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(d)
Apocarotenal Intermediate Solution
Transfer 3.0 mL apocarotenal stock solution to a 50 mL volumetric flask and dilute to volume
with sample solvent.
(e)
Mixed Carotenoid Intermediate Solution
Combine 2.0 mL each of lutein and β-carotene standard stock solutions in a 100 mL volumetric
flask and dilute to volume with sample solvent.
(f)
Calibration Solutions
Transfer apocarotenal intermediate solution and mixed carotenoid intermediate solution to
volumetric flasks according to the table below and dilute to volume with sample solvent.
Calibration
Solution
Apocarotenal
Intermediate
(mL)
Mixed
Carotenoid
Intermediate
(mL)
Total
volume
(mL)
Lutein
conc
(μg/100
mL)
Apocarotenal
conc (μg/100
mL)
β-
Carotene
conc
(μg/100
mL)
C1
2.0
15.0
25
240
96
240
C2
2.0
8.0
25
128
96
128
C3
2.0
5.0
25
80
96
80
C4
2.0
2.0
25
32
96
32
C5
8.0
1.0
100
4
96
4
(g)
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. Store in refrigerator.
(h)
Lutein System Suitability Solution
Transfer 10 mg USP lutein to a 100 mL volumetric flask, dilute to volume with ROH, and mix.
Transfer 1 mL to a 50 mL volumetric flask. Add ca. 35 mL IPA, stopper loosely, and heat in a
water bath at 80 °C for two hours. Cool to room temperature, add 250 μL apocarotenal stock
solution, and dilute to volume with IPA. Store in refrigerator.
(i)
Internal Standard Solution (ISTD)
Prepare immediately before use.
(1)
For infant formula and samples with low carotenoid concentrations (up to 100 μg of an
individual carotenoid per 100 g as fed):
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.
(2)
For samples with individual carotenoid concentrations > 100 μg/100 g:
Transfer 4.0 mL apocarotenal intermediate solution to a 50 mL volumetric flask and dilute to
volume with pyrogallol solution.
METHOD
FOR ERP USE ONLY
DO NOT DISTRIBUTE