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Silliker
Chem, Res. Center Crete, IL – Report of A Validation of LC-MS/MS Method for Folate Analys
24
The above results of total folate as folic acid (sum of the detected folate compounds after calculation as
folic acid) in the SPIFAN samples obtained using the trienzyme extraction including the conjugase
treatment for all 12 SPIFAN samples and four check samples are based on analysis on 3 separate days
in triplicates (SPIFAN samples). Averages and interday-repeatability value/precision (%RSD) for the
analysis are presented.
Total folate values obtained for the reference materials are within the specified range of total folate for
respective sample.
Total folate concentration determined in the reconstituted liquid samples is in the range of 16-34
mcg/100g and in the range of 16.5 to 68 mcg/100g in the RTF samples. The inter-day-precision or
repeatability (%RSD) obtained from the analysis in of all the analyzed SPIFAN II samples was found
to be less than 5%. The precision RSD
r
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). Thus the method meets the precision requirement of the Folate SMPR for all the
SPIFAN samples (analyzed on 3 days in triplicates).
Intermediate precision or inter-day precision as % RSD not clarified in the SMPR requirements. Inter-
day precision as % RSD obtained values are well below the reproducibility requirement (as %RSD
R
) of
the SMPR (< 16%). The range of the inter-day precision (as % RSD) obtained by the method are 7 –
12.7% .
Averages and Precision of folate analysis in SPIFAN II samples using protease and amylase but
no conjugase treatment:
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 extracted using
protease and amylase but the conjugase treatment was excluded during the extraction. Results 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 of the 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 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 17.
Table 17: Results of folate analysis in the SPIFAN II samples using the protease and amylase for
extraction but excluding the conjugase treatment. Total folate (mcg/100g) calculated as sum of
the detected folate compounds as folic acid in reconstitute beverages/RTF liquid samples.
Averages and precision (as % RSD) of replicates analyzed on separate 3 days (total n=9).
Sample
Matrix
Lot#
Analyzed
AS
Total Folate as Folic Acid
n
Repeat
days
#
Av. Mcg/100g
Liquid or RTF*
Stdev
% RSD
NIST SRM
1849a**
Infant Formula
-
Powder
210.6
3.3
1.6
3
1
BCR 485** Dried Vegetable
Powder
-
Powder
259.2
11.4
4.4
3
1
NIST SRM
3233**
Breakfast
Cereal
-
Powder
1233.6
41.1
3.3
3
1
LCS**
Breakfast
Cereal
-
Powder
39.3
4.4
11.1
5
3
2011.06/Fol-22 (February 2016)
FOR ERP USE ONLY
DO NOT DISTRIBUTE