Extraction

Gregor Kos and Rudolf Krska

Together with an effective clean-up step, extraction of samples is an integral step in optimum analysis for mycotoxins. Extraction can be performed when two immiscible liquid phases or a solid and a liquid phase are present. Depending on the conditions, the analyte (and any compounds with similar properties) will migrate into the extraction solvent until equilibrium is established. This way, desired compounds can be concentrated in a solvent and interferences can be removed. This is essential, when dealing with biological samples, which usually are complex systems with numerous (often similar) compounds present. It is the main objective in mycotoxin sample preparation to remove interferences in order to make a chromatographic separation and subsequent detection and identification as unambiguous as possible.

Liquid-solid Extraction

This technique is one of the basic operations in mycotoxin analysis. It is performed, if the sample is available in a solid form, which is the case for cereals including maize, and most other agricultural products. If solid samples are not available, freeze-drying or dehydration is sometimes an option for making the sample easier to handle.

It is the purpose of this purification step to dissolve the analyte quantitatively (i.e. the mycotoxin, which is to be analysed) in the solvent, with as few little additional compounds as possible in order to avoid interferences. Therefore the choice of solvent is an important question consideration. Polar analytes favour polar solvents and also the pH plays a key role during extraction, because it determines the species in which the analyte is present in. Consideration of pH is necessary, especially for polar mycotoxins.

In the eluotropic list, solvents are sorted according to their polarity, starting with the lowest. Examples are given in the on-line training course on Sample Preparation Techniques for the Determination of Mycotoxins

Ideally the solvent should extract the analyte quantitatively and it should easily be recoverable, non-toxic and non-flammable. Depending on the conditions other aspects may be important as well, such as:

• Volatility
  Volatility makes it easier to reduce the volume after extraction, which is a necessary preconcentration step for clean-up and analysis. Highly volatility requires special safety precautions (e.g. working in a fume cupboard), especially if the solvent is toxic.

• Stability
  Extraction and clean up procedures take up most of the analysis time and therefore the solvent must be stable for the duration of the entire process. If heating steps are included, thermal stability is also an aspect.

• Transparency to UV light
  No absorbance at the analytical wavelength of the analyte has to occur, if the solvent is to be used for chromatography with a UV detector.

Shaking or blending are the most common procedures used for extraction. Mechanical tube rollers or gentle end over end mixing are widely used. A compromise between the ideal time (leading to quantitative extraction) and what is practical can easily be found by plotting the transfer of the analyte into the solvent against time. Trenholm et al [3] have investigated the extraction time for deoxynivalenol and found that naturally contaminated samples needed a longer extraction time than spiked samples. Therefore it is recommended that the extraction procedure is evaluated using samples that match, as closely as possible, the actual conditions of analysis.

Ionisable compounds (e.g. moniliformin) can also be extracted into organic solvents as neutral ion pairs in the presence of ion-pairing agents. A suitable buffer, which controls the pH is necessary.

Common extraction solvents are:

Aflatoxins: Acetonitrile/water, methanol/water

Type A Trichothecenes: Acetonitrile/water, methanol/water

Type B Trichothecenes: Acetonitrile/water, water/PEG, chloroform/methanol

Zearalenone: Ethyl-acetate, methanol, acetonitrile, chloroform and mixtures thereof

Moniliformin: Methanol,acetonitrile/water, water, water/tetra-butyl ammonium hydroxide (TBAH)

Beauvericin: Acetonitrile/water, methanol

Ochratoxin A: MTBE, chloroform, acetonitrile/water, mixtures of toluene/HCl/MgCl₂

Fumonisins: Methanol/water (3:1), acetonitrile/water (1:1)

Patulin: Ethyl-acetate, acetone

Liquid-liquid Extraction

Solvent Extraction is a useful technique for the analysis of liquid samples, such as milk and apple juice. The method is based on the relative solubility of an analyte in two immiscible liquids and is used to remove interferences and concentrate the analyte prior to analysis. Examples of use of this technique include the analysis for patulin in fruit juices or aflatoxin M₁ in milk. Usually multiple extractions are necessary. In the end all fractions are pooled for further treatment (e.g. clean-up).

During extraction good mixing is essential, but if shaking is too vigorous, the tendency to form emulsions increases. Gentle mixing is therefore recommended.

References

[1] Ahuja, S., Trace and Ultra-trace Analysis by HPLC, in: Chemical Analysis Series, Vol. 115, Wiley, New York, chapter 5 (1999)

[2] De Levie, R., Principles of Quantitative Chemical Analysis, McGraw-Hill, New York, chapter 10 (1997)

[3] Trenholm H.L., Warner R.M., Prelusky, D.B., Assessment of extraction procedures in the analysis of naturally contaminated grain products deoxynivalenol (vomitoxin), Journal of the Association of Official Analytical Chemists, 68 (4), 645-649 (1985)

[4] Krska R., Baumgartner S., Josephs R., The State-of-the-art in the Analysis of Type-A and -B Trichothecene Mycotoxins in Cereals, Fresenius Journal of Analytical Chemistry, 371, 285-299 (2001)