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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 9 of 10
25
0.25 x
NLWS
CS3
D2conc
D2conc
25
0.25 x
NLWS
CS3
D3conc
D3conc
25
0.5 x
NLWS
CS4
D2conc
D2conc
25
0.5 x
NLWS
CS4
D3conc
D3conc
25
1.25 x
NLWS
CS5
D2conc
D2conc
25
1.25 x
NLWS
CS5
D3conc
D3conc
where:
CS1-5
D2conc
= concentration of vitamin D
2
in calibration standards (
units
: ng mL
-1
)
CS1-5
D3conc
= concentration of vitamin D
3
in calibration standards (
units
: ng mL
-1
)
NLWS
D2conc
= concentration of vitamin D
2
in working standard (
units
: ng mL
-1
)
NLWS
D3conc
= concentration of vitamin D
3
in working standard (
units
: ng mL
-1
)
Concentration of stable isotope labelled vitamin D
3
in calibration standards
CS1–5
:
25
0.25 x
SILIS
5- CS1
D2conc
D2conc
25
0.25 x
SILIS
5- CS1
D3conc
D3conc
where:
CS1-5
D2conc
= concentration of SIL d6-vitamin D
2
in calibration standards (
units
: ng mL
-1
)
CS1-5
D3conc
= concentration of SIL d6-vitamin D
3
in calibration standards (
units
: ng mL
-1
)
SILIS
D2conc
= concentration of SIL d6-vitamin D
2
in internal standard (
units
: ng mL
-1
)
SILIS
D3conc
= concentration of SIL d6-vitamin D
3
in internal standard (
units
: ng mL
-1
)
Determine the linear regression curve y =
m
x +
c
(using the "least squares" method) for the ratio of peaks areas
(non-labelled vitamin D/stable isotope labelled vitamin D) vs. the ratio of concentrations (non-labelled vitamin
D/stable isotope labelled vitamin D) for five calibration standards with the y-intercept forced through zero.
The concentration of vitamin D
3
in the sample is calculated as:
1000
100 x
mass S
alqt
SILIS
x
L
D2conc
SILIS
x
SILD2 PA
NLD2 PA
D
Result
2
where:
Result D
2
= vitamin D
2
concentration in sample (
units
: μg hg
-1
)
PA
NLD2
= peak area of vitamin D
2
in sample (
units
: none)
PA
SILD2
= peak area of SIL d6-vitamin D
2
in sample (
units
: none)
SILIS
conc
= concentration of SIL d6-vitamin D
2
in
SILIS
(
units
: ng mL
-1
)
L = slope of calibration curve (
units
: none)
SILIS
alqt
= volume of
SILIS
aliquot spiked to sample (
units
: mL); [0.5 mL]
1000 = mass conversion factor (
units
: μg g
-1
to mg g
-1
)
100 = mass conversion factor (
units
: mg g
-1
to mg hg
-1
)
S
mass
= mass of sample (
units
: g)
The concentration of vitamin D
3
in the sample is calculated as:
1000
100 x
mass S
alqt
SILIS
x
L
D3conc
SILIS
x
SILD3 PA
NLD3 PA
D
Result
3
where:
Result D
3
= vitamin D
3
concentration in sample (
units
: μg hg
-1
)
PA
NLD3
= peak area of vitamin D
3
in sample (
units
: none)
VitD-18 (February 2016)
FOR ERP USE ONLY
DO NOT DISTRIBUTE