2015.14 Vitamin B (July 2018)

2015.14 (JULY 2018) BVit‐02 Total Bs ERP Evaluation Form

FOR ERP USE ONLY DO NOT DISTRIBUTE

Overall the method performs well, and meets SMPR's for each of the four vitamins.

SRM statistical analysis was done even though the SRM values are not "totals" for vitamins B1,  B2, and B3.  However, there is very little  of the phosphated forms of thiamine  and very little nicotinic acid in the SRM, so the results are in good agreement.  There is a high bias for riboflavin, but that is expected due to the release of the phosphated forms  by the enzymatic digestion.  For B6, only the pyridoxine results were used for statistical analysis, and they compare well. Both repeatability (RSDr) and intermediate precision (RSD iR) are given.  There are no SMPR criteria for intermediate precision, but they are less than the RSDR requirement  which is promising for this method meeting RSDR if it proceeds to multilab evaluation (Table 3).  Intermediate precision was collected by three different analysts, on two  different instruments of the same make and model, and on six separate days over the course of about a month. Linearity criteria are not contained in the SMPR, but linearity is a daily method suitability criteria.  Table 4 shows the 5‐day averages of the standard solutions calculated as  samples against the curve. Typically, 95‐105% recovery is desired, but larger deviations can be acceptable depending on circumstances.  Pyridoxal recovery range is a little larger  (90‐110%) because the signal is more than 10x lower than the other forms.  This is by method design because pyridoxal is a minor component.  Therefore, the curve must be of  low concentration to keep product levels within the curve.  The larger deviation does not negatively impact Total B6 quantitaion becuase pyridoxal is a minor component.  If the  levels were higher, the precision would be similar the other compounds. Table 5 contains the practical LOQ (PLOQ) data derived from the Linearity study.  PLOQ's is set as the low standard of the curve after appropriate unit conversion and application  of the dilution factor of 500.  The PLOQs meet the SMPR's except for B6 by a narrow margin.  However, the LOD for B6 is nearly 200x lower than the PLOQ, so a slight  adjustment to the working standard concentration range would allow the B6 LOQ to be easily met. Spike recoveries for all 4 vitamins are in the 90‐110% range as required except for B3 recovery of 110.7% for one sample.  All five placeboes and three of the fortified matrixes  were studied.  The remaining five matrixes were not studied because they are not different enough from the eight matrixes tested to warrant additional scrutiny.  The relative  amount of each vitamin form in the spiking solution is given below:    B1: Thiamine (~80%), thiamine monophosphate (~12%, and thiamine pyrophosphate (~8%).    B2: Riboflavin (~73%), riboflavin phosphate (~18%), and FAD (~9%).    B3: Nicotinamide (~93%), nicotinic acid (~7%).    B6: Pyridoxine (~80%), pyridoxal (~5%), pyridoxal‐5'‐phosphate (~4%), pyridoxamine (~6%), pyridoxamine‐5'‐phosphate (~5%). Spikes were done at approximately 50 and 200% of fortification levels.

Table 1

Table 2a‐2d

Table 3

Table 4

Table 5