610 / 670
ethanol.
2.1.6 Working standard solutions
˖
In 7 volumetric flasks (10 mL), different amount of VD2/VD3
standard mixtures (shown in Table 1) and 0.05 mL of ID3 stock internal standard solution (20
μ
g/mL, 800 IU/mL) were added, bring to volume with acetonitrile to prepare working standard
solutions.
2.1.7 Derivatization of working standard solutions
Working standard solutions (0.1 mL) were accurately transferred into small vials, and the
solvent was removed by nitrogen flushing. PTAD acetonitrile solution (0.6 mL, 1.0 mg/mL) was
then added, followed with vortexing (5 s) and sonication (5 min). The reaction solution was
incubated at room temperature for 30 min. After the derivatization reaction, 0.4 mL of lab water
was added and mixed well. The solutions were filtered with 0.22
μm P
VDF membranes to obtain
derivatized working standard solutions to prepare calibration curve.
Table 1. The concentration of working standard solutions before and after the derivatization.
Level
Preparation of working
Standard solutions
Concentration of working
Standard solutions
Concentration of derivatized working
Standard solutions
VD2/VD3
Standard mixture,
mL
ID3
Stock solution,
mL
VD2/VD3
ng/mL
ID3
ng/mL
VD2/VD3
ng/mL
ID3
ng/mL
1
0.05
a
0.05
c
5
100
0.5
10
2
0.2
a
0.05
c
20
100
2
10
3
0.5
a
0.05
c
50
100
5
10
4
0.1
b
0.05
c
100
100
10
10
5
0.2
b
0.05
c
200
100
20
10
6
0.5
b
0.05
c
500
100
50
10
7
1.0
b
0.05
c
1000
100
100
10
Note
˖
a
VD2/VD3 standard mixture 1 (1.0
μg/mL,
40 IU/mL);
b
VD2/VD3 standard mixture 2 (
10 μg/mL, 400
IU/mL)
˗
c
ID3 stock internal standard solution (
20 μg/mL
, 800 IU/mL).
2.1.8 Pyrogallic acid ethanol solution
᧤
2 g/mL
᧥᧶
Accurately weigh 2 g of pyrogallic acid into a
100mL volumetric flask, dissolve and bring to volume with anhydrous ethanol.
2.1.9 BHT hexane solution
᧤
12.5 mg/L
᧥᧶
Accurately weigh 12.5 mg of BHT into a 1 L volumetric
flask, dissolve and bring to volume with hexane.
2.2 Sample Preparation
The sample preparation procedure was performed according to the AOAC First Action
method 2011.11 with some modifications. Powder samples were reconstituted by dissolving 25 g
of powder into 200 g of lab water. Reconstituted samples or liquid samples (10 g) were accurately
weighed (sensitive to 0.0001 g) into a 100 mL flask, followed by adding 40 mL of 2 g/100 mL
pyrogallic acid ethanol solution, 1 mL of 0.1
μ
g/mL ID3 working internal standard solution (0.1
VitD-17 (February 2016)
FOR ERP USE ONLY
DO NOT DISTRIBUTE