Clean-up

Gregor Kos and Rudolf Krska

Sample clean-up is the removal of substances, which may interfere with the detection of the analyte. Usually it is also used for preconcentration of the analyte by reducing the amount of solvent. Matrix components such as lipids, carbohydrates and peptides that are usually present in the raw extract (see Extraction fact sheet) make an additional purification step necessary prior to the ultimate separation and detection step.

Liquid-liquid Separation

Liquid-liquid partitioning is a well known and well established clean-up technique. It is based on the partition between immiscible solvents, one of which contains the analyte. The analyte then migrates into the other phase until an equilibrium has been reached. This step can be performed several times with fresh solvent in order to extract the analyte quantitatively. After the partitioning step rotary evaporation is performed to reduce the amount of solvent and to pre-concentrate the analyte. The method is simple and easy to perform with standard laboratory equipment and often still forms part of official methods. However, the method is used less frequently nowadays, because it is labour intensive and large volumes of (sometimes chlorinated) solvents are required. Contamination and sample losses are also common due to adsorptions to glassware). Liquid-liquid partitioning is a batch method and can not be automated. Therefore, the method is now often replaced by less labour intensive techniques such as Solid Phase Extraction (SPE).

Solid Phase Extraction (SPE)

SPE is suitable for the analysis of aqueous samples and can be performed on- or off-line. A typical SPE sequence starts with conditioning of the column (i.e. activating it with solvent). The aqueous sample is then applied and the analyte is trapped together with the matrix. Most of the interferences are removed by a rinsing step, with the analyte staying on the column. Consequently, the analyte is eluted and a further preconcentration step is employed evaporating excess solvent with nitrogen. Vacuum manifolds enable the simultaneous preparation of batches with up to 24 samples. For a number of mycotoxins SPE methods have been developed as a convenient alternative to liquid-liquid separation. Analytes include A and B trichothecenes, zearalenone (ZON), ochratoxin A (OTA) and fumonisins.

C₈ and C₁₈ bonded silica columns are most frequently used as they are very pressure resistant and give reproducible results. SPE does not have any significant drawbacks compared to conventional liquid-liquid separation, but advantages include the consumption of less solvent and the possibility of automation. Less time is needed and percolation of samples in the field is possible.

Ion Exchange Columns

Ion exchange mechanisms are employed, if the analyte can be made present as an ion (e.g. moniliformin, MON). Anionic compounds can be isolated on SAX (strong anion exchange) bonded silica columns. The retention is based on the electrostatic attraction of a charged functional group of the analyte to the charged group on the silica surface of the column. Other species from matrix components of the same charge may interfere with the adsorption and reduce the selectivity. For the elution of the analyte, the bond to the sorbent must be broken. The electrostatic force is disrupted and the compound is eluted. Alternatively, a solution with high ionic strength is used for elution, because of its higher affinity to the sorbent. Ion exchange columns are well suited for the clean-up of samples containing moniliformin. SAX columns are used to isolate strong anionic (very low pKa, <1) or weak anionic (low pKa, >2) compounds (mostly strong or weak acids) and should be conditioned with the sample solvent. It is important to note that the packing should not dry up between conditioning and sample addition and that, after regeneration, columns can be used again several times. SAX columns are used for the determination of ochratoxin A and fumonisin.

Immunoaffinity Columns

Immunoaffinity columns (IACs) for clean-up purposes have become increasingly popular in recent years, because they offer high selectivity. They are easy to use for purification of samples that are contaminated with different mycotoxins. The analyte molecules (i.e. the mycotoxin) are bound selectively to the antibodies on the column after a preconditioning step. As matrix components do not interact with the antibodies, a rinsing step removes most of the possible interferences and the toxin can be eluted by antibody denaturation. Columns feature a higher recovery than standard liquid-liquid partitioning. Single analyte columns include those for aflatoxins, zearalenone, ochratoxin A, fumonisins and deoxynivalenol (DON). Multifunctional columns for the simultaneous determination of ochratoxin A and zearalenone are also available. The fact that columns can only be used once and their relative high costs are major disadvantages. Columns are commercially available.

