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H
ostetler
:
J
ournal of
AOAC I
nternational
V
ol
.
100, N
o
.
3, 2017
7
above; 1000 = the conversion of milligrams to micrograms;
(3/50) = the dilution of stock solution to apocarotenal intermediate
solution; V
AI
= the volume of apocarotenal intermediate solution,
E(d)
, used; and V
Total
= the dilution volume.
(d)
For each calibration solution in
E(f),
calculate (
1
)
the peak area ratio for each analyte: (peak area of all-
trans
lutein or β-carotene)/(peak area of internal standard); and
(
2
) the concentration ratio: (concentration of all-
trans
lutein
or β-carotene)/(concentration of internal standard). Build a
five-point calibration curve with internal standard by plotting
peak area ratios against concentration ratios, with relative
concentration on the
x
-axis.
The accuracy on calibration points should be 100 ± 10%,
and the coefficient of determination (R
2
) should be greater than
0.995.
The calibration and calculation may be achieved through data
processing within the instrument software or off-line.
(e)
Calculate the mass (μg) of apocarotenal (M
A
) added to
the test samples:
(
) (
)
= × ×
M C V 4 50
A A A
where C
A
= the concentration (μg/100 mL) of apocarotenal in
the intermediate or working solution used in the ISTD; V
A
= the
volume (mL) of ISTD added to each sample; 4 = the volume (mL)
of apocarotenal intermediate or working solution used in the
ISTD; and 50 = the total volume (mL) of ISTD made.
(f)
Calculate the contents of all-
trans
-lutein,
cis
isomers of
lutein, and total lutein in the test samples. For peak identification,
refer to relative retention times of peaks in Figures
2016.13A
,
2016.13C
, and
2016.13D
.
(
)
(
)
(
)
=
×
− ×
Lut
M M A A I
100 RF
trans
A S
Lut
A Lut
Lut
where Lut
trans
= the concentration (μg/100 g) of all-
trans
-lutein
in the sample; M
A
= the mass (μg) of apocarotenal added to the
test sample; M
S
= the sample weight (g); A
Lut
= the peak area
(AU) of all-
trans
-lutein in the sample chromatogram; A
A
= the
peak area (AU) of apocarotenal in the sample chromatogram;
I
Lut
= the
y
-intercept of the calibration curve for all-
trans
-lutein;
and RF
Lut
= the slope of the calibration curve for all-
trans
-
lutein.
(
)
(
) (
)
(
)
=
×
+
+
+
−
×
Lut
M M A A A A A I
100 RF
cis
A S
13cisLut
13'cisLut
9cisLut
9'cisLut
A Lut
Lut
where Lut
cis
= the concentration (μg/100 g) of
cis
isomers of
lutein in the sample; M
A
= the mass (μg) of apocarotenal added
to the test sample; M
S
= the sample weight (g); A
13cisLut
= the
peak area (AU) of 13-
cis
-lutein in the sample chromatogram;
A
13′cisLut
= the peak area (AU) of 13′-
cis
-lutein in the sample
chromatogram;A
9cisLut
= the peak area (AU) of 9-
cis
-lutein in the
sample chromatogram; A
9′cisLut
= the peak area (AU) of 9′-
cis
-
lutein in the sample chromatogram; A
A
= the peak area (AU) of
apocarotenal in the sample chromatogram; I
Lut
= the
y
-intercept
of the calibration curve for all-
trans
-lutein; and RF
Lut
= the
slope of the calibration curve for all-
trans
-lutein.
=
+
Lut
Lut
Lut
Total
trans
cis
(g)
Calculate the contents of all-
trans
-β-carotene,
cis
isomers
of β-carotene, and total β-carotene in the test samples. For
peak identification, refer to relative retention times of peaks in
Figures
2016.13B
-
D
.
(
)
(
)
(
)
=
×
− ×
BC M M A A I
100 RF
trans
A S
BC A BC
BC
where BC
trans
= the concentration (μg/100 g) of all-
trans
-β-
carotene in the sample; M
A
= the mass (μg) of apocarotenal
added to the test sample; M
S
= the sample weight (g);
A
BC
= the peak area (AU) of all-
trans
-β-carotene in the sample
chromatogram; A
A
= the peak area (AU) of apocarotenal in the
sample chromatogram; I
BC
= the
y
-intercept of the calibration
curve for all-
trans
-β-carotene; and RF
BC
= the slope of the
calibration curve for all-
trans
-β-carotene.
(
)
(
(
)
)
(
)
=
×
×
+
×
+
+
− ×
BC M M A 1.4 A 1.2
A A A I
100 RF
cis
A S
15cisBC
13cisBC
9cisBC XcisBC A BC
BC
where BC
cis
= the concentration (μg/100 g) of
cis
isomers of
β-carotene in the sample; M
A
= the mass (μg) of apocarotenal
added to the test sample; M
S
= the sample weight (g); A
15cisBC
=
the peak area (AU) of 15-
cis
-β-carotene in the sample
chromatogram; A
13cisBC
= the peak area (AU) of 13-
cis
-β-
carotene in the sample chromatogram; A
9cisBC
= the peak area
(AU) of 9-
cis
-β-carotene in the sample chromatogram; A
XcisBC
= the peak area (AU) of unidentified
cis
isomers of β-carotene
in the sample chromatogram; A
A
= the peak area (AU) of
apocarotenal in the sample chromatogram; I
BC
= the
y
-intercept
of the calibration curve for all-
trans
-β-carotene; and RF
BC
= the
slope of the calibration curve for all-
trans
-β-carotene.
=
+
BC BC BC
Total
trans
cis
Validation
Selectivity
SMPR 2014.014 calls for the determination of all-
trans
and
cis
isomers of lutein and β-carotene, as well as the separation
of lutein from zeaxanthin. Selectivity was evaluated with visual
inspection of chromatograms and by measuring the resolution
of system suitability standard mixtures. Because apocarotenal
is used as an internal standard, samples were prepared without
internal standard to ensure there were no interfering peaks. To
identify major
cis
isomers of α-carotene, β-carotene, and lutein,
standard mixtures were isomerized by heating at 80°C for 2 h.
The separation of all-
trans
-lutein,
cis
isomers of lutein,
zeaxanthin, and apocarotenal is shown in Figure
2016.13A
,
whereas the separation of geometric isomers of α-carotene and
β-carotene is shown in Figure
2016.13B
. Peak assignments
were based on relative retention times from previous studies
using C30 columns and methanol–MTBE as the mobile
phase (11, 14–16). A chromatogram showing separation of
lutein and β-carotene from lycopene is shown in Figure 1,
and isomerized standard solutions showing major
cis
isomers
of the carotenoids are shown in Figures 2 and 3. One of
the minor
cis
isomers of β-carotene elutes before 15-
cis
-β-
carotene, and this peak has a similar retention time to one
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