AOAC SPIFAN ERP & Working Group Method Reviews (July 10, 2019)

Figure 2011.06. Chromatogram of folate analysis providing elution pattern of folate vitamers. 5-Methyltetrahydrofolic acid peak is marked as 5-CH 3 - THF, 5-formyltetrahydrofolic acid peak is marked as 5-CHO-THF, and folic acid peak is marked as FA. Color images are available online at http://aoac. publisher.ingentaconnect.com/content/aoac/jaoac.

Blank-corrected folic acid (ng/mL) in the assayed mixture = folic acid (ng/mL) in assayed mixture – folic acid (ng/mL) in conjugase test blank

[sample aliquot, g × 10]

The amount of CH 3 -THF (mcg/100 g) in the samples are calculated as folic acid by multiplying (mcg/100 g) concentration of the folate compound by the corresponding conversion factors provided in Table 2011.06E . Total folate in the reconstituted or RTF sample is calculated by the sum of all folate compound as folic acid (mcg/100 g). Total folate as folic acid mcg/100 g in reconstituted or RTF samples = folic acid (mcg/100 g) + [5-CH 3 – THF (mcg/100 g) × 0.9606] + [5-CHO-THF (mcg/100 g) × 0.9324] ( f )  Calculation of total folate in dry samples (mcg/100 g) as-is basis .—The calculation of total folate (mcg/100 g) in dry samples as-is basis from total folate (mcg/100 g) in reconstituted beverage samples is based on the following relationship: -THF and CH 0 where C (mcg/100 g) is total folate in the reconstituted sample, V (g) is the total weight of the reconstituted beverage sample, and W (g) is the weight of aliquot of the powder sample used for reconstitution. ( g )  Calculation of folic acid nanograms released in the conjugase test. —The ratios of peak areas of folic acid and folic acid internal standard are calculated in the assayed solution. Folic acid (ng/mL) in the solution is calculated based on its calibration curve. Folic acid (ng/mL) is also calculated similarly in the conjugase test blank. Total folate as-is basis in dry sample analyzed as reconstituted (mcg/100 g) = [ C (mcg/100 g) × V (g)]/ W (g)

Adjusted folic acid released in whole assayed mixture = blank-corrected folic acid (ng/mL) × final volume of assay mixture after purification (SPE elution volume = 1.05 mL) ( h )  Calculation of percent conversion of Pte-Glu3 to folic acid in the conjugase assay. —Folic acid (ng) released in conjugase assay is converted to folic acid (nanomoles) by using the following equation: Folic acid (nanomoles) in the assayed mixture = folic acid (ng) in assay/441.4 (folic acid molecular weight)

Pte-Glu3 (ng) in conjugase assay = 0.03 mL × 20 mcg/mL (in conjugase activity substrate solution) = about 600 ng

Pte-Glu3 (nanomoles) in conjugase assay = Pte-Glu3 (ng) in the assay/699.6 (Pte-Glu3 molecular weight) Each molecule of Pte-Glu3 on hydrolysis yields one molecule of folic acid.

% conversion of Pte-Glu3 to folic acid = [folic acid (nanomoles) released in the assay/Pte-Glu3 (nanomoles) in the assay] × 100

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