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were much higher than typically expected in product to ensure the enzyme was working
adequately.
Procedure
Briefly, powder samples were first reconstituted in water at 10% (w/w). Two
grams of the reconstituted sample was weighed into 50 mL disposable centrifuge tubes,
internal standard was added, and 5mL of a three enzyme cocktail was added. The
enzyme cocktail was prepared in 50 mM ammonium formate solution adjusted to a pH
between 4.0 and 4.5. The samples were incubated in a 37° water bath and shaken for a
minimum of 12 hours. Upon removal from the bath, the samples were filled to 30 mL
with 50 mM ammonium formate solution. The samples were then mixed thoroughly and
filtered through 0.20 µm PTFE syringe filters into autosampler vials. The full procedural
method is included with this report.
Results
Precision:
Three fortified samples SRM 1849, SRM 1846, and a milk based infant formula,
and a placebo infant formula were tested in triplicate on six days with freshly prepared
intermediate, and working standards. The same stock solutions were used for the
standards on all six days. The same mobile phase was used for three consecutive days
and then made fresh for the next three days.
Total
B
6
:
The Total
B
6
amount is made up of a sum of the active forms of pyridoxine
(fortified), pyridoxal (inherent), and pyridoxamine (inherent). The results for Total
B
6
are
reported in Tables 1, 2, and 3. The precision criteria was met for two of the three matrices
tested, with the third exhibiting a 5.5% RSD. The slightly higher RSD is consistent with
the increased error introduced by summing three results. Despite the slightly high RSD
of the SRM 1849 data, the overall method precision for this Total
B
6
is acceptable.
Total Thiamin:
Total thiamin has one form and two phosphorylated analogs. Milk generally has
some bound thiamin, but in scant amounts
3 .The RSDs for all three products tested were
under 2% which is well below the 5% criteria (Tables 1, 2, and 3).
Total Riboflavin:
Total riboflavin has one form and two phosphorylated analogs. The majority of
inherent Riboflavin is in the free form
3
. The RSDs for all three products tested were
approximately 2% which is well below the 5% criteria (Tables 1, 2, and 3).
9
BVIT-02
FOR ERP/WORKING GROUP USE ONLY
DO NOT DISTRIBUTE