AOAC Final Action Methods in 2018

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.

Folate vitamer concn, mcg/100 g, in reconstituted or RTF sample = [vitamer concn, ng/mL, in extract × 1.05 (final volume, mL)]/ [sample aliquot, g × 10] The amount of CH 3 -THF and CH 0 -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:

Folic acid (ng/mL) is also calculated similarly in the conjugase test blank. 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 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)

Total folate as-is basis in dry sample analyzed as reconstituted (mcg/100 g) = [ C (mcg/100 g) × V (g)]/ W (g)

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

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.

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 =

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