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 phase to an empty test tube containing a small amount of anhydrous sodium sulfate) (6) 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. (g) Preparation of the calibration curve for 2-MCPD and 3-MCPD. (1) Prepare 7 calibration samples by pipetting 100 µL of the working standard solution of stable isotope F(f) (labelled MCPD in ethyl acetate, 1 µg/mL) and the volume of working standard solution F(d) or F(e) as indicated in Table 2018.03D . (2) Add a volume of ethyl acetate corresponding to extraction approach chosen at G(e) (1.8 mL or 4 mL). Derivatize as described in sample preparation G(e). H. Determination by GC-MS/MS (a) Sequence setup: Inject solutions in the following order: one solvent blank (n-heptane), calibrants, one solvent blank (n-heptane), reference material, samples. Repeat injection of Cal 4 in the middle and at the end of the sequence to monitor stability of analytical system. (b) Reference material with known value (from collaborative study or Proficiency test) should be included within each batch. I. Calculations (a) Prepare a calibration curve by plotting the ratio of the amount of standard (free 3-MCPD, 2- MCPD and glycidol equivalent, respectively) to the amount of internal standard (free 2-MCPD- d5, 3-MCPD-d5 and deuterated glycidol equivalent, respectively) on the x-axis, against the ratio of the corresponding peak areas on the y- axis. Transitions 196→147.1 (3 -MCPD), 150→93 (3 -MCPD-d5), 19 6→104 (2 - MCPD), 201→104.1 (2 -MCPD- d5), 240→147.1 (3 -MBPD) and 150→93 (3 -MBPD-d5) are used for the quantifications. Typical calibration curve a given in Figure 2018.03B .

(b) Calculate the regression line as follows:

y = ax + b

where a is the slope and b is the y-intercept. Regression with a 1/x weighing factor is recommended to ensure accuracy along the calibration curve. Confirm that the linearity is good (R² > 0.99) and accuracy of each calibration point is within 80-120%. (c) Determine the concentration of 3-MCPD, 2-MCPD, 3-MCPD esters, 2-MCPD esters and glycidyl esters, respectively in the test sample (mg/kg) by applying the following formula:

c = [(A a /A IS ) – b] x IS x 1/a x 1/W