43 / 258
H
ostetler
:
J
ournal of
AOAC I
nternational
V
ol
.
100, N
o
.
3, 2017
3
separate 100 mL volumetric flasks and dilute each to volume
with MTBE. Prepare fresh when needed.
(c)
Individual
carotenoid
working
solutions
for
chromatographic purity check (200 μg/100 mL).—
(
1
)
For lutein
and β-carotene
.—Transfer 100 μL aliquots of each standard stock
solution,
E(a)
, to separate 10 mL volumetric flasks and dilute each
to volume with sample solvent. Prepare fresh when needed.
(
2
)
For apocarotenal
.—Transfer 1.0 mL standard stock
solution,
E(a)
, to a 100 mL volumetric flask and dilute to
volume with sample solvent. Store at −20°C for up to 1 month
and use for internal standard,
E(i)
.
(d)
Apocarotenal intermediate solution (1200 μg/100 mL).—
Transfer 3.0 mL apocarotenal stock solution,
E(a)
, to a 50 mL
volumetric flask and dilute to volume with sample solvent.
Store at −20°C for up to 1 month.
(e)
Mixed carotenoid intermediate solution (400 μg/100 mL).—
Combine 2.0 mL each of lutein and β-carotene standard stock
solutions,
E(a)
, in a 100 mL volumetric flask and dilute to volume
with sample solvent. Store at −20°C for up to 1 month.
(f)
Calibration solutions.—
Transfer apocarotenal intermediate
solution,
E(d)
, and mixed carotenoid intermediate solution,
E(e)
,
to volumetric flasks according to Table
2016.13B
and dilute to
volume with sample solvent. Store at −20°C for up to 1 month.
(g)
β-Carotene system suitability solution.—
Transfer 20 mg
β-carotene system suitability reference standard,
D(e)
, to a
50 mL volumetric flask. Add 1 mL water and 4 mL THF and
sonicate for 5 min. Dilute to volume with IPA and sonicate for
5 min. Cool to room temperature, filter the cloudy suspension
through 0.2 μm PTFE, and dilute the clear filtrate 1:4 with IPA.
Store in the refrigerator for up to 3 months.
(h)
Lutein system suitability solution.—
Transfer 10 mg USP
lutein,
D(d)
, to a 100 mL volumetric flask, dilute to volume
with ROH, and mix. Transfer 1 mL to a 50 mL volumetric flask.
Add approximately 35 mL IPA, stopper loosely, and heat in a
water bath at 80°C for 2 hours. Cool to room temperature, add
250 μL apocarotenal stock solution,
E(a)
, and dilute to volume
with IPA. Store in the refrigerator for up to 3 months.
(i)
Internal standard solution (ISTD).—
Prepare immediately
before use.—(
1
)
For infant formula and samples with low
carotenoid concentrations (up to 100 μg individual carotenoid per
100 g)
.—Transfer 4.0 mL apocarotenal working solution,
E(c)
, to
a 50 mL volumetric flask and dilute to volume with pyrogallol
solution,
C(n)
. This is enough solution for nine samples.
(
2
)
For samples with individual carotenoid concentrations
>100 μg/100 g
.—Transfer 4.0 mL apocarotenal intermediate
solution,
E(d)
, to a 50 mL volumetric flask and dilute to volume
with pyrogallol solution,
C(n)
.
F. Sample Preparation
Note on range:
Although this method can quantify
carotenoids in the range of 1–1300 μg/100 g, it is recommended
to quantify only a 100-fold difference with a single preparation.
For example, the range of 1–100 μg/100 g works well for infant
formula, but the range of 15–1500 μg/100 g would work best for
samples with the highest carotenoid concentrations.
(a)
Prepare up to nine samples at a time.
(b)
Weights
.—Weigh all samples (powders and liquids) to
0.1 mg.—(
1
)
Powders
.—Weigh approximately 625 mg powder
sample into a 50 mL centrifuge tube. Add 5 mL water, cap, and
vortex-mix until dissolved. Let sit for up to 15 min at room
temperature.
Note
: For nonhomogeneous powder samples, first dissolve
25 g powder sample with 200 mL water (record weights of both
powder sample and water) and then transfer approximately 5 g
reconstituted sample into a 50 mL centrifuge tube.
(
2
)
Liquid RTF with individual carotenoid concentrations
≤200 μg/100 g.
—Shake bottle or can on a reciprocating shaker
10 min before opening. Transfer approximately 5 g sample into
a 50 mL centrifuge tube.
Table 2016.13A. Composition and nominal concentrations of carotenoid standard solutions
Analyte
Stock solution
(20000 μg/100 mL) in
MTBE/vitamin E
UV-Vis solution
(200 μg/100 mL) in MTBE
Working solution
(200 μg/100 mL) in sample
solvent
Intermediate solution in sample solvent
Standard,
mg
Total volume,
mL
Stock solution,
mL
Total volume,
mL
Stock solution,
mL
Total volume,
mL
Stock solution,
mL
Total volume,
mL
Concn,
μg/100 mL
Lutein
10
50
1.0
100
0.1
10
2.0
100
400
β-Carotene
10
50
1.0
100
0.1
10
2.0
400
Apocarotenal
10
50
—
—
1.0
100
3.0
50
1200
Table 2016.13B. Composition and nominal concentrations of carotenoid calibration solutions
Calibration solution
Apocarotenal
intermediate
solution, mL
Mixed carotenoid
intermediate
solution, mL
Total volume, mL
Lutein concn,
μg/100 mL
Apocarotenal concn,
μg/100 mL
β-Carotene concn,
μg/100 mL
1
2.0
15.0
25
240
96
240
2
2.0
8.0
25
128
96
128
3
2.0
5.0
25
80
96
80
4
2.0
2.0
25
32
96
32
5
8.0
1.0
100
4
96
4
43