AOAC Final Action Methods in 2017

in purity standard (μg/mL); and 1000 = concentration conversion factor (μg/mL to ng/mL). (i)  Concentrations of vitamin D 2 and vitamin D 3 in CS1–CS5. — = × CS1 NLWS 0.01 25 Dconcn Dconcn

SILD SS

λ

=

×

3 abs( max)

10000

SILD SS

3 D3concn

1 cm 1%

E

where SILD 3 SS D3concn = concentration of d6 -vitamin D 3 in the stock standard (μg/mL); SILD 3 SS abs(λ max) = UV absorbance of the stock standard at 265 nm (cm −1 ); E 1cm 1% = extinction coefficient for vitamin D 3 in ethanol (485 dL/g·cm); and 10000 = concentration conversion factor (g/dL to μg/mL). (c)  Concentration of SIL vitamin D 2 in SILIS. — = × × SILIS SILD SS 1.0 10 1000 D2concn 2 D2concn where SILIS D2concn = concentration of d6 -vitamin D 2 in the internal standard (ng/mL); SILD 2 SS D2concn = concentration of d6 -vitamin D 2 in the stock standard (μg/mL); and 1000 = concentration conversion factor (μg/mL to ng/mL). (d)  Concentration of SIL vitamin D 3 in SILIS. — = × × SILIS SILD SS 1.0 10 1000 D3concn 3 D3concn where SILIS D3concn = concentration of d6 -vitamin D 3 in the internal standard (ng/mL); SILD 3 SS D3concn = concentration of d6 -vitamin D 3 in the stock standard (μg/mL); and 1000 = concentration conversion factor (μg/mL to ng/mL). (e)  Concentration of nonlabeled vitamin D 2 in NLD 2 PS. — = × λ 10000 NLD PS NLD PS E 2 D2concn 2 abs( max) 1 cm 1% where NLD 2 PSD 2concn = concentration of vitamin D 2 in the purity standard (μg/mL); NLD 2 PS abs(λ max) = UV absorbance of the purity standard at 265 nm (cm −1 ); E 1 cm 1% is the extinction coefficient for vitamin D 2 in ethanol (461 dL/g·cm); and 10000 is the concentration conversion factor (g/dL to μg/mL). (f)  Concentration of nonlabeled vitamin D 3 in NLD 3 PS. — = × λ 10000 NLD PS NLD PS E 3 D3concn 3 abs( max) 1 cm 1% where NLD 3 PS D3concn = concentration of vitamin D 3 in the purity standard (μg/mL); NLD 3 PS abs(λ max) = UV absorbance of the purity standard at 265 nm (cm −1 ); E 1 cm 1% = extinction coefficient for vitamin D 3 in ethanol (485 dL/g·cm); and 10 000 = concentration conversion factor (g/dL to μg/mL). (g)  Concentration of nonlabeled vitamin D 2 in NLWS. — = × × NLWS NLD PS 1.0 10 1000 D2concn 2 D2concn where NLWS D2concn = concentration of vitamin D 2 in the working standard (ng/mL); NLD 2 PS D2concn = concentration of vitamin D 2 in the purity standard (μg/mL); and 1000 = concentration conversion factor (μg/mL to ng/mL). (h)  Concentration of nonlabeled vitamin D 3 in NLWS. — = × × NLWS NLD PS 1.0 10 1000 D3concn 3 D3concn

0.05

=

×

CS2

NLWS

Dconcn

Dconcn

25

0.25

=

×

CS3

NLWS

Dconcn

Dconcn

25

0.5

=

×

CS4

NLWS

Dconcn

Dconcn

25

1.25

=

×

CS5

NLWS

Dconcn

Dconcn

25

where CS1–CS5 Dconcn = concentration of vitamin D 2 or vitamin D 3 in the CSs (ng/mL); and NLWS Dconcn = concentration of vitamin D 2 or vitamin D 3 in the working standard (ng/mL). (j)  Concentrations of SIL d6-vitamin D 2 and d6-vitamin D 3 in CS1–CS5. — − = × CS1 5 SILIS 0.25 25 Dconcn Dconcn where CS1–CS5 Dconcn = concentration of d6 -vitamin D 2 or d6 - vitamin D 3 in the CSs (ng/mL); and SILIS Dconcn = concentration of d6 -vitamin D 2 or d6 -vitamin D 3 in the internal standard (ng/mL). (k)  Mass of powder in slurried sample. — = + ´ S D (D W ) A mass mass mass mass mass where S mass = mass of powder in the slurried sample (g); D mass = mass of the dry powder used to make the slurry (g) W mass = mass of water used to make the slurry (g); and A mass = mass of the aliquot of slurried sample used in the analysis (g). (l)  Determine the linear regression curve y = m·x + c (using the least-squares method) for the ratio of peak areas (nonlabeled vitamin D/SIL d6 -vitamin D) versus the ratio of concentrations (nonlabeled vitamin D/SIL d6 -vitamin D) for five CSs with the y -intercept forced through zero. (m)  The concentration (w/w) of vitamin D 2 or vitamin D 3 in dry powders is calculated as where Result D = vitamin D 2 or vitamin D 3 concentration in the sample (μg/hg); PA NLD = peak area of vitamin D 2 or vitamin D 3 in the sample; PA SILD = peak area of d6 -vitamin D 2 or d6 -vitamin D 3 in the sample; SILISD concn = concentration of d6 -vitamin D 2 or d6 - vitamin D 3 in the SILIS (ng/mL); L = slope of the calibration curve; SILIS alqt = volume of the SILIS aliquot spiked into the sample (0.5 mL); S mass = mass of the sample (g); 1000 = concentration conversion factor (ng/g to μg/g); and 100 = concentration conversion factor (μg/g to μg/hg). (n)  The concentration (w/v) of vitamin D 2 or vitamin D 3 in ready-to-feed (RTF) liquids is calculated as = × × × Result D PA PA SILIS L SILIS S 100 1000 NLD SILD Dconcn alqt mass

where NLWS D3concn = concentration of vitamin D 3 in working standard (ng/mL); NLD 3 PS D3concn = concentration of vitamin D 3

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