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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
E.
PROCEDURE
NOTE: It is recommended that all samples be prepared under UV shielded fluorescent lights
(A.L.P. Protect-A-Lamp, UV-cutoff at 460 nm).
1.
Liquid Product Sample Preparation
Liquid product samples, i.e. Ready-to-feed (RTF), ready-to-hang (RTH) or concentrated liquids (CL)
are prepared directly. See Section E.3 below for sample preparation details.
2.
Powder Product Sample Preparation
Powder products are reconstituted into room temperature laboratory water prior to sampling and
preparation. Refer to the Method Sample Table, 001s, for sample sizes and final reconstituted sample
weights.
a)
Using a tarred beaker, weigh the appropriate sample amount of sample.
b)
Add room temperature laboratory water to bring the total weight reconstituted sample weight (to
include the product weight) to the appropriate weight as defined in the Sample Table.
c)
Carefully add a stir bar so as not to splash the liquid from the beaker and place the beaker onto a
stir plate. Set the stir plate to stir the sample as fast as possible without causing the sample to
splatter or froth. Powder samples should stir for at least 10 minutes but not more than 30 minutes.
d)
Proceed to Section F.3 and to complete sample preparation.
3.
Using a tarred 50-mL, self-standing centrifuge tube, weigh an appropriate sample amount (2 grams for
typical infant formula). Record the weight to 0.1 mg.
4.
Add 50 µL of the internal standard mixture (ISSM) via Gilson positive-displacement pipette.
NOTE: The consistent addition of the ISSM to the samples is a critical component of an
analysis using stable isotope internal standards. It is strongly recommended that the
same Gilson pipette be used to add the ISSM to all samples and working standard
solutions within a given sample set.
5.
Carefully add 5mL of Enzyme Solution.
6.
Carefully add a small stir bar or glass beads to each sample to aid with agitation.
7.
Screw the cap on the sample and put samples in a 37° shaker bath, and set shaker to gently agitate
samples for 12-18 hours.
8.
Remove samples from water bath and fill to 30 mL with 50mM Ammonium Formate.
NOTE: Due to the addition of an internal standard for each compound to be analyzed, the
final volume (or weight) of the sample preparation is not required and is not
recorded.
37
BVit-02
FOR ERP USE ONLY
DO NOT DISTRIBUTE