372 / 670
(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-8ʹ-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
Carot-02 (February 2016)
FOR ERP USE ONLY
DO NOT DISTRIBUTE