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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.
11
BVIT-02
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