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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