AOAC SPIFAN Review Team Meeting Book-July 22, 2015

BVIT-02 FOR ERP/WORKING GROUP USE ONLY DO NOT DISTRIBUTE

Abbott Nutrition Division Simultaneous Determination of Total Vitamin B 6, B 2, B 3 in Infant Formula Products by LC-MS/MS Using Enzymatic Digestion and B 1

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.

Vol

Vit

Vit

WS ] [ 4

IWSS ] [

=

×

IWSS mL

25

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 30 250 .0 ] [ mL

ng

6215 *

PN

mL ng

/ 49.51

=

=

WS

5

mL

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.

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