AOAC SPIFAN Nutrients ERP-Final Review (Oct. 2018)

B handari et al . : J ournal of AOAC I nternational V ol . 101, N o . x , 2018  7

( e )  Calculation of folate compounds and total folate in reconstituted or RTF samples (mcg/100 g) .—Estimated folate vitamer concentration (nanograms per milliliter) in the extract is multiplied by fold dilutions (if any) and the final volume of the relevant step (volume after SPE elution, 1.05 mL). The total amount of folate vitamer in the sample is adjusted for the aliquot of the sample used for the extraction. The folate vitamer then calculated as μg/100 g of the reconstituted or RTF sample. Folate vitamer concentration mcg/100 g in reconstituted or RTF sample = [vitamer concentration in nanograms per milliliter in extract × 1.05 (final volume in milliliters)] / [sample aliquot (grams) × 10]. The amount of CH3-THF and CH0-THF (mcg/100 g) in the samples are calculated as folic acid by multi­ plying (mcg/100 g) concentration of the folate compound by the following 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-CH3 – 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: total folate as-is basis in dry sample analyzed as reconstituted (mcg/100 g) = [ C (mcg/100 g) × V (grams)] / W (grams), where C (mcg/100 g) is total folate in the reconstituted sample, V (grams) is the total weight of the reconstituted beverage sample, and W (grams) is the weight of aliquot of the powder sample used for reconstitution. ( g )  Calculation of folic acid nanograms released in the conjugase test. —The ratio of peak areas of folic acid and folic acid internal standard are calculated in the assayed solution. Folic acid (nanograms per milliliter) in the solution is calculated based on its calibration curve. Folic acid (nanograms per milliliter) is also calculated similarly in the conjugase test blank. Blank-corrected folic acid (nanograms per milliliter) in the assayed mixture = folic acid (nanograms per milliliter) in assayed mixture – folic acid (nanograms per milliliter) in conjugase test blank. Adjusted folic acid released in whole assayed mixture = blank-corrected folic acid (nanograms per milliliter) × 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 (nanograms) released in conjugase assay is converted to folic acid (nanomoles) by using the following equation: folic acid (nanomoles) in the assayed mixture = folic acid (nanograms) in assay / 441.4 (folic acid molecular weight). Pte-Glu3 (nanograms) in conjugase Table 2011.06E. Conversion of amount of 5-CH3-THF and 5-CHO-THF to folic acid equivalent

Table 2011.06D. MRM transitions for different folates and MS parameters for their detection

Q1 a , m/z Q3 b , m/z DP c , V EP d , V CXP e , V CE f , V

Folate

FA

442.2 295.2 75 442.2 176.2 75 460.2 313.2 75 460.2 194.2 75 474.2 327.2 75 474.2 299.2 75 447.2 295.2 75

6 6 6 6 6 6 6 6 6

6 6 6 6 6 6 6 6 6

20 45 28 46 25 40 20 28 25

5-CH3-THF

5-CHO-THF

13 C 5 13 C 5 13 C 5

-FA

-5-CH3-THF 465.2 313.2 75 -5-CHO-THF 479.2 327.2 75

a  Q1 = Precursor ion. b  Q3 = Product ion. c  DP = Declustering potential. d  EP = Entrance potential. e  CXP = Collision cell exit potential. f  CE = Collision cell potential.

G. Calculations ( a )  Calculation of ratios of peak areas of the folate compounds to the respective internal standard peak areas .— Calculate the ratios of peak area of each folate vitamer against respective internal standard area for all calibration standards, method blank, and all of the sample extracts. ( b )  Calibration curve .—A calibration curve is drawn by plotting the concentration of each calibration standard (nanograms per milliliter) of a specific folate compound against the corresponding ratio of its peak area to the relevant internal standard peak area. The calibration curves are prepared for all of the folate compounds. The calibration curves are not forced through zero (weighting 1/x). This calculation is usually done by the software used for the processing of MS-acquired data. ( c )  Calculation of concentration of folate compounds in the method blank .—The ratios of peak areas of the detected folate compounds to the respective internal standards are calculated in the method blank. The concentration of each of the detected folate compounds as nanograms per milliliter is calculated based on the respective calibration curve. This is usually done by the software used for the processing of the MS data. ( d )  Calculation of concentration of folate compounds in the sample extracts .—The ratios of peak areas of the detected folate vitamers to the respective internal standard areas for every sample is calculated. The concentration of each of the folate compounds as nanograms per milliliter is calculated based on the calibration curve of the respective folate compound. This calculation is usuallyperformedby the softwareused for processing MS data. The concentration of each of the folate compounds as nanograms per milliliter in a sample extract is adjusted by subtracting the amount of respective folate compound detected in the method blank ran in the batch. Corrected folate compound concentration in a sample extract (nanograms per milliliter) = concentration of specific folate compound (nanograms per milliliter) – concentration of respective specific folate compound in the method blank (nanograms per milliliter).

Multiply by to convert to folic acid

Example value, mcg/100 g

Value as folic acid, mcg/100 g

Formula weight

Folate vitamers

Folic acid

441.4 459.5 473.4

1

100.0 100.0 100.0

100.0

5-CH3-THF 5-CHO-THF Total folate as folic acid

0.9606 0.9324

96.1 93.2

Sum as folic acid

289.3