned into one of the solvents. Care must be taken in the choice of solvents in order to minimise the risk of emulsion formation.
5. Thin layer chromatography (TLC) -although this technique is used mainly for detection and quantification of toxins it is also useful for separating the mycotoxins from interfering compounds in the extract. Sometimes it is necessary to try a range of developing solvents in order to separate the toxins of interest from the interfering compounds. If this proves impossible, or too time consuming, then twodimensional TLC may be applicable.
6. Other clean-up procedures.
   The clean-up procedures outline above are those employed in officially approved methods for aflatoxin analysis, and they are also used in analytical methods for many other mycotoxins. Other clean-up procedures, including reverse dialysis and base extraction are explained in the next period.

Work-Up

After the clean-up step the extract must be "worked-up" in order to prepare it for the detection and/or quantification step. Following clean-up the extract is often dissolved in a large volume of aqueous solvent, so it must be transferred into a small volume (10-50ml) of a volatile solvent, such as chloroform. The chloroform solution often requires drying and this is achieved by passing it through a bed of anhydrous sodium sulphate. The solvent is evaporated off, as described below, to near dryness. Care must be taken at this stage as some mycotoxins, eg the aflatoxins, can break-down if the dry extract is heated at 100°C. To avoid this the evaporation is best carried out using a rotary evaporator at 30.C to reduce the volume to a few ml, followed by evaporation to dryness in an inert atmosphere using a sample concentrator. If such items of equipment are not available, then evaporation should be carried out using a steam-bath, preferably under a stream of nitrogen, and great care should be taken to ensure that flasks are removed from the steam-bath just before they go dry, and that vials are removed on the point of dryness. The extracts are now ready for detection and quantification.

Detection and Quantification

It is fortunate that the aflatoxins and many of the other important mycotoxins are fluorescent under ultraviolet light. This enable them to be detected at very low levels (parts per billion), and this method of detection is used in the majority of analytical methods for mycotoxins.

Detection or "qualitative assay" is usually by TLC or mini-column and only requires the use of a qualitative (not accurately determined) standard. Such a standard is also sufficient to enable a semi-quantitative assessment to be made by TLC, using a "dilution-toextinction" principle. Fully quantitative determinations, which require the use of a standard of known concentration, can be carried out by a variety of techniques including:TLC high performance liquid chromatography (HPLC), high performance thin layer chromatography (HPTLC), and the fluorotoxinmeter (FTM). These, and other quantitative techiques are discussed in subsequent sections of this manual.

Confirmation

It is essential that confirmatory tests be carried out if a mycotoxin is thought to have been detected, especially when a new commodity, or new source of commodity is being analysed. Failure to do this could easily lead to false-positive results (ie., an interfering compound is mistaken for a known mycotoxin).

Most confirmatory tests involve the formation of a derivative which has different properties (eg., colour of fluorescence or polarity), than the presumptive mycotoxin. When detection and/or quantification is by TLC, then derivatives can be formed "on-the-plate" either by spotting a reagent on the plate before development or by spraying on a reagent after development. An example of the former approach is the use of trifluoroacetic acid (TFA) to form the hemi-acetal derivatives of aflatoxin B1, G1 amd M1 which are identified, if present, as slower running fluorescent spots. A commonly used spray reagent for the aflatoxins is 50% sulphuric acid which reacts with the blue and green-blue fluorescent aflatoxins to give yellow fluorescent derivatives.

DETERMINATION OF THE PURITY AND CONCENTRATION OF MYCOTOXIN STANDARDS

Pure mycotoxins are available from several manufacturers (listed in Appendix: 2) and these are used to prepare standard solutions for use in mycotoxin analytical procedures. It is essential that the exact concentrations of the standard solutions are known; these may be determined by UV absorbance measurements on a spectrophotometer.

THEORETICAL ASPECTS OF CONCENTRATION MEASUREMENTS

There are two laws which describe the absorption of light by matter.—

Lambert's Law: The proportion of monochromatic light absorbed by a homogeneous medium is independent of the intensity of the incident light, and each successive unit layer absorbs an equal fraction of the light incident upon it.

Beer's law: The fraction of the incident light absorbed by a solute in a transparent solvent depends on the concentration of the solution (c) and on the path length (1).

