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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