Review Team (February 2016).pdf

Silliker

Chem, Res. Center Crete, IL – Report of A Validation of LC-MS/MS Method for Folate Analys

The above results of total folate as folic acid (sum of the detected folate compounds after calculation as

folic acid) in the SPIFAN samples and the check samples obtained using no cojugase treatment during

the extraction. All the SPIFAN samples were analyzed on 3 separate days in triplicates. Averages and

interday-repeatability value/precision (%RSD) for the analysis are presented. Total folate

concentration determined in the reconstituted liquid samples were in the range of 14-33 mcg/100g and

in range of 15 to 57 mcg/100g in the RTF samples. The values are some what lower than those obtained

in the analysis with the conjugase treatment for corresponding samples.

The interday-precision or repeatability (%RSD) obtained from the analysis of SPIFAN II samples with

no conjugase treatment was found to be in the range of 2-17%. The precision RSD

requirement of the

folate SPIFAN SMPR (2011.006) is 11% or less for concentration range 0.5 to 21.4 mcg/100g and 7%

or less for higher concentration samples (21.5-85 mcg/100g). Seven of the samples when analyzed

with no conjugase treatment provided %RSD values within the folate SPIFAN SMPR specifications.

But five of the analyzed samples for some reason provided % RSD values somewhat higher than the

SMPR specifications. It may be stated that the intermediate precision or inter-day precision as % RSD

is not clarified in the SMPR requirements. Inter-day precision as % RSD values obtained in SPIFAN II

samples with no conjugase treatment are well below the reproducibility requirement (as %RSD

) of

the SMPR (< 16%) with exception of one sample which provided unusually higher variability in

replicate analysis (% RSD value of 17%).

Comparison of folate analysis by the method in SPIFAN II samples with and without conjugase

treatment during extraction

Nine beverages prepared from the respective powdered SPIFAN samples and three RTF samples were

analyzed for all the targeted folate compounds. The folate in these samples was analyzed in one

experiment by employing trienzyme extraction including the conjugase treatment. In another

experiment the same samples were analyzed by the method employing the amylase and protease but no

conjugase treatment during the extraction. Results in both cases in the reconstituted samples were

calculated and reported on reconstituted beverage basis (mcg/100g). The folate compounds in the RTF

samples were calculated on as is basis (mcg/100g). Each detected folate compound was calculated as

folic acid and sum of that provided total folate as folic acid and reported as mcg/100g. Each sample in

both experiments (with conjugase and without conjugase) was analyzed in three replicates on three

separate days. The averages and inter-day repeatability/precision (%RSD) calculated and are presented

in the following Table 18.

2011.06/Fol-22 (February 2016)

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