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© 2013 AOAC INTERNATIONAL
(
c
)
dl-α-Tocopherol acetate stock standard (E1)
.—Weigh (to
0.01 mg) approximately 180 mg dl-α-tocopherol acetate into a
50 mL Class A certified volumetric flask. Dissolve in and dilute to
volume with iso-octane.
(
d
)
dl-α-Tocopherol stock solution (E2)
.—Weigh (to 0.01 mg)
100 mg α-tocopherol into a 50 mL Class A certified volumetric
flask. Dissolve in and dilute to volume with iso-octane.
(
e
)
Combined working standard (S3)
.—Transfer by pipet 4 mL
Pl, 4 mLAl, 7 mL El, and 20 mL E2 into a 50 mL volumetric flask.
Dilute to volume with iso-octane, S2. Transfer by pipet 4 mL S2
into a 50 mL volumetric flask and dilute to volume with iso-octane.
Store in a refrigerator for up to 7 days.
(
f
)
Level 1 calibration standard.—
Prepare as follows: Into a 50
mL volumetric flask, transfer by pipet 0.5 mL S3, and dilute to
volume with iso-octane. Transfer 2 mL into a 10 mL volumetric
flask and dilute to volume with iso-octane.
Level 2 calibration standard
.—Prepare as follows: Into a 50 mL
volumetric flask, transfer by pipet 8 mL S3, and dilute to volume
with iso-octane.
Level 3 calibration standard
.—Prepare as follows: Use S3
solution.
The standard curve calibration standards for levels 1 and 2
should be prepared daily.
G. Concentration of Stock Standards
(
a
)
Vitamin A palmitate
.—(
1
) Pipet 3 mL stock solution P1 into
a l00 mL volumetric flask and make up to volume with iso-octane.
Pipet 3 mL this solution into a l00 mL volumetric flask and dilute
to volume with iso-octane.
(
2
) Determine the absorption at 325 nm, zeroed against
iso-octane in a 1 cm quartz cell.
(
3
) Repeat the reading twice, rinsing the sample cuvet with the
solution before each reading. Calculate the average absorbance
reading. The potency of the vitamin A palmitate stock solution is
then calculated as follows:
IU
/
mL
=
Abs
975 × 100 3 × 100 3 × 10000 × 1.817
where
Abs
= average absorbance reading, determined above;
975 = extinction coefficient of retinyl palmitate at 325 nm;
10000 = conversion of percent to μg/mL; and 1.817 = conversion
from μg to IU for retinyl palmitate.
(
b
)
Vitamin A acetate
.—(
1
) Pipet 3 mL stock solution A1 into
a l00 mL volumetric flask and make up to volume with iso-octane.
Pipet 3 mL this solution into a l00 mL volumetric flask and dilute
to volume with iso-octane.
(
2
) Determine the absorption at 325 nm, zeroed against
iso-octane, in a 1 cm quartz cell.
(
3
) Repeat the reading twice, rinsing the sample cuvet with the
solution before each reading. Calculate the average absorbance
reading. The potency of the vitamin A acetate stock solution is then
calculated as follows:
904 .2
10000
3
100
3
100
1560
/
u
u u u
Abs
mL IU
where
Abs
= average absorbance reading, determined above;
1560 = extinction coefficient of retinyl acetate at 325 nm;
10000 = conversion of percent to μg/mL; and 2.904 = conversion
from μg to IU for retinyl acetate.
(
c
)
dl-α-Tocopherol acetate.—
(
1
) Pipet 3 mL stock solution E1
into a l00 mL volumetric flask and make up to volume with iso-
octane.
(
2
) Determine the absorption at 284 nm, zeroed against
iso-octane, in a 1 cm quartz cell.
(
3
) Repeat the reading twice, rinsing the sample cuvet with the
solution before each reading. Calculate the average absorbance
reading. The potency of the dl-α-tocopherol acetate stock solution
is then calculated as follows:
Table 2012.10. Pump gradient elution cycle
Time, min Flow, mL/min % Mobile phase A % Mobile phase B
0.0
1.5
95
5
3.0
1.5
95
5
12.0
1.5
5
95
14.0
1.5
5
95
15.0
1.5
95
5
20.0
1.5
95
5
Figure 2012.10A. VitaminApalmitate and vitaminA acetate calibration standard.
Candidates for 2016 Method of the Year
358