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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