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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 USE ONLY

DO NOT DISTRIBUTE