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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/WORKING GROUP USE ONLY
DO NOT DISTRIBUTE