AOAC SPIFAN Nutrients ERP Review (March 14, 2019)

Carot-02 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

Combine 1.0 mL of each standard stock solution in a 100 mL volumetric flask and dilute to volume with sample solvent. (e) β-Carotene System Suitability Solution Transfer 20 mg β-carotene system suitability reference standard to a 50 mL volumetric flask. Add 1 mL water and 4 mL THF and sonicate 5 minutes. Dilute to volume with IPA and sonicate 5 minutes. Cool to room temperature, filter the cloudy suspension through 0.2 μm PTFE, and dilute the clear filtrate 1:4 with IPA. (f) Qualitative cis/trans Lutein Solution Transfer 0.5 mL lutein stock solution to a 50 mL volumetric flask. Add 25 mL IPA, stopper loosely, and heat in a water bath at 80 °C for two hours. Cool to room temperature and dilute to volume with IPA. (g) Internal Standard Solution (ISTD) Prepare immediately before use. Transfer 4.0 mL apocarotenal working solution to a 50 mL volumetric flask and dilute to volume with pyrogallol solution. This is enough solution for 9 samples. (a) Prepare up to 9 samples at a time. (b) Weights – Weigh all samples (powders and liquids) to 0.1 mg. (1) Powders- Weigh approximately 625 mg powder sample into a 50 mL centrifuge tube. Add 5 mL water, cap, and vortex 10 sec. Let sit up to 15 min at room temperature. (2) Liquid ready-to-feed (RTF) – Shake bottle or can on a reciprocating shaker 10 min before opening. Pipette 5.0 mL sample into a 50 mL centrifuge tube. (3) Liquid concentrate – Shake bottle or can on a reciprocating shaker 10 min before opening. Pipette 2.5 mL sample into a 50 mL centrifuge tube. Add 2.5 mL water, cap, and vortex 10 sec. Let sit up to 15 min at room temperature. (c) Pipette 5.0 mL ISTD solution to each tube. (d) Add 1.5 mL KOH solution to each tube with a repeater pipette. (e) Shake on reciprocating shaker 5 min. (f) Add 8 mL extraction solution to each tube with a repeater pipette. (g) Shake 10 min. (h) With a repeater pipette or dispenser, add 10 mL hexane and 10 mL water to each tube. (i) Shake 1 min. (j) Centrifuge at 3000 rpm for 5 min. (k) Depending on the sample carotenoid concentration, transfer 3-10 mL supernatant to a 12 mL scintillation vial. Dry under nitrogen at ≤40 °C. (l) Reconstitute dried extract in 500 μL sample solvent. Filter through 0.2 μm PTFE syringe filter prior to injection.

F. Sample Preparation