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H
ostetler
:
J
ournal of
AOAC I
nternational
V
ol
.
100, N
o
.
3, 2017
coefficient (18; lutein in MTBE, 2589 at 444 nm; β-carotene in
MTBE, 2588 at 450 nm); and W = the weight (mg) of reference
standard. Spectrophotometric purity is typically greater than
0.90 (i.e., 90%).
(
2
)
Chromatographic purity.
—Inject standard working
solutions,
E(c)
, at least three times. The chromatographic purity
(CP) is calculated as
(
)
(
)
=
CP area of theall -
- carotenoid peak
sumof areas of allrelevant peaks
trans
Relevant peaks include all peaks in the HPLC chromatogram,
with the exception of solvent peaks. Chromatographic purity is
typically greater than 0.95 (i.e., 95%).
(
3
)
Reference standard purity
.—Calculate the purity (P) of
each reference standard:
= × ×
P SP CP 100
where SP = the spectrophotometric purity; CP = the
chromatographic purity; and 100 = the factor for converting
decimal to percent.
(b)
Calculate the concentration (μg/100 mL) of each
carotenoid analyte (e.g., C
Lut
for lutein) in the all-
trans
form in
each calibration solution,
E(f)
:
(
)
(
)
= × ×
× ×
×
C W 2 P 100 1000 (2/100) V V
Lut
Lut
Lut
MC Total
where W
Lut
= the weight (mg) of lutein used to make the stock
solution; 2 = the conversion of 50 mL to 100 mL; P
Lut
= the
reference standard purity of all-
trans
-lutein calculated in
H(a)
(
3
) above; 100 = the conversion from percent to decimal;
1000 = the conversion of milligrams to micrograms; (2/100) =
the dilution of stock solution to mixed carotenoid intermediate
solution; V
MC
= the volume of mixed carotenoid intermediate
solution,
E(e)
, used; and V
Total
= the dilution volume.
(c)
Calculate the concentration (μg/100mL) of the apocarotenal
internal standard (C
A
) in each calibration solution,
E(f)
:
(
)
= × × × ×
×
C W 2 CP 1000 (3/50) V V
A A
A
AI
Total
where W
A
= the weight (mg) of apocarotenal used to make the
stock solution; 2 = the conversion of 50 mL to 100 mL; CP
A
= the
chromatographic purity of apocarotenal calculated in
H(a)(
2
)
Figure 2016.13D. Chromatogram of a toddler formula sample. Lut = lutein, Zea = zeaxanthin, Apo = apocarotenal, AC = α-carotene, and
BC = β-carotene.
Figure 2016.13E. Chromatogram of an RTF adult nutritional sample. Lut = lutein, Apo = apocarotenal, AC = α-carotene, and BC = β-carotene.
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