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Analysis of Vitamin D

2

and Vitamin D

3

by LC-MS/MS in Milk

Powders, Infant Formulas, and Adult Nutritionals

Page 4 of 10

1

Pipette 1.0 mL of

NLD

3

SS

into a 100 mL volumetric flask. Make to volume with ethanol.

2

Measure the absorbance of an aliquot of this solution at the 265 nm. The

spectrophotometer should be zeroed against an ethanol blank solution. Record

absorbance and calculate concentration.

Non-Labelled Working Standard (NLWS) ~1 μg mL

-1

— expiry 1 day

Step

Action

1

Pipette 1.0 mL of

NLD

2

PS

and 1.0 mL of

NLD

2

PS

into a single 10 mL volumetric flask.

Dilute to volume with acetonitrile.

Calibration Standards (CS)— expiry 1 day

Step

Action

1

Prepare calibration standards fresh each run.

2

CS

1

Pipette 250 μL of

SILIS

and 10 μL of

NLWS

in a 25 mL volumetric flask.

CS

2

Pipette 250 μL of

SILIS

and 50 μL of

NLWS

in a 25 mL volumetric flask.

CS

3

Pipette 250 μL of

SILIS

and 250 μL of

NLWS

in a 25 mL volumetric flask.

CS

4

Pipette 250 μL of

SILIS

and 0.5 mL of

NLWS

in a 25 mL volumetric flask.

CS

5

Pipette 250 μL of

SILIS

and 1.25 mL of

NLWS

in a 25 mL volumetric flask.

3

Add 5 mL of acetonitrile

4

Add 75 μL of

PTAD Solution

to each calibration standard; shake to mix.

5

Leave calibration standards in dark for 5 mins.

6

Make each volumetric flask to volume with acetonitrile; shake to mix.

7

Transfer ~1 mL of each calibration standard to an HPLC vial ready for analysis.

Slurry Sample Preparation

Powder Sample Preparation

Step

Action

1

Accurately weigh 19.0

21.0 g of powder to a disposable slurry container. Record weight on

spreadsheet.

2

Accurately weigh ~80 mL water to container. Record weight on spreadsheet.

3

Shake thoroughly until mixed. Place in dark at room temperature for 15 mins and shake to

mix every 5 mins.

4

Accurately weigh 9.5

10.5 g of slurry or reconstituted powder sample into a boiling tube.

Record weight on spreadsheet.

Step

Action

1

Accurately weigh 1.8

2.2 g of powder sample into a boiling tube. Record weights on

spreadsheet.

VitD-18 (February 2016)

FOR ERP USE ONLY

DO NOT DISTRIBUTE