6. AOACSPIFANMethods-2018Awards

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(3) Centrifuge samples 1–5 min at 3000 to 4000 RPM and then transfer the liquid supernatant into a new 12 mL screw cap vial using a Pasteur pipette. Place the extract under a nitrogen stream on a base plate tempered to 70°C in order to remove the solvent. (4) Add 6 mL of a methanol–tBME (1 + 1, v/v) mixture to the corresponding insoluble residue from the first extraction step and mix vigorously (while the organic phase of the sample is evaporating). If the sediment clumps, make an attempt to manually break up the sediment, for example using a spatula. If it is still impossible to achieve a fine-grained slurry by any manual handling, use a disperser at 24000 rpm equipped with a particularly small stator to fit the reaction vessel. Similar to the first extraction, perform the second extraction for 15 min in the ultrasonic bath, but at a starting temperature of 65 to 70°C. (5) After sonication, centrifuge the mixture and separate the liquid supernatant in the same way as the first extraction step. Combine the supernatant with the first extract, which may still be under the nitrogen stream. (6) Finally, perform a third extraction of the insoluble residue with 6 mL tBME, where the starting temperature of the ultrasonic bath may vary between 65 and 75°C. Centrifuge the sample, separate the supernatant, and add it to the previously combined extracts under the nitrogen stream. Discard the insoluble sediments after the third extraction step. Continue evaporation of the combined organic extracts under nitrogen flow until the solvents have been evaporated to a large extent. Do not overdo the solvent evaporation procedure in order to avoid altering the extracted compounds. (7) Add 4 mL saturated sodium sulphate solution (solution 5) to the extracted residue and carry out a double l/l-extraction with 2.5 mL (x 2) of an isohexane–tBME (4 + 1, v/v) mixture in order to separate the polar and non-polar contents. Centrifuge the sample and transfer the upper organic isohexane–tBME phases from each extraction to a previously weighed 8 mL screw cap vial. Pay close attention not to carry over any part of the lower aqueous layer into the organic phase. (8) Place the combined isohexane–tBME extracts under a stream of nitrogen. Accelerate this process as needed by heating a base plate between 40 and 50°C, but do not overdo this solvent evaporation procedure in order to avoid altering the lipid phase. Further preparation of the non-polar isohexane–tBME fraction containing lipids and bound analytes is described in section ( e ). The preparation of the polar aqueous phase containing the free analytes is presented in the following section ( b ). Note : As no g-forces are given, all centrifugation steps may be carried out for 1 to 5 min at 3000 to 4000 rpm depending on the matrix and devices available. It is recommended to evaluate appropriate conditions by empirical testing. (b) Powdered samples.--Sample preparation to determine free 2- and 3-MCPD .— (1) Wash the aqueous phase obtained from the fraction separation detailed in section ( a ) with 2.5 mL of a mixture of isohexane– tBME (4 + 1, v/v); discard the organic phase. (2) Subsequently, extract the aqueous phase three times using 2 mL diethyl ether or a mixture of diethyl ether–ethyl acetate (9 + 1, v/v). Following centrifugation, separate the organic phases using a Pasteur pipette (paying close attention not to carry over any part of the aqueous phase into the organic phase) and combine them in an 8 mL screw cap vial containing a spatula tip of anhydrous sodium sulphate. Add 100 µL of phenylboronic acid solution (solution 7) and concentrate to dryness under a stream of nitrogen.