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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