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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 5 of 10
Liquid Sample Preparation
Extraction and Derivatisation
Chromatography: Instrument Parameters
Action
Set up reverse phase UHPLC system and allow to equilibrate:
Mobile Phase:
A = Formic acid, 0.1%
B = Methanol, 100%
Column:
Kinetex C
18
core-shell, 2.6
P
m, 2.1 x 50 mm
Oven Temp:
40 °C
Chiller Temp:
15 °C
Injection Volume:
3
P
L
Initial flow rate:
0.6 mL min
-1
(see gradient details below)
Step
Action
1
Accurately weigh 10 mL of liquid milk into a boiling tube. Record weight on spreadsheet.
Step
Action
1
To reconstituted powder sample, slurry sample, or liquid sample in a boiling tube.
2
Add 10 mL
Ethanolic Pyrogallol Solution
, cap and mix on vortex mixer.
3
Add 0.5 mL of internal standard
SILIS
, cap and mix on vortex mixer.
4
Add 2 mL of
Potassium Hydroxide Solution
, cap and mix on vortex mixer.
5
Place in water bath at 70 °C for 1 hour with regular mixing on vortex mixer every 15 mins.
6
Cool in water bath at room temperature.
7
Add 10 mL isooctane.
8
Cap tube tightly and mix on horizontal shaker for 10 mins.
9
Add 20 mL of water and invert tube 10 times.
10
Centrifuge at 250 x g for 15 mins.
11
Transfer a 5 mL aliquot of the upper isooctane layer into a 15 mL disposable centrifuge tube
using a disposable pipette, taking care NOT to transfer any of the lower layer.
12
Add 5 mL of water, cap and mix on vortex mixer.
13
Centrifuge at 2000 x g for 5 mins.
14
Transfer 4
–
5 mL of upper layer to a new 15 mL disposable centrifuge tube using a
disposable pipette, taking care NOT to transfer any of the lower layer (discard tube with
lower layer).
15
Add 75 μL of PTAD reagent, cap and immediately vortex mix.
16
Allow to stand in dark for 5 mins to allow for derivatisation reaction to complete.
17
Add 1 mL acetonitrile, cap and mix on vortex mixer.
18
Centrifuge at 2000 rcf for 5 mins.
19
Using a 1 mL pipette, transfer 0.5 mL of the lower layer into an Eppendorf vial taking care
NOT to transfer any of the upper layer.
20
To the Eppendorf vial, add 167 μL of water; cap and vortex mix.
21
Using syringe filter, transfer an aliquot to an amber LC vial, cap ready for analysis.
VitD-18 (February 2016)
FOR ERP USE ONLY
DO NOT DISTRIBUTE