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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 2 of 10

Reagents

x

PTAD (4-Phenyl-1,2,4-triazoline-3,5-dione), reagent grade [store in desiccator at 2-8 °C]

x

Formic acid, LC-MS grade

x

Potassium hydroxide, reagent grade

x

Magnesium Chloride anhydrous, reagent grade

x

Pyrogallol, reagent grade

x

Vitamin D

2

(ergocalciferol), Sigma 47768 or equivalent

x

Vitamin D

3

(cholecalciferol), Sigma C1357 or equivalent

x

SIL d6-vitamin D

2

(26,26,26,27,27,27-d6 ergocalciferol), Chemaphor CHE018 or equivalent

x

SIL d6-vitamin D

3

(26,26,26,27,27,27-d6 cholecalciferol), Chemaphor CHE012 or equivalent

x

Ethanol, LC grade

x

Methanol, LC-MS grade

x

Isooctane, LC grade

x

Acetone, LC grade

x

Acetonitrile, LC-MS grade

x

Water, reagent grade (

18 M

Ω)

Solutions

x

Acetone (dry):

To 50 mL acetone add ~10 g magnesium chloride to remove traces of moisture. Seal

container with parafilm and wait for magnesium chloride to settle before use (~24 h). Expiry 1 month.

x

PTAD solution

(10 mg mL

-1

): To a 5 mL volumetric flask add 50 mg of PTAD, add 4 mL of dry acetone

and dissolve; make to volume with acetone and sealed in capped vial with parafilm. Expiry: 1 day.

x

Potassium hydroxide solution

(50% w/v): Dissolve 500 g potassium hydroxide in 1 L water. Expiry:

1 month.

x

Ethanolic pyrogallol solution

(1% w/v): Dissolve 5 g pyrogallol in 500 mL of ethanol. Expiry 1 day.

x

Mobile phase A

(Formic acid 0.1% v/v): To a measuring cylinder, add 0.5 mL formic acid, make to 500 mL

with water. Expiry 1 week.

Safety

x

Wear safety glasses and protective gloves particularly when handling potassium hydroxide, PTAD, and

organic solvents.

x

Handle solvents in the fumehood.

x

Refer to MSD sheets for detailed safety instructions for each chemical used.

Procedure

Vitamin D is sensitive to light; perform all steps under low-level incandescent lighting.

Stable Isotope Labelled Vitamin D

2

Stock Standard (SILD

2

SS) ~10 μg mL

-1

—expiry as per CoA

Step

Action

1

Weigh accurately the contents of a 1 mg vial of SIL d6-vitamin D

2

into a 100 mL

volumetric flask.

2

Dissolve in ~90 mL of ethanol. To promote dissolution, sonicate if necessary. Mix

thoroughly, make up to volume with ethanol.

3

Measure the absorbance of an aliquot of

SILD

2

SS

at 265 nm. The spectrophotometer

should be zeroed against an ethanol blank solution. Calculate and record concentration.

4

Immediately dispense aliquots (~1.3 mL) into cryogenic vials and freeze at <-15 °C.

Stable Isotope Labelled Vitamin D

3

Stock Standard (SILD

3

SS) ~10 μg mL

-1

—expiry as per CoA

Step

Action

VitD-18 (February 2016)

FOR ERP USE ONLY

DO NOT DISTRIBUTE