528 / 670
Silliker
Chem, Res. Center Crete, IL – Report of A Validation of LC-MS/MS Method for Folate Analys
28
* Total Folate Results are calculated based on sum of all the folate compounds in the samples, after conversion
of each compound as folic acid. Results are provided as mcg/g per 100 g of reconstituted beverage or RTF.
The total folate concentration determined in the reconstituted liquid SPIFAN samples employing the
conjugase in extraction is in the range of 16-34 mcg/100g and 16.5 to 68 mcg/100g in the RTF samples.
The interday-precision (%RSD) in all of the SPIFAN II samples was found to be less than 5% in the
analysis employing conjugase treatment. Total folate concentration obtained by the method which
excluded conjugase treatment in the same SPIFAN reconstituted liquid samples were in the range of
14-33 mcg/100g and 15 to 57 mcg/100g in the RTF samples. The conjugase treatment during the
extraction in folate analysis provided total folates in the samples about 4-19% higher than the method
which excluded the conjugase. The samples analyzed employing conjugase treatment provided % RSD
of <5% where as the method with no conjugase exhibited higher variation with %RSD in the range of
4-15% and in one exceptional case of 17%. There is no obvious explanation available for higher
variability observed in folate analysis of the SPIFAN samples by the method with no conjugase.
Minor Folates contribution to total folate in the SPIFAN II samples:
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 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 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.
The targeted folate compound in the analysis include folic acid, 5-CH3-THF, 5-CHO-THF, THF, 10-
CH3-folic acid, 10-CHO-folic acid and 5,10-CH=THF (5,10-methenyl-THF). Minor folates in the
current study are those contributing to total folate other than folic acid and 5-CH3-THF. Contribution
of minor folates to total folate is calculated using the following relationship.
Minor folates as folic acid = Total folate as folic acid – sum of folic acid and 5-CH3-THF as folic acid
in the respective sample.
Minor folate in a sample can be contributed by all or any of the following detected folate compounds.
Minor Folate as folic acid (Minor folate) = 5-CHO-THF + THF + 10-CH3-folic acid + 10-CHO-folic
acid + 5,10-CH=THF (sum of all or any of the detected compound calculated as folic acid).
The contents of folate due to minor folates (as folic acid) determined in the SPIFAN II samples by the
analysis with and without conjugase treatment is presented in the following Table 19.
17
as Beverage
Adlt Nutr RTF
High Fat
00729RFOO
Recd 1/22/16
RTF
68.1
1.8
57.4
1.7
118.8
2011.06/Fol-22 (February 2016)
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