AOAC SPIFAN Review Team Meeting Book-July 22, 2015

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

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. 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). 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

9