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ostetler
:
J
ournal of
AOAC I
nternational
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ol
.
100, N
o
.
3, 2017
9
of the major
cis
isomers of α-carotene. Although this could
potentially cause error in the β-carotene calculation, even in
a sample with high α-carotene (Figure
2016.13D
), the
cis
-
α-carotene/
cis
-β-carotene peak accounted for only 5% of the
total β-carotene peak area.
To test whether the all-
trans
isomers of lutein and β-carotene
were isomerized during the sample preparation, chromatograms
from spike and recovery experiments (
n
= 3) were used.
Samples were spiked with all-
trans
carotenoid standards along
with internal standard and carried through the preparation. No
cis
isomers of lutein were detected in the standard mixture,
and none were detected in the spiked sample. In the β-carotene
standard solution,
cis
isomers of β-carotene accounted for 3.4%
of the total peak area in the standard mixture and 3.8% of the
total β-carotene peak area in the spiked sample. This indicates
that any isomerization of all-
trans
-lutein or β-carotene during
the sample preparation is negligible.
Linearity
Linearity of the relative responses of analyte concentrations
was measured using a five-point standard curve on 3 different
days. Coefficients of determination, visual inspection, residuals,
and relative errors of back-calculated concentrations were used
for evaluation. Linearity of the internal standard was also tested.
Regression lines for all-
trans
-lutein, all-
trans
-β-carotene,
and apocarotenal are shown in Figure 4. Regression data for
residuals and back-calculated concentrations are shown in
Tables 1–3. The determination coefficients (R
2
) for each curve
were >0.999. The
y
-intercepts for all of the curves appeared
insignificant; to test this assumption, sample calculations for
all-
trans
-lutein and all-
trans
-β-carotene were performed by
using both the
y
-intercept and forcing the
y
-intercept through
zero. Two infant formulas were used: one with typical lutein and
β-carotene concentrations and one with concentrations near the
LOQ. The results (Tables 4 and 5) indicate that even for very low
concentrations (3–4 μg/100 g) the difference between the two
calculations was not more than 3%. Only when concentrations
were near 1 μg/100 g did the calculations differ by as much
as 9%. Based on these data, the
y
-intercept was forced through
zero to simplify the calculations.
In accordance with SMPR 2014.014, all data for
infant formula and adult nutritionals are presented on a
reconstituted basis (as is for RTF liquids, 25 g powder/225 g
reconstituted weight for powder samples, or 1:1 by weight
for liquid concentrates). The ranges for lutein and β-carotene
(4–240 μg/100 mL) correspond to approximately 0.8–45 μg/100 g
for samples prepared for the lowest sample concentrations. With
dilutions specified in the method, the range can be extended to
approximately 2250 μg/100 g. This range extends beyond that of
1–1300 μg/100 g specified in the SMPR.
LOD/LOQ
The LOD and LOQ were extrapolated from the S/N
calculated in ChemStation software (Agilent Technologies,
Santa Clara, CA) when measuring analyte concentrations
of 1.4–1.7 μg/100 g in spiked NIST SRM 1849a. The LOD
was calculated as (3 × measured concentration)/(S/N). The
LOQ was calculated as (10 × measured concentration)/(S/N).
Results from three different spiked samples were averaged. The
determined LOQ for both lutein and β-carotene (Tables 6 and 7)
meet the LOQ requirement of ≤1 μg/100 g in SMPR 2014.014.
Precision
Precision experiments were performed using the full
SPIFAN sample kit, designed to represent current infant
formulas and adult nutritional drinks on the market, in addition
Figure 4. Linearity plot for (A) lutein, (B) β-carotene, and
(C) apocarotenal.
Table 1. Residuals for the internal standard
Apocarotenal concn, μg/100 mL
Residual
203.5
4.119148067
101.8
–7.399430255
40.7
–3.177165248
20.4
1.435593088
4.07
2.222225757
2.04
2.79962859
49