AOAC 2018 Methods

(Note: it is also possible to extract the aqueous phase with 2 times 2 mL of ethyl acetate, shake each time for 10 s – vortex – and transfer the upper phases to an empty tube containing a small amount of anhydrous sodium sulfate). (8) Derivatization .- Add 150 µl of the derivatization reagent D(f) (PBA solution in diethyl ether) to the organic solvent (1.8 mL or 4 mL of ethyl acetate depending on extraction approach)and shake vigorously for 15 seconds and further place into ultrasonic bath or rotating shaker for 5 minute at room temperature. To complete the derivatization reaction, evaporate the extracts to about 50 µL at 40 °C under a low stream of nitrogen. Add 1 mL of n-Heptane, vortex 15 s and centrifuge at 1000 rpm. Transfer supernatant into an empty GC vial (containing a glass insert if only an aliquot is injected) and proceed with GC-MS/MS quantification. (e) Preparation of the calibration curve for bound Glycidol, 2-MCPD and 3-MCPD. (1) Prepare 7 calibration samples by pipetting 50 µL of Mixed Internal Standard F(c) and the volume of calibration solutions F(a) or F(b) indicated in Table 2018.03C . Add one drop of a blank oil if such sample is available (e.g. organic rapeseed oil, olive oil or any other blank oil) (2) Add 2 mL of tetrahydrofuran and shake vigorously for 10 s (vortex). (3) Treat the calibration samples according to the procedure used for the test samples G(d) . (f) Sample preparation for determination of free 2-MCPD and 3-MCPD. (1) Weigh 2 g of sample with accuracy over 0.01 g into a 50-mL conical Polypropylene tube. Add with a pipette 100 µl of the working standard solution of stable isotope F(f) (labelled MCPD in ethyl acetate, 1 µg/mL). For powder sample, add a ceramic homogenizer and 2 mL of water C(u) and shake vigorously to fully dissolve the powder. Add for all samples 10 ml of solution D(g) (n-hexane-acetone) (2) Shake the sample on a vertical shaker (e.g. Genogrinder) for 3 min at 1300 rpm. Place into an ultrasonic bath for 5 minutes at room temperature. After sonication, centrifuge for 10 minutes at 4500 x g at room temperature. If an intermediate fatty layer is observed between the two liquid phases, reiterate shaking and centrifugation process another time. (3) Transfer the whole supernatant to a new 50-mL Polypropylene tube and discard the solid residues. Add 2 ml of water and shake vigorously (e.g Genogrinder for 1 min at 1500 rpm). To speed up the phase separation centrifuge the vials for 5 minutes at 4500 x g at room temperature. (4) After phase separation transfer aqueous phase (going through the organic phase) into a new tube (10-mL glass vial or a 15-mL polypropylene conical tube) and discard organic phase. Evaporate the remaining traces of hexane acetone fromwater phase at 40 °C under a stream of nitrogen until remaining volume is about 2 mL. (5) Add around 1 g of sodium sulfate and extract the free form of 2- and 3-MCPD from the aqueous phase with 3 times 0.6 ml of ethyl acetate. Shake each time for 10 s (vortex) and centrifuge 2 min at 1000 x g. Addition of sodium sulfate facilitate salting out of analytes into the ethyl acetate phase and avoid gel formation with certain samples. Transfer the upper phase to an empty test tube (10-mL glass vial or a 15-mL polypropylene conical tube) containing a small amount of anhydrous sodium sulfate.