AOAC SPIFAN Review Team Meeting Book-July 22, 2015

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

pyridoxamine, the impact of underecovery is negligible for Total B 6 determination. Given the concentration of inherent pyridoxamine 5’-phosphate, 80% recovery is acceptable for this analyte.

Thiamin and Riboflavin: The recovery data for both thiamin and riboflavin in the nonphosphorylated form was excellent (97-103%) (Table 4). The phosphoylated recoveries for these two analytes also meet the 85-115% criteria (Table 5). Both of these compounds recoveries are derived from a spike solution containing two different phosphorylated forms of the analyte. The day 2 recovery of phosphorylated thiamin in SRM 1849 is 83.2% which is below the validation criteria. However, the average recovery was acceptable, indicating suitable method performance. Linearity: The standard range for pyridoxine, thiamin and riboflavin was approximately 5 ng/mL to 200 ng/mL; and for pyridoxal and pyridoxamine it was 1 ng/mL to 40 ng/mL. Six standard levels were used for each run and all curves had a correlation of R= 0.998 or better. The method is considered valid in the linear range used for each compound. Limits of Quantitation: True limits of detection were not determined in this validation exercise. However the practical limits of quatitation for each analyte are approximately +10% of the highest standard response and -10% of the lowest standard response.

Ruggedness:

Retention times for all analytes were stable throughout pre-validation and validation of this method. During this time, 3 different columns from two different packing lots were used. Because of the enzymatic treatment, a guard column was used and needs to be changed on a regular basis. The useful lifetime for the guard column will need to be determined over a longer period of time and will be documented in a separate report. With the evidence demonstrated through the pre-validation and validation, this method is determined to be adequately rugged.

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