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Page
5
of
9
G.
Chromatography
(a)
Conditions
Analytical column
YMC C30 3 μm, 250 x 2.0 mm
Guard column
YMC C30 3 μm, 10 x 2.0 mm
Column temperature 30 °C
Mobile phases
A: 20 mM ammonium acetate in 98:2 MeOH/Water
B: MTBE
Gradient
Time (min)
%B
0
5
1
8
8
15
25
100
25.5
5
32
5
Flow rate
0.25 mL/min
Backpressure
ca. 185 bar
Injection volume
5 μL
UV/Visible detection 450 nm, ref = off
(b)
Resolution between isomers
(1)
Resolution between lutein
cis
and
trans
isomers. – Inject the lutein system suitability
solution and determine the resolution between the two major
cis
isomers and all-
trans
-
lutein. Typical resolution between 13-
cis
and 13′-
cis
lutein is ≥1.5, between 13′-
cis
and all-
trans
-lutein is ≥2.6.
(2)
Resolution between all-
trans
-lutein, zeaxanthin, and apocarotenal. – From the
chromatogram of the lutein system suitability solution, determine the resolution between
all-
trans
-lutein, zeaxanthin, and apocarotenal. Typical resolution between all-
trans
-lutein
and zeaxanthin is ≥3.7, between zeaxanthin and apocarotenal is ≥3.0.
(3)
Resolution between β-carotene
cis
and
trans
isomers and α-carotene. – Inject the β-
carotene system suitability solution and determine the resolution between the two major
cis
isomers, all-
trans
-β-carotene, and α-carotene. Typical resolution between 13-
cis
-β-
carotene and
cis/trans
α-carotene is ≥1.7, between all-
trans
- β-carotene and 9-
cis
- β-
carotene is ≥2.6.
(c)
Calibration. – Inject the calibration solutions before and after each set of sample injections (up
to 12 samples in each set). Average the peak areas from the two standard curves and calculate
the slope relative to the internal standard, using zero as the y-intercept. See H.(d) for
calculation. The slopes from the two curves should not differ by more than 2.0%.
H.
Calculations
(a)
Determine the purity of lutein and β-carotene standards by first determining the
spectrophotometric purity and then the chromatographic purity of each. The overall purity is
calculated as the product of the two measured purities
(1)
Spectrophotometric Purity (SP)
METHOD
FOR ERP USE ONLY
DO NOT DISTRIBUTE