AOAC SPIFAN Nutrients ERP Review (March 14, 2019)

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

(e) Reagent alcohol (ROH). – denatured ethanol, HPLC grade (f) α-Tocopherol (Vitamin E). – 95% (g) Pyrogallic acid (Pyrogallol). – ACS grade (h) 2-Propanol (IPA). – HPLC grade

(i) Tetrahydrofuran (THF). – 99.9%, stabilized with BHT (j) Ammonium acetate. – HPLC grade, 98%, FW 77.08 (k) Potassium hydroxide solution, 50% (w/w) Add 50 mL water to a 250 mL beaker. Weigh 50 g KOH and slowly transfer to the beaker under constant stirring. When dissolved and cooled, transfer to a media bottle. (l) Standard Diluent, 10 mM Vitamin E in MTBE Weigh 2.2 g α-tocopherol into a 500 mL media bottle. Add 500 mL MTBE and mix on a spin plate to dissolve. Store in refrigerator. (m) Pyrogallol Solution (0.2 M Pyrogallic Acid in Ethanol) Weigh 3.2 g pyrogallic acid into a 500 mL beaker. Add 250 mL ROH and mix on a spin plate to dissolve. Transfer to a media bottle. (n) Extraction Solution (10 mM Vitamin E in 1:1 MTBE/THF) Weigh 2.2 g α-tocopherol into a 1 L beaker. Add 250 mL MTBE and 250 mL THF and mix on a spin plate to dissolve. Store in refrigerator. (o) Sample Solvent (10 mM Vitamin E in IPA) Weigh 2.2 g α-tocopherol into a 500 mL media bottle. Add 500 mL IPA and mix on a spin plate to dissolve. Store in refrigerator. (p) Mobile Phase for LC System (1) Phase A. – 20 mM Ammonium Acetate in Methanol Combine 1.5 g ammonium acetate with 1 L methanol and mix. (2) Phase B. - MTBE D. Standards (a) Lutein. – USP p/n 1370804 (b) β-Carotene. – USP p/n 1065480 (c) Apocarotenal (β-8′-Apocarotenal). – USP p/n 1040854 (d) β-Carotene System Suitability Reference Standard. – USP p/n 1065491 E. Standards Preparation (a) Carotenoid Stock Solutions For each carotenoid, weigh (to 0.1 mg) approximately 25 mg reference standard into a 100 mL volumetric flask. Add ca. 90 mL standard diluent, sonicate 2-3 minutes, and dilute to volume. The solutions should be clear, with no particulates. (b) Standard Measuring Solutions for UV-Visible Spectroscopy For each carotenoid, transfer 1.0 mL standard stock solution to a separate 100 mL volumetric flask and dilute to volume with MTBE. (c) Individual Carotenoid Working Solutions Transfer 1.0 mL of each standard stock solution to separate 100 mL volumetric flasks and dilute each to volume with sample solvent. (d) Mixed Carotenoid Working Solution (WS) for Calibration