179 / 258
M
c
M
ahon
:
J
ournal of
AOAC I
nternational
V
ol
.
99, N
o
.
1, 2016
227
(
3
) Calculate the average absorbance reading. Calculate the
spectrometric purity as a decimal, SP
AA
, of retinyl acetate using
Equation 2:
SP
AA
=
01
1
100 05
560 1
× × ×
ts
AA
m
A
r
(2)
where
A
= average absorbance reading, determined above;
1560 = extinction coefficient of retinyl acetate at 325 nm; and
m
st
= mass of the reference standard in mg.
(
c
)
Spectrometric purity of α-tocopherol acetate stock
solution.—
(
1
) Pipet 3 mL α-tocopherol acetate stock standard
solution into a l00 mL volumetric flask and make up to volume
with ethanol.
(
2
) Determine the absorption at 284 nm, zeroed against
ethanol, in a 1 cm quartz cell. Repeat the reading twice,
rinsing the sample cuvet with the solution before each reading.
Calculate the average absorbance reading.
(
3
) Calculate the spectrometric purity as a decimal, SP
TA
, of
α-tocopherol acetate using Equation 3:
SP
TA
=
01
3
100 05
6.34
× × ×
ts
m
A
r
(3)
where
A
= average absorbance reading, determined above;
43.6 = extinction coefficient of tocopherol acetate at 284 nm;
and
m
st
= mass of the reference standard in mg.
(
d
)
Spectrometric purity of α-tocopherol stock solution
.—
(
1
) Pipet 3 mL α-tocopherol stock standard solution into a l00 mL
volumetric flask and make up to volume with ethanol.
(
2
) Determine the absorption at 292 nm, zeroed against
ethanol in a 1 cmquartz cell. Repeat the reading twice, rinsing the
sample cuvet with the solution before each reading.
(
3
) Calculate the average absorbance reading. Calculate the
spectrometric purity as a decimal, SP
T
, of α-tocopherol using
Equation 4:
SP
T
=
01
3
100 05
8.57
× × × =
ts
T
m
A
r
(4)
where
A
= average absorbance reading, determined above; 75.8 =
extinction coefficient of tocopherol at 292 nm; and
m
st
= mass
of the reference standard in mg.
Figure 2012.10B. HPLC chromatogram of vitaminA
palmitate test sample. Peak 1,
13
cis
-isomer; peak 2,
cis
-
isomer; peak 3,
trans
-isomer.
Figure 2012.10C. HPLC chromatogram of vitaminA
acetate calibration standard. Peak 1,
13
cis-
isomer; peak 3,
trans
-isomer.
Figure 2012.10A. HPLC chromatogramof vitaminA
palmitate calibration standard. Peak 1,
13
cis
-isomer; peak 2,
trans
-isomer.
179