Mycosep™ Columns

The Mycosep™ multifunctional cleanup columns (Romer Labs) consist of adsorbents, which are packed in a plastic tube. A rubber flange, a porous frit and a one-way valve on the lower end ensure that the extract is forced through the packing material, when the column is inserted into the culture tube. On top of the plastic tube, the purified extract appears within seconds. No additional rinsing steps are required and almost all interfering substances are retained on the column, while the analyte does not show an affinity to the packing material. Columns are usually suitable for one analyte only and are available for a range of mycotoxins such as deoxynivalenol (DON) and patulin.

Other Techniques

A number of alternative clean-up techniques for mycotoxins have been described in the literature: A Microwave-assisted extraction (MAE) method has been developed by Pallaroni et al. for zearalenone (ZON) in wheat and corn and subsequent determination by LC-MS with an atmospheric pressure chemical ionization interface (APCI). Extraction was performed with 1:1 (v/v) methanol-acetonitrile at 80 Deg C for 5 min. Extraction and clean-up is performed in one step.

Supercritical fluid extraction (SFE) is another possibility to reduce the number of sample preparation steps. An SFE method has been published for DON by Krska, where extraction and clean-up are performed with supercritical CO₂, which is non-toxic, non-flammable and chemically inert. An extraction thimble is filled with sample and a modifier (e.g. methanol) is added to the extraction solvent. The analyte is trapped in an SPE silica trap and detected by HPLC-DAD. Obtained recoveries for DON in wheat flour were 53.0±3.2% (n=5). The LOD was calculated to be 250 micrograms/kg.

Summary of Common Clean-up Procedures

Aflatoxins: IAC, SPE

Type A Trichothecenes: SPE, Mycosep columns

Type B Trichothecenes: liquid-liquid separation, IAC (DON), SPE, Mycosep

Zearalenone: liquid-liquid separation, IAC, SPE, Mycosep

Moniliformin: Ion exchange column

Beauvericin: Liquid-liquid separation, SPE, Mycosep

Ochratoxin A: IAC, SPE, Ion exchange column

Fumonisins: IAC, SAX, SPE

Patulin: liquid-liquid separation, Mycosep, SPE

A more detailed approach towards several clean-up techniques can be found in the on-line training course: Sample Preparation Techniques for the Determination of Mycotoxins - Training Course 1

References

[1] R. Krska, Performance of Modern Sample Preparation Techniques in the Analysis of Fusarium Mycotoxins in Cereals, Journal of Chromatography A 815 (1998) 49-57.

[2] S. Ahuja, Trace and Ultra-trace Analysis by HPLC, Chapters 5 (Sample Preparation) and 8.4 (Derivatisation), John Wiley and Sons, New York (1992) 121-160 and 291-294.

[3] A. Visconti; M. Pascale, Determination of Zearalenone in Corn by Means of Immunoaffinity Clean-up and High-performance Liquid Chromatography with Fluorescence Detection, Journal of Chromatography A 818 (1998) 133-140.

[4] W. Langseth, T. Rundberget, Instrumental Methods for Determination of Non-macrocyclic Trichothecenes in Cereals, Foodstuffs and Cultures, Journal of Chromatography A 815 (1998), 103-121.

[5] T.R. Romer, Use of Small Charcoal/Alumina Cleanup Columns in Determination of Trichothecene Mycotoxins in Foods and Feeds. J.Assoc.Off.Anal.Chem., 69 (4) (1986) 699-703.

[6] L. Pallaroni, C. von Holst, C.S. Eskilsson, E. Bjorklund, Microwave-assisted Extraction of Zearalenone from Wheat and Corn, Analytical and Bioanalytical Chemistry 374 (1) (2002), 161-166.

[7] Trucksess M. W., Stack M. E., Nesheim S., Romer T. H.,. Multifunctionnal Column Coupled with Liquid Chromatography for Determination of Aflatoxin B₁, B₂, G₁, G₂ in Corn, Almonds, Brazil Nuts, Peanuts and Pistachio Nuts : Collaborative Study. Journal of the Association of Official Analytical Chemists 77 (6) (1995), 1512-1521