45 / 258
H
ostetler
:
J
ournal of
AOAC I
nternational
V
ol
.
100, N
o
.
3, 2017
5
≥3.7 between all-
trans
-lutein and zeaxanthin and ≥2.5 between
zeaxanthin and apocarotenal.
See
Figure
2016.13A
.
(
3
)
Resolution between β-carotene cis and trans isomers and
α-carotene
.—Inject the β-carotene system suitability solution,
E(g)
, and determine the resolution between the two major
cis
isomers of β-carotene, all-
trans
-β-carotene, and α-carotene.
Resolution should be ≥1.7 between 13-
cis
-β-carotene and
cis/
trans
-α-carotene and ≥2.6 between all-
trans
-β-carotene and
9-
cis
-β-carotene.
See
Figure
2016.13B
.
(
4
) Inject the calibration solutions before and after each set
of sample injections (up to 12 samples in each set). Calculate
the slope relative to the internal standard as shown in
H(d)
.
The coefficient of determination (R
2
) for each curve should be
>0.995. The slopes from the two curves should not differ by
more than 2.0%.
(
5
) Representative sample chromatograms are shown in
Figures
2016.13C
-
E
.
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
.—Measure each standard measuring
solution,
E(b)
, against an MTBE blank at its absorbance
maximum (444 nm for lutein and 450 nm for β-carotene).
Calculate the spectrophotometric purity (SP) of each reference
standard as the observed absorbance over the expected
absorbance:
SP Abs
50000 E
W
MS
1%,1cm
(
)
(
)
=
×
×
where Abs
MS
= the absorbance of the standard measuring
solution; 50000 = the dilution factor; E
1%,1cm
= the extinction
Figure 2016.13B. Chromatogram of β-carotene system suitability solution, E(g). AC = α-carotene and BC = β-carotene.
Figure 2016.13C. Chromatogram of a milk-based infant formula sample. Lut = lutein, Zea = zeaxanthin, Apo = apocarotenal, and
BC = β-carotene.
45