6. AOACSPIFANMethods-2018Awards

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(3) Re-dissolve the soluble residue in 300 µL isooctane and transfer a 200 µL aliquot into a 2 mL GC vial (containing a glass insert) for GC-MS analysis. Centrifuge the aliquots intended for GC-MS analysis. If sediment is present, decant the clear supernatant into a new vial insert prior to GC-MS analysis in order to prevent contamination of the injection system. (c) Liquid samples.—Extraction procedure for free 2-MCPD and free 3-MCPD.—(1) Weigh between 4.975 and 5.025 g of liquid sample in a 12 mL screw cap vial. Add 200 µL standard working solution 2d (with 1.0 µg/mL 2-MCPD equivalent and 1.0 µg/mL 3-MCPD equivalent in methanol, resulting in a sample concentration of 40 µg/kg each). (2) Mix the sample with 5 mL of acidified sodium bromide solution (solution 3) and wait until flocculation has occurred. Centrifuge the sample for 1 to 5 min at 3000 to 4000 rpm and transfer the lower clear aqueous phase into a new 12 mL screw cap vial. Vigorously shake the remaining insoluble precipitation, centrifuge once more, separate the lower clear aqueous phase and combine it with the first extract. Discard the remaining insoluble precipitation. (3) Extract the aqueous phase with 2.5 mL of an isohexane–tBME (4 + 1, v/v) mixture, centrifuge the sample, and transfer the aqueous phase into a new 12 mL screw cap vial using a Pasteur pipette. Discard the organic phase. Repeat this washing step, which serves as a matrix removal, once or twice more. Finally, extract the aqueous phase three times with 2.5 mL of diethyl ether or a mixture of diethyl ether–ethyl acetate (9 + 1, v/v). Following centrifugation, separate the organic phases using a Pasteur pipette and combine them in an 8 mL screw cap vial containing a spatula tip of anhydrous sodium sulphate. (4) Transfer the lower aqueous phase (from step 3) into a new screw cap vial. If the organic phase cannot be pipetted due to emulsion formation, break the remaining emulsion by adding anhydrous sodium sulphate. Pay close attention not to carry over any part of the aqueous phase into the organic phase. Add 100 µL of phenylboronic acid solution (solution 7) to the combined diethyl ether or diethyl ether–ethyl acetate extracts and concentrate to dryness under a stream of nitrogen. (5) Re-dissolve the soluble residue in 300 µL isooctane and transfer a 200 µL aliquot into a 2 mL GC vial (containing a glass insert) for GC-MS analysis. Centrifuge the aliquots intended for GC-MS analysis. If sediment is present, decant the clear supernatant into a new vial insert prior to GC-MS analysis in order to prevent contamination of the injection system. (d) Liquid samples.—Extraction procedure for ester-bound 2-MCPD, ester-bound 3-MCPD and ester-bound glycidol.—(1) Weigh between 13.93 and 14.04 g of liquid sample in a 50 mL screw cap centrifuge tube. Add 140 µL standard working solution 3d (with 5 µg/mL d 5 -2-MCPD equivalent, resulting in a sample concentration of 50 µg/kg; 10 µg/mL d 5 -3-MCPD equivalent, resulting in a sample concentration of 100 µg/kg and 5 µg/mL d 5 -glycidol equivalent in toluene, resulting in a sample concentration of 50 µg/kg). (2) Add 4.2 mL 25% ammonium hydroxide solution, mix well, and allow the sample to remain at room temperature for 15 min. Subsequently add 20 mL of ethanol and extract the mixture three times with 10 mL of a mixture of diethyl ether–petrol ether (1 + 1, v/v). Following centrifugation, separate the organic phases using a Pasteur pipette and combine them in a suitable disposable snap cap vial. Take care not to transfer any part of the aqueous phase into the organic phase. Place the first organic extract under a nitrogen stream. Perform the two subsequent extractions and then combine the organic extracts with the first extract under the nitrogen stream. Concentrate the combined extracts under nitrogen until the