These two laws can be combined to give the "socalled" Beer-Lambert Law which leads to the expression

A = log₁₀ (l_o/l) cl = kcl

where

A = The absorbing capacity, absorbance or optical density of the solution
I = intesity of the transitted radiation
l_o = intesity of the transmitted radiation
c = concentration of the absorbing substance
I = path length of the solution
k = extinction coefficient of the absorbing substance (a constant)

In practice, the quantities actually measured are the relative intensities of the radiation beam transmitted by a cell full of pure solvent, and by an indentical cell full of the solution. When these intensities are taken as lo and I respectively (see Figure 1), the resulting absorbance, which is generally read directly from the logarithmic chart paper, is that of the dissolved solute only. In manual UV single-beam spectrophotometers the absorbance scale is first 'zeroed' with the solvent blank alone. The solvent blank is then replaced by the cell containing the solution and the absorbance read off.

FIGURE 1: Measurement of absorbance (A). The right hand diagram represents an absorption band as recorded by a double-beam spectrophotometer A = log₁₀ (l_o/l)

The extinction coefficient (k), at a given wave length, is a constant, characteristic of the substance under examination. When 1 is expressed in centimetres and the concentration as a percentage, k is called A, cm 1% cm; the optical density for a 1% w/v solution in a 1cm cell. If the molecular weight of the substance under examination is known, the concentration can be expressed in g. moles/litre and k is then called the molar absorptivity and is designated E (the optical density for a molar solution in a Icm cell). It follows that:

CALIBRATION OF THE SPECTROPHOTOMETER

In order to avoid errors in determining the concentration of the mycotoxin standards, the spectrophotometer which is to be used should be checked to ensure that it is functioning correctly. This is done using three serially diluted solutions of potassium bichromate to confirm that absorbance of the solutions is directly proportional to the concentration. The correction factor (CF) should be as near to 1.0 as possible (ie no correction to any calculation being necessary).

Method

Make up the solutions of potassium dichromate in 0.018N sulphuric acid as described in Appendix:1. Determine the absorbance of the three solutions at the wavelength of maximum absorption (about 350 nm) using 0.018N sulphuric acid as the reference blank.

Calculate the absorptivity (E) for each of the solutions using the equation.

Take an average of the three values obtained to give E

Determine the correction factor (CF) for the particular instrument and cells used by applying the equation:

If CF is less than 0.95 or greater than 1.05 check the instrument or the technique.

DETERMINATION OF CONCENTRATION OF MYCOTOXIN STANDARD SOLUTIONS

If the standard is received as a dry film in a vial or as a crystalline sample, add a small known volume of appropriate solvent by means of a syringe through the rubber septum of the vial to dissolve the standard. Quantitatively withdraw a known volume of the concentrated solution and transfer it to a volumetric flask and dilute accordingly to give the required concentration.

This method of dissolving the pure standard minimises the hazards of handling highly toxic material. Measure the absorbance of the mycotoxin standard solution at the wavelength of the maximum absorption, the majority of mycotoxins have a maximum absorption at wavelength between 200-400 nm and details of these and other spectrophotometric parameters are given in Table 1. Calculate the concentration using the equation:

Where CF is the correction factor obtained above and E is the absorptivity of the particular toxin being examined in the Particular solvent used.

Table 1: Spectorphotometric Parameters for Various Mycotoxins

Nb If the standard is dissolved in benzene: acetonitrike 98:2, then use this solvent as the reference blask.

MYCOTOXIN STANDARD SOLUTION CONCENTRATIONS

Although the list in Table 2 below does not necessarily correspond with the concentrations quoted in the literature, we have found them to be most useful.

1. Stock solutions - these are made directly from the pure crystals or dry film.
2. UV solutions - these are direct dilutions from the stock solution and are those which are used for determining the correct concentration of standard by UV spectrophotometric measurement.
3. TLC working solutions - these are made by dilution from either the stock or the UV solutions.

TABLE 2: CONCENTRATIONS OF MYCOTOXIN STANDARD SOLUTIONS

0.05 65 Available from suppliers.

REFERENCES

1. "Microbial Toxins" Vols. Vl, Vll, VIII (ed Ciegler, Kadis and Aji) Academic Press, New York, 1917
2. "Mycotoxins" (ed Purchase), Elsevier, Amsterdam, 1974.

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