Ochratoxin Immunoaffinity HPLC Column
The Ochratoxin Immunoaffinity HPLC Column is For Research Use Only
Size: 3ml, 10 columns
Assay Principle
These instructions for Ochratoxin determination in food and feed focuses on the enrichment step of extract using immunoaffinity column (Immunoaffinity Chromotography (IAC)) in combination with quantification with HPLC (High Performance Liquid Chromotography). Accepted laboratory extraction methods could be maintained. If the criteria of organic solvent tolerance, elution process of analyte, and working range of column are followed you will see full performance with the IAC column. If problematic matrices are applied, many pretreatment methods of Ochratoxin determination in food and feed will show low sensitivity because of interfering substances. This method of content determination of ochratoxins combines the high selectivity of an immunoaffinity column (IAC) with its potential to concentrate eluate with the 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 please note that a literature method is provided. The given apparatus (e.g. HPLC system) might serve as example among other possibilities.
Extraction (Literature method given):
Assuming that 25g sample are extracted by a total of 100ml methanol/water (80/20 v/v). 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. Grains could be prepared by the literature method of Olsson et al. For milk as an example of a problematic matrix, method of Zimmerli et al. could be applied.
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Product Developed and Manufactured in Germany by BioTeZ Berlin-Buch GmbH
Enrichment Step IAC:
4ml extract (see above, contains the quantity of ochratoxins of 1g sample) are diluted with 16ml 50mM PBS (pH=7.4) and then applied in a reservoir on top of the B-TeZ IAC Ochratoxin 3ml column. To maintain full performance of the column, please make sure that proportion of dilution buffer in the solution on top of the column is not too small. A proportion of 16% methanol, resulting in this example enrichment, does not affect column performance.
Caution: The proportion of organic solvent of PBS diluted extract, which is applied on the column, should not exceed 20% methanol and 10% 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 in the gel or between gel and the lock outlet of column. This can 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 insert) must be adapted.
Wash:
After whole sample has passed through the gel the latter is washed with 5ml of 10mM PBS/Methanol (90/10 v/v). Remaining liquids in the gel are removed by applying either pressure from top of the column or pressure from bottom.
Elution:
Sample reservoir on top of anhe B-TeZ IAC Ochratoxin 3ml column is removed and a appropriate vial is placed below the affinity column. The bound toxins are eluted by using a total of 1.5ml of methanol/acetic acid (98/2 v/v) 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-acetic acid 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. There is no danger of loss of analytes even though the elute is evaporated to complete dryness. The residue then is redissolved in HPLC solvent and an aliquot is finally injected into the system.
