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4
H
ostetler
:
J
ournal of
AOAC I
nternational
V
ol
.
100, N
o
.
3, 2017
(
3
)
RTF sample with individual carotenoid concentrations
>200 μg/100 g
.—Shake bottle or can on a reciprocating shaker
10 min before opening. Transfer approximately 2 g sample into
a 50 mL centrifuge tube. Add 3 mL water, cap, and vortex-mix
10 s. Let sit for up to 15 min at room temperature.
(
4
)
Infant formula concentrate
.—Shake bottle or can
on a reciprocating shaker 10 min before opening. Transfer
approximately 2.5 g sample into a 50 mL centrifuge tube. Add
2.5 mL water, cap, and vortex-mix 10 s. Let sit for up to 15 min
at room temperature.
(c)
Pipet a 5.0 mL aliquot of the appropriate ISTD from
E(i)
to each tube.
(d)
Add 1.5 mL KOH solution,
C(l)
, to each tube with a
repeater pipet.
(e)
Shake on reciprocating shaker for 5 min.
(f)
Add 8 mL extraction solution,
C(o)
, to each tube with a
repeater pipet.
(g)
Shake for 10 min.
(h)
With a repeater pipet or dispenser, add 10 mL water and
10 mL hexane to each tube.
(i)
Shake for 1 min.
(j)
Centrifuge at 1000 rpm (or equivalent to 200 ×
g
) for
5 min.
(k)
Supernatant volume
.—Transfer a portion of the
supernatant to a 12 mL scintillation vial.—(
1
)
For samples
with individual carotenoid concentrations ≤50 μg/100 g
.—Use
10 mL supernatant.
(
2
)
For samples with individual carotenoid concentrations
>50 μg/100 g
.—Use 3 mL supernatant.
(l)
Dry under nitrogen at ≤40°C.
(m)
Reconstitution volume
.—Reconstitute dried extract
in sample solvent and vortex-mix.—(
1
)
For samples with
individual carotenoid concentrations ≤100 μg/100 g
.—Add
0.5 mL.
(
2
)
For samples with individual carotenoid concentrations
of 100–500 μg/100 g
.—Add 1 mL.
(
3
)
For samples with individual carotenoid concentrations
of 500–1000 μg/100 g
.—Add 2 mL.
(
4
)
For samples with individual carotenoid concentrations
of 1000–1500 μg/100 g
.—Add 3 mL.
(n)
Filter through 0.2 μmPTFE syringe filter before injection.
G. Chromatography
(a)
Chromatographic conditions.—
Set up the UHPLC
system according to the specifications in Table
2016.13C
.
Follow the manufacturer’s instructions for column installation,
cleaning, and storage.
(b)
System suitability checks
.—(
1
)
Resolution between
lutein cis and trans isomers.—
Inject the lutein system suitability
solution,
E(h)
, and determine the resolution between the two
major
cis
isomers and all-
trans
-lutein. Resolution should
be ≥1.4 between 13-
cis
- and 13′-
cis
-lutein and ≥2.2 between
13′-
cis
- and all-
trans
-lutein using the half-width method.
See
Figure
2016.13A
.
(
2
)
Resolution between all-trans-lutein, zeaxanthin, and
apocarotenal
.—From the chromatogram of the lutein system
suitability solution,
E(h)
, determine the resolution between all-
trans
-lutein, zeaxanthin, and apocarotenal. Resolution should be
Table 2016.13C. Chromatographic conditions
Parameter
Condition
Analytical column
YMC C30 3 μm, 250 × 2.0 mm
Guard column
YMC C30 3 μm, 10 × 2.0 mm
Column temperature
30°C
Mobile phases
A: 20 mM ammonium acetate in MeOH–water
98 + 2; B: MTBE
Time, min
Mobile phase B, %
0
5
1
8
Gradient
8
15
25
100
25.5
5
32
5
Flow rate
0.25 mL/min
Backpressure
∼
185 bar
Injection volume
5 μL
UV/Vis detection
450 nm, ref = off
Figure 2016.13A. Chromatogram of lutein system suitability solution, E(h). Lut = lutein, Zea = zeaxanthin, and Apo = apocarotenal.
44