AOAC 2018 Methods

(2) For liquid samples with known fat content between 3-5%: Into a 50-mL polypropylene conical tube, weigh to the nearest 0.05 g, 3 g of sample (3) For concentrated liquid with known fat content between 6-12%: Into a 50-mL polypropylene conical tube, weigh to the nearest 0.05 g, 1.5 g of sample (4) Add 50 µL of Mixed ISTD (1 µg/ml in toluene) F(c) . (5) For both type of samples, add 12 mL of ethyl acetate C(v) and 12 mL of water C(u) . (6) Vortex to homogenize. (7) Incubate for 15 minutes in sonicator at 50°C ± 10°C. (8) Shake for 3 min at 1500 rpm using a GenoGrinder. (9) Centrifuge for 10 minutes at 4500 x g. (10) Add 10 g (± 0.5 g) of Na2SO4. (11) Shake for 3 min at 1500 rpm using a GenoGrinder. (12) Centrifuge for 10 minutes at 4500 x g. (13) Weigh an empty 60-mL collection vial (or any other glassware suitable for evaporation of solvent at step (19)) and record its weight to the nearest 0.001 g (14) Transfer the upper phase (ethyl acetate) in the collection vial (it is recommended to leave some ethyl acetate in the extraction tube in order not to take any water phase). (15) Add 12 ml of ethyl acetate on the solid residue to perform a second extraction. (19) Transfer upper phase (ethyl acetate) into the collection vial (combine extracts). (20) Evaporate the extract under vacuum (e.g. with Rocket evaporator). Alternatively, evaporate solvent under a N2 evaporator at 40°C. (21) Weigh the collection vial containing the fat and calculate fat extracted (Weight of vial after extraction minus weight of vial before extraction). (22) For samples known to contain significant levels of diacylglycerides and monoacylglycerides, proceed with G(c) , otherwise re-suspend the fat extracted with 2 mL of tetrahydrofuran and proceed with G(d) . (c) SPE clean-up for sample containing partial acylglycerol (DAGs,MAGs) (1) For samples known to contain significant levels of diacylglycerides and monoacylglycerides, a cleanup using aminopropyl SPE is necessary. Results for Glycidyl ester above regulatory limits have to be generated with this cleanup step to eliminate risk of analytical artefact (if a sample analyzed without SPE cleanup step lead to Glycidyl ester result above regulatory limit, analysis must be repeated with a SPE clean up step). For other samples, step (c) can be omitted. (2) Condition SPE cartridge with 2 mL elution solvent D(h) n-hexane:ethyl acetate (85:15,v/v) (3) Re-suspend fat extracted in G(b) (21) in 1 mL elution solvent, and load on the cartridge (4) Elute and collect with 10 mL elution solvent (5) Evaporate solvent under a N2 evaporator at 40°C. (6) Re-suspend the fat extracted in 2 mL tetrahydrofuran and proceed with G(d) . If needed, transfer the re-suspended solution to an adequate tube in order to proceed for analysis (10-mL glass vial or a 15-mL polypropylene conical tube). (16) Shake for 3 min at 1500 rpm using a GenoGrinder. (17) Sonicate for 15 minutes in a sonicator at 50°C ± 10°C. (18) Centrifuge for 10 minutes at 4500 x g.

Note: For samples with unknown fat amount, extract fat using 2 g powder sample (14 g liquid sample), add 50 µL of Mixed ISTD (1 µg/ml in toluene) F(c) , add 12 mL of ethyl acetate and