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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 3 of 10
1
Weigh accurately the contents of a 1 mg vial of SIL d6-vitamin D
3
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
3
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 Internal Standard (SILIS) ~1 μg mL
-1
—expiry 1 day
Step
Action
1
Depending on the number of samples that need to be analysed in a run, more or less
SILIS
needs to be made up.
2
For every 15 samples (or part thereof) in an analytical run, remove 1 vial of
SILD
2
SS
and
1 vial of
SILD
3
SS
from the freezer and allow to warm to room temperature.
3
Pipette 1.0 mL each of
SILD
2
SS
and
SILD
3
SS
vial into a 10 mL volumetric flasks (use a
separate 10 mL volumetric flask for each pair of
SILD
2
SS
and
SILD
3
SS
vials used).
Dilute to volume with acetonitrile and mix thoroughly.
4
Pool all of
SILIS
together and mix thoroughly.
Non-Labelled Vitamin D
2
Stock Standard (NLD
2
SS) ~1 mg mL
-1
—expiry 3 months
Step
Action
1
Weigh accurately, approximately ~50 mg of vitamin D
2
into a 50 mL volumetric flask.
2
Dissolve ~40 mL of ethanol. To promote dissolution sonicate if necessary. Mix
thoroughly, make up to volume with ethanol. Store in freezer at <-15 °C for maximum of
3 months.
Non-Labelled Vitamin D
3
Stock Standard (NLD
3
SS) ~1 mg mL
-1
—expiry 3 months
Step
Action
1
Weigh accurately, approximately ~50 mg of vitamin D
3
into a 50 mL volumetric flask.
2
Dissolve ~40 mL of ethanol. To promote dissolution sonicate if necessary. Mix
thoroughly, make up to volume with ethanol. Store in freezer at -15 °C for maximum of
3 months.
Non-Labelled Vitamin D
2
Purity Standard (NLD
2
PS) ~10 μg mL
-1
—expiry 1 day
Step
Action
1
Pipette 1.0 mL of
NLD
2
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 Vitamin D
3
Purity Standard (NLD
3
PS) ~10 μg mL
-1
— expiry 1 day
Step
Action
VitD-18 (February 2016)
FOR ERP USE ONLY
DO NOT DISTRIBUTE