AOAC 2018 Methods

(g) 2-MCPD, 3-MCPD, d5-3-MCPD and d5-2-MCPD stock solutions (1 mg/mL) .-Prepare individual stock solutions at 1 mg/mL by weighing 10 mg each of 2-MCPD, 3-MCPD, d5-3-MCPD and d5- 2-MCPD into separate 10 mL volumetric flasks. Dilute to volume with ethyl acetate and mix well. (h) d5-2-MCPD and d5-3-MCPD pooled ISTD intermediate solution in ethyl acetate (10 µg/mL) .- Transfer 200 µL each of d5-2-MCPD and d5-3-MCPD stock solutions (1 mg/mL) to a 20 mL volumetric flask. Dilute to volume with ethyl acetate and mix well. The resulting concentration is 10 µg/mL for both d5-2-MCPD and d5-3-MCPD. F. Working Standard Preparation (a) PP-3-MCPD, PP-2-MCPD and Gly-P Mix Cal 1 at 5 µg/mL (free form eq.) in toluene .-Transfer volume indicated in the Table 2018.03B below of PP-3-MCPD, PP-2-MCPD and Gly-P stock solution in a 20-mL volumetric flask. Dilute to volume with Toluene and mix well. The resulting concentration is 5 µg/mL for PP-3-MCPD, PP-2-MCPD and Gly-P expressed as free form equivalent. (b) PP-3-MCPD, PP-2-MCPD and Gly-P Mix Cal 2 at 0.1 µg/mL (free form eq.) in toluene -Transfer 400 µL of PP-3-MCPD, PP-2-MCPD and Gly-P Mix Cal 1 at 5 µg/mL in a 20-mL volumetric flask. Dilute to volume with Toluene and mix well. The resulting concentration is 0.1 µg/mL for PP- 3-MCPD, PP-2-MCPD and Gly-P. (c) d5- PP-3-MCPD, d5-PP-2-MCPD and d5-Gly-P Mix ISTD at 1 µg/mL (free form eq.) in toluene .- Transfer volume indicated in Table 2018.03B of d5-PP-3-MCPD, d5-PP-2-MCPD and d5-Gly-P stock solution in a 20-mL volumetric flask. Dilute to volume with Toluene and mix well. The resulting concentration is 1 µg/mL for d5-PP-3-MCPD, d5-PP-2-MCPD and d5-Gly-P expressed as free form equivalent. (d) 2-MCPD and 3-MCPD pooled working solution I in ethyl acetate (5 µg/mL) .-Transfer 100 µL each of 2-MCPD and 3-MCPD stock solutions (1 mg/mL) to a 20 mL volumetric flask. Dilute to volume with Ethyl acetate and mix well. The resulting concentration is 5 µg/mL for both 2- MCPD and 3-MCPD. (e) 2-MCPD and 3-MCPD pooled working solution II in Ethyl acetate (0.5 µg/mL).- Transfer 1 mL of each of 2-MCPD and 3-MCPD pooled working solution I at 5 µg/mL to a 10 mL volumetric flask. Dilute to volume with ethyl acetate and mix well. The resulting concentration is 0.5 µg/mL for both 2-MCPD and 3-MCPD. (f) d5-2-MCPD and d5-3-MCPD pooled ISTD working solution in ethyl acetate (1 µg/mL) .-Transfer 1 mL pooled intermediate solution of d5-3-MCPD and d5-2-MCPD (10 µg/mL) to a 10 mL volumetric flask. Dilute to volume with ethyl acetate and mix well. The resulting concentration is 1 µg/mL for both d5-2-MCPD and d5-3-MCPD. G. Sample Preparation (a) Test sample preparation for oil and fat samples. (1) Weigh 100-110 mg of oil/fat (± 0.01 mg) in a 10-mL glass vial or a 15-mL polypropylene conical tube. (2) Add 50 µL of Mixed Internal Standard (1 µg/ml in toluene) F(c) and 2 mL of tetrahydrofuran C(k) . (3) Mix for 15 s (or until complete dissolution) by means of a vortex mixer. Proceed with G(d). (b) Fat extraction for infant formula (1) For Infant/Nutritional samples with fat content between 20-30%: Into a 50-mL polypropylene conical tube, weigh to the nearest 0.05 g, 0.5 g of sample