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M
c
M
ahon
:
J
ournal of
AOAC I
nternational
V
ol
.
99, N
o
.
1, 2016
225
A. Principle
This procedure utilizes the proteolytic enzyme papain to
hydrolyze the hydrophilic protein coating of fat micelles in
milk- or soy-based infant formulations in an aqueous solution.
The hydrophobic contents of the micelles are then extracted
quantitatively into iso-octane in a single extraction and
chromatographed by normal-phase HPLC using a Zorbax
®
NH2 analytical column. The analytes are eluted with a gradient
and α-tocopherol and α-tocopherol acetate quantified using
fluorescence detection, excitation/emission, 280/310 nm.
Vitamin A palmitate (
cis
and
trans
) and vitamin A acetate (
cis
and
trans
) are quantified using UV detection at 325 nm.
B. Apparatus
Common laboratory glassware and equipment and, in
particular, the following:
(
a
)
HPLC system.—
Consisting of pump, autosampler,
programmable UV detector operating at 325 nm for vitamin A,
and a fluorescence detector (FLD) at an excitation wavelength
of 280 nm and an emission wavelength of 310 nm for vitamin E.
(
b
)
HPLC column
.—Analytical normal-phase column, e.g.,
Zorbax NH2, 5 µm, 150 × 4.6 mm, or equivalent.
(
c
)
Water bath
.—Set at 37 ± 2°C.
(
d
)
Centrifuge
.—With adapters for 50 mL centrifuge tubes,
capable of 4000 min
–1
.
(
e
)
UV-Vis spectrophotometer
.—With 1 cm quartz cells.
(
f
)
Analytical balance
.—Weighing to four decimal places.
(
g
)
Amber HPLC
vials
.—2 mL, with plastic caps and
polytetrafluoroethylene (PTFE) seals.
(
h
)
Disposable centrifuge tubes
.—50 mL, e.g., Falcon
(Fisher, Pittsburgh, PA), or equivalent.
(
i
)
Laboratory mechanical test tube shaker
.
(
j
)
Sonic bath
.
(
k
)
One-mark volumetric flasks
.—50 and 100 mL.
(
l
)
Vacuum filtration apparatus
.—With 0.45 μm nylon
membrane.
(
m
)
Laboratory glass bottles
.—250 mL and 1 and 2 L, e.g.,
Duran (Wertheim/Main, Germany), or equivalent.
(
n
)
Pipettors and tips.—
Gilson P10002, or equivalent.
C. Standards
(
a
)
Vitamin A palmitate reference standard.—
Primary
standard, U.S. Pharmacopeial Convention (USP; Rockville, MD,
USA), or equivalent. The standard shall contain antioxidant.
Table 2012.10A. Method performance requirements: Single-laboratory validation (SLV) and multilaboratory testing (MLT)
results summary—Vitamin A
a
Parameter
Method performance
requirements
Retinyl palmitate
Retinyl acetate
Analytical range
7.0–382.6
b
2–450
2–450
Limit of detection (LOD)
≤2.0
b
0.099
0.85
Limit of quantitation (LOQ)
≤7.0
b
0.33
2.83
Repeatability (RSD
r
) (SLV)
7–300
b
≤8%
≤4.03%
≤6.56%
Intermediate precision (RSD
r
) (SLV)
7–300
b
—
≤6.23%
≤10.63%
Recovery (SLV)
90–110% (mean spiked recovery
over the range of the assay)
99.13%
96.53%
Reproducibility (RSD
R
) (MLT)
10–383
b
≤16%
6.51–16.25%
11.73–22.61%
a
Concentrations apply to (
1
) ‘ready-to-feed’ liquids; (
2
) reconstituted powders (25 g into 200 g water); and (
3
) liquid concentrate diluted 1:1 by weight.
b
µg/100 g reported separately as
cis
-13 retinol and all-
trans
retinol in reconstituted final product.
Table 2012.10B. Method performance requirements: Single-laboratory validation (SLV) and multilaboratory testing (MLT)
results summary—Vitamin E
a
Parameter
Method performance
requirements
α-Tocopherol
α-Tocopherol acetate
Analytical range
0.2–8
b
0.03–8
0.02–9.4
Limit of detection (LOD)
≤0.1
b
0.01
0.023
Limit of quantitation (LOQ)
≤0.2
b
0.035
0.075
Repeatability (RSD
r
) (SLV)
0.5–2.0
b
≤8%
≤4.25%
≤4.39
4–8
b
≤6%
≤3.78%
≤3.53%
Intermediate precision (RSD
r
) (SLV)
<2.0
b
—
≤17.31%
≤10.54%
>2.0
b
—
≤9.24%
≤8.25%
Recovery (SLV)
90–110% (mean spiked
recovery over the range of
the assay)
100.60%
102.92%
Reproducibility (RSD
R
) (MLT)
0.5–1.0
b
≤22%
3.84–43.56%
—
1.0–8.0
b
≤16%
—
4.15–11.25%
Reproducibility (RSD
R
) (MLT) total vitamin E 0.5–1.0
b
≤22%
3.84–10.78%
1.0–8.0
b
≤16%
≤12.47%
a
Concentrations apply to (
1
) ‘ready-to-feed’ liquids; (
2
) reconstituted powders (25 g into 200 g water); and (
3
) liquid concentrate diluted 1:1 by weight.
b
mg/100 g α-tocopherol and α-tocopheryl acetate in reconstituted final product.
177