6. AOACSPIFANMethods-2018Awards

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12 mL of water (only 12 mL ethyl acetate for liquid samples), and proceed as described above. After calculation of fat extracted, re-suspend with a calculated volume of tetrahydrofuran (± 0.01 mL) to achieve a solution at 0.05 g/mL of fat, and transfer a 2 mL portion in a 10-mL glass vial to proceed with G(d) .: ( ) = ( ) 0.05 ( ) In case a SPE cleanup is necessary, re-suspend with the calculated volume of elution solvent n-hexane:ethyl acetate (85:15,v/v) to achieve a solution at 0.1 g/mL (which correspond to 100 mg fat loaded on the cartridge in a 1 mL volume) and proceed with G(c): . ( ) = ( ) 0.1 ( ) (d) Sample preparation for determination of bound 2-MCPD, 3-MCPD and glycidol in oil samples and extracted fat (1) Following G(a), G(b) or G(c) , add 30 µL of acid aqueous solution of sodium bromide D(a) to the sample, shake vigorously (vortex) and incubate the mixture at 50 ºC (± 5 ºC) for 15 min. (2) Stop the reaction by addition of 3 mL of 0.6% aqueous solution of sodium hydrogen carbonate D(b) . In order to separate the oil/fat from the water phase, add 2 mL of n-heptane and shake vigorously (e.g. vortex 15 s). It is advisable to centrifuge test tubes to improve phase separation (e.g: 4500 x g for 2 minutes for 15 mL polypropylene tubes). (3) After separation of the two phases, transfer the upper layer to an empty tube (10-mL glass vial or a 15-mL polypropylene conical tube) and evaporate the collected organic phase solution under a nitrogen stream at 40ºC until an oily residue remains. Dissolve the residue in 1 mL of tetrahydrofuran. (4) Add 1.8 mL of sulfuric acid/methanol solution D(c) to the sample and shake vigorously (vortex, 10 s). Ensure test tube is closed tightly and incubate the mixture at 40ºC for 16-20 h. (5) After the incubation period, stop the reaction by adding 0.5 mL of sodium hydrogen carbonate saturated solution D(d) to the sample. Shake (vortex) for 10 s. Evaporate the organic solvents of the mixture under a nitrogen stream (stop when the remaining volume is about 1 mL). (6) Add 2 mL of sodium sulfate solution D(e) and 2 mL of n-heptane. Shake (vortex) for 10 s. The two phases will spontaneously separate in a few seconds. To ensure a clear and fast separation, centrifuge 1 min at 1000 x g. Discard the upper phase (that contains fatty acid methyl esters dissolved in n-heptane) and repeat the extraction with n-heptane (remove completely the upper organic phase during each extraction). (7) Extract the free form of 2- and 3-MCPD as well as 3-MBPD from the aqueous phase with 3 times 0.6 ml of ethyl acetate. Shake each time for 10 s (vortex) and transfer the upper phases to an empty tube (10-mL glass vial or a 15-mL polypropylene conical tube) containing a small amount of anhydrous sodium sulfate.