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Abbott Nutrition Division

Simultaneous Determination of Total Vitamin B

6,

B

2,

B

3

and B

1

in Infant

Formula Products by LC-MS/MS Using Enzymatic Digestion

1mL/1000µL and 1000ng/µg: Units Conversion Factors

Example:

800 µL of Riboflavin, 250 µL of Thiamin, 250 µL of Pyridoxine, 50 µL of Pyridoxal, and

50 µL of Pyridoxamine stock solution are combined into a 5-mL volumetric flask. Fill the

flask to the mark with Mobil Phase A and invert to mix. The concentrations of each

vitamin in the IWSS are calculated as follows:

Pyridoxine, Stock Solution Concentration = 235.52

m

g/mL:

mL ng

µg

ng

µL

mL

mL

µg

mL

µL

PN

IWSS

/

6215

1

1000

1000

1

31. 124

5

250

] [

=

×

×

×

=

5.

Calculation of Working Standards

Specified volumes (see Table 8) of the VSSM are combined with Internal Standard to produce the

Calibration Standards used for the calibration curve. All calibration solutions are prepared in 25-mL

volumetric flasks.

IWSS

IWSS

WS

Vit

mL

Vol

Vit

] [

25

] [

4

×

=

Example:

Per Table 8, 250

m

L of IWSS containing 6215 ng/mL Pyridoxine is combined with 50

m

L

of ISSM into a 30-mL centrifuge tube and QS to volume with 50mM Ammonium Formate

solution. The concentration of Pyridoxine in WS4 is:

mL ng

mL

ng

mL

mL

PN

WS

/

49.51

6215 *

30

250 .0 ] [

5

=

=

6.

Analyte Quantitation: Stable-Isotope Labeled Internal Standards

a)

The peak area for both the analyte of interest and its stable-isotope labeled analog internal

standard are measured for each Working Standard concentration.

b)

The ratio of the peak areas (native/internal standard) is calculated and used together with the

analyte concentration to generate a linear regression line. The regression is calculated by plotting

peak area response ratios for each Working Standard solution vs. the known native vitamin

concentration.

Example:

Linear Regression line (Pyridoxine response ratio vs. Pyridoxine concentration) is

presented below. In this example, a single standard bracket was used.

40

BVit-02

FOR ERP USE ONLY

DO NOT DISTRIBUTE