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© 2013 AOAC INTERNATIONAL
I. HPLC Analysis
(
a
) Use the pump gradient elution cycle (Table
2012.10
).
(
Note
: The gradient parameters can be altered as required to
maximize the analytical separation and avoid interferences.)
(
b
) Set the detectors to the following wavelengths: UV detector,
325 nm for vitamin A palmitate and vitamin A acetate. FLD
(excitation/emission) 280/310 nm for dl-α-tocopherol acetate and
dl-α-tocopherol (Figures
2012.10A–D
).
J. System Suitability
The following system suitability and standard checks are to be
met when running this method.
(
a
)
Linearity
.—The coefficient of determination, R
2
, of each
calibration curve shall be ≥0.995.
(
b
)
Standard injection precision
.—When a stable baseline
is obtained, inject a medium reference standard three times in
succession and determine the RSD. The RSD determined must be
≤2%.
(
c
)
Standard response accuracy
.—Determine the slope change
between successive calibrations and the difference must be ≤3%.
The appropriate number of samples between successive calibrations
is typically 6–8.
(
d
)
Tailing/asymmetry factor
.—The target limits of the tailing
factor for meeting system suitability are 0.8–1.2. The system should
be closely monitored if outside the target limits, and action taken if
the value is outside 0.5–1.5 (e.g., column cleaning, reconditioning,
or replacement).
K. Calculations of Sample Concentrations
(
a
)
Powder samples
.—
ܸ݅
ݐ
ܽ݉݅݊
ܥ
݊ܿ (
IU/100 g
ݎ )
݁ܿ݊
ݐݏ
݅
ݐݑݐ
݁݀
ܨ
݈݅݊ܽ ܲ
ݎ
݀
ݑ
ܿ
ݐ
= (
Area/Height of Sample Peak
െ
C
)
݈ܵ݁
× ܻ ܹ × 10 × 100 × ܹ225
where
C
=
y
-intercept;
Y
= dilution volume of test portion;
10 = volume (mL) of iso-octane; 100 = conversion to per 100 g;
W
= sample amount in g; and 225 = weight of dilution water.
(
b
)
Liquid samples
.—
ܸ݅
ݐ
ܽ݉݅݊
ܥ
݊ܿ. (
IU/100 g reconstituted Final Product
) =
= (
Area/Height of Sample Peak
െ
C
)
݈ܵ݁
× ܻ ܹ × 10 × 100 ×
ܦ
where C=
y
-intercept,
Y
=dilution volume of test portion,
10=volume (mL) of iso-octane, 100=conversion to per 100 g,
W
= sample amount in mL for liquid ready-to-feed and concentrates,
and
D
=density of liquid product. Total vitamin A is the sum
of the
trans
vitamin A concentration and the 13-
cis
vitamin A
concentration in IUs.
One IU is equal to 0.30 μg
all-trans
retinol. Retinyl palmitate
or retinyl acetate in IU/100 g reconstituted final product can
be converted to μg/100 g reconstituted final product in retinol
equivalents (REs) by multiplying by 0.30.
One RE is defined as 1 μg retinol.
Total vitamin E (sum of dl-α-tocopherol and dl-α-tocopherol
acetate) in IU/100 g reconstituted final product can be converted
to mg/100 g reconstituted final product in α-TEs by multiplying
by 0.671.
References:
J. AOAC Int
.
96
, 1073(2013)
DOI: 10.5740/jaoacint.13-103
AOAC SMPR 2011.003
J. AOAC Int
.
95
, 291(2012)
DOI: 10.5740/jaoac.int.11-0439
AOAC SMPR 2011.010
J. AOAC Int
.
96
, 485(2013)
DOI: 10.5740/jaoac.int.SMPR2011.010
Candidates for 2016 Method of the Year
361