BS 15835:2010 pdf download – Foodstuffs — Determination of ochratoxin A in cereal based foods for infants and young children — HPLC method with immunoaffinity column cleanup and fluorescence detection
6 Procedure
6.1 Extraction Stir the sample thoroughly before removing an analytical test portion. Weigh, to the nearest 0,1 g, a 25 g test portion of baby food sample into a centrifuge bottle (5.9). Add 100 ml of the mixture of phosphoric acid solution and sodium chloride solution (4.15). Mix for 1 min on a Vortex mixer (5.20). Add 50 ml of tert-butyl methyl ether (4.22). Blend for 2 min with a high speed blender (5.2). Centrifuge for 10 min at 15 300 g with cooling at approximately 4 °C. Transfer the upper organic layer to a capped conical flask or measuring cylinder. Re-extract with a second 50 ml portion of tert-butyl methyl ether, blending again for 2 min. After centrifugation under the same conditions, combine the two organic phases. Pour an aliquot of 75 ml of the combined organic phases into a round bottomed flask (5.15) and evaporate at 35 °C to 40 °C in a rotary evaporator (5.10) until no more solvent distils. Re-dissolve the remaining fatty residue as follows to obtain an extract in a mixture of 15 parts per volume of methanol (4.19) and 85 parts per volume of PBS solution (4.14). Add 3 ml of methanol and thoroughly rinse the walls of the flask. Transfer the methanol extract into a separating funnel (5.19) by using a Pasteur pipette. Repeat this step once again with a new portion of 3 ml of methanol. Combine both methanol extracts in the separating funnel. Dilute by addition of 34 ml of PBS solution (4.14) to the separating funnel and shake vigorously for 1 min. Add 50 ml of hexane (4.18) to the separating funnel and shake gently for 1 min. Allow the layers to separate, then pour off the lower layer into a second separating funnel and discard the top hexane layer. Repeat this operation with a second portion of 50 ml of hexane. Pour off the lower layer into a centrifuge bottle (5.9) and centrifuge for 10 min at 15 300 g and approximately 4 °C in order to separate any fatty residue. Drain into a measuring cylinder through a funnel and filter paper (5.6).
NOTE Try to avoid the re-mixing of aqueous and organic phases. If draining is difficult, collect and filter at least 35 ml of the aqueous layer by pipetting.
6.2 Immunoaffinity column cleanup Connect the immunoaffinity column (4.26) to the vacuum manifold (5.5), and attach a reservoir of 50 ml or 20 ml capacity (5.11) to the immunoaffinity column. Columns should be allowed to reach room temperature prior to conditioning. For conditioning apply 20 ml of PBS solution (4.14) to the top of the column and let it pass at a speed of 2 ml/min to 3 ml/min through the column by gravity. Make sure that a small portion (e.g. 0,5 ml) of the PBS remains on the column until the sample solution is applied. Transfer 30 ml of the sample extract as obtained in 6.1 to the reservoir and pass through the immunoaffinity column. Do not exceed a flow rate of 3 ml/min. Let it pass by gravity or pushing down slightly with a syringe or applying little vacuum. CAUTION — If using a vacuum manifold, extra care is necessary to avoid increasing the flow rate through the column to the point where recovery is adversely affected.
6.3 Preparation of the sample test solution Wash the column with 10 ml of water. Remove the reservoir and eliminate any residual water on the inside of the upper part of the column with a piece of absorbing paper or a cotton stick. Dry the column by applying vacuum for 1 min or blowing air with a syringe. Attach to the column a reservoir of 5 ml capacity (5.11) and elute the ochratoxin A in a two step procedure:  Apply 4 ml of methanol on the column and let it pass through by gravity until elution of the first drop. Close the luer lock. Wait for 1 min and slowly elute ochratoxin A from the column into a glass tube;