Deoxynivalenol and Zearalenon Immunoaffinity HPLC Column

$240.00

The Eagle Biosciences Deoxynivalenol (DON) and Zearalenon (ZON) immunoaffinity column determination in food and feed focuses on the enrichment step of extract using immunoaffinity column (IAC) and quantification with HPLC. The Deoxynivalenol (DON) and Zearalenon (ZON) immunoaffinity column is for research use only.

SKU: BTCD323005 Categories: , ,

Deoxynivalenol and Zearalenon Immunoaffinity HPLC Column

3ml, 10 columns

For Research Use Only

Product Developed and Manufactured in Germany by BioTeZ Berlin-Buch GmbH

Additional Information

Assay Instructions

This instruction of Deoxynivalenol (DON) and Zearalenon (ZON) determination in food and feed focuses on the enrichment step of extract using immunoaffinity column (IAC) and quantification with HPLC. Accepted laboratory extraction methods could be maintained. Full performance of the IAC column is given if pronounced criteria regarding organic solvent tolerance, elution process of analyte and working range of column is followed. Many pretreatment methods of DON and ZON determination in food and feed show low sensitivity because of interfering substances if problematic matrices are applied.

This method of content determination of DON and ZON combines the high selectivity of an immunoaffinity column (IAC) with its potential to concentrate elute and additional step of purification by HPLC column.

Please notice that this instruction focuses on the handling with the IAC column. For the commodity extraction step a literature method is given. The given apparatus (e.g. HPLC system) might serve as example among other possibilities.

Extraction (Literature method given)
Assuming that 25g ground corn sample are extracted by a total of 100ml acetonitrile/water (75/25, v/v), as reported by Krska et al. If organic solvent proportion is varied the dilution of extract with PBS should be adapted accordingly in the enrichment step. On the other hand, if proportion of sample quantity and volume of extraction solvent is altered, calculation of gram equivalents must be corrected.

Enrichment Step Immunoaffinity Chromatography (IAC)
An aliquot of 1ml extract (see above, contains the quantity of toxins of 0.25g commodity) are diluted with 9ml 50mM PBS (pH=7.4) and then applied in a reservoir on top of the B-TeZ IAC DonZon column.

To maintain full performance of the column, please take care that proportion of dilution buffer in the solution on top of the column is not to small. A proportion of 7.5% acetonitrile, resulting in this example enrichment, does not affect column performance.

The proportion of organic solvent of PBS diluted extract, which is applied on the column, should not exceed 15% methanol or 15% acetonitrile.

If organic solvent proportion lies above these limits, recovery rates are diminished. Increase of diluted extract volume by diluting extract with additional PBS, on the other hand, has almost no consequences to column performance.

If samples are to be prepared simultaneously, manifold of J.T. Baker for 12 samples has proven of value. Rate of flow through the affinity gel is 1 to 3 ml/min. In case of problematic matrices rate of flow should lie below 2ml/min.

Caution! Be aware that no big air bubbles are neither in the gel nor between gel and luer lock outlet of column which prevent a permanent flow or necessary exchange of matter.

Depending on application and on expected contents, larger or smaller extract aliquots can be applied. In such cases the sample calculation (see below) must be adapted.

Wash
After whole sample has passed through the gel the latter is washed with 20ml of deionized water. Remaining liquids in the gel are removed by applying either pressure from top of the column or underpressure from bottom.

Elution
Sample reservoir on top of the B-TeZ IAC DonZon column is removed and an appropriate vial is placed below the affinity column. The bound toxins are eluted by using a total of 1.5ml of methanol as elution solvent. The elution process is performed in two steps to ensure complete release of analytes. First, a volume of 0.5ml elution solvent is applied. After that volume has passed through column half a minute is waited before the second portion of 1ml of elutions solvent is eluted through the column. Remaining solvent solutions should be eluted by application of slight under- or overpressure. All methanolic fractions are unified to give the column eluate.

The column eluate may be injected into the HPLC directly or in case concentrations are low it may be concentrated by evaporation, e.g. using VLM evaporator at 50°C under a permanent stream of nitrogen.

Caution: As long as evaporation process is performed moderately, it is not necessary to add a keeper. If that is not the case, it is recommended to use a keeper, e.g. 100μl deionized water or PBS.

The residue then is redissolved in HPLC solvent (e.g. 0.5ml) and an aliquot is finally injected into the system.

Cross Reactivity

DON: 100%
Nivalenol (NIV): 31%
15-Acetyl-DON: 33%
3-Acetyl-DON: <1%
ZON: 100%
α-Zearalanol: 99%
β-Zearalanol: 94%
α-Zearalenol: 97%
β-Zearalenol: 86%

Manual

Product Manual