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(e)
Potassium hydroxide (KOH). – pellets, ACS grade
(f)
Reagent alcohol (ROH). – denatured ethanol, HPLC grade
(g)
α-Tocopherol (Vitamin E). – 95%
(h)
Pyrogallic acid (Pyrogallol). – ACS grade
(i)
2-Propanol (IPA). – HPLC grade
(j)
Tetrahydrofuran (THF). – 99.9%, stabilized with BHT
(k)
Ammonium acetate. – HPLC grade, 98%
(l)
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.
(m) MTBE/Vitamin E
Dissolve 2.2 g α-tocopherol in 500 mL MTBE. Store in refrigerator.
(n)
Pyrogallol solution (0.2 M Pyrogallic Acid in Ethanol)
Dissolve 6.3 g pyrogallic acid in 250 mL ROH. Store in refrigerator. Solution should be clear;
discard if colored.
(o)
Extraction solution (10 mM Vitamin E in 1:1 MTBE/THF)
Dissolve 2.2 g α-tocopherol in 250 mL MTBE and 250 mL THF. Store in refrigerator.
(p)
Sample Solvent (10 mM Vitamin E in IPA)
Dissolve 4.4 g α-tocopherol in 1000 mL IPA. Store in refrigerator.
(q)
Mobile Phase for LC System
(1)
Phase A. – 20 mM Ammonium Acetate in 98:2 Methanol/Water
Combine 980 mL MeOH, 20 mL water, and 1.54 g ammonium acetate and mix.
(2)
Phase B. - MTBE
D.
Standards
(a)
Lutein. – ChromaDex p/n ASB-00012453 or equivalent
(b)
β-Carotene. – USP p/n 1065480 or equivalent
(c)
Apocarotenal (β-8′-Apocarotenal). – USP p/n 1040854 or equivalent
(d)
Lutein containing ca. 10% zeaxanthin. – USP p/n 1370804
(e)
β-Carotene System Suitability Reference Standard. – USP p/n 1065491
E.
Standards Preparation
(a)
Carotenoid Stock Solutions
Weigh (to 0.1 mg) approximately 10 mg each of lutein, β-carotene and apocarotenal reference
standard into separate 50 mL volumetric flasks. Add ca. 40 mL MTBE/Vitamin E to each, sonicate
2-3 minutes, and dilute to volume.
(b)
Standard Measuring Solutions for UV-Visible Spectroscopy
Transfer 1.0 mL of each standard stock solution to separate 100 mL volumetric flasks and dilute
each to volume with MTBE. Measure against an MTBE blank.
(c)
Individual Carotenoid Working Solutions
For lutein and β-carotene: Transfer 100 μL of each standard stock solution to separate 10 mL
volumetric flasks and dilute each to volume with sample solvent.
For apocarotenal: Transfer 1.0 mL standard stock solution to a 100 mL volumetric flask and
dilute to volume with sample solvent.
METHOD
FOR ERP USE ONLY
DO NOT DISTRIBUTE