AOAC Final Action Methods in 2017

260 G ILL & I NDYK : J OURNAL OF AOAC I NTERNATIONAL V OL . 101, N O . 1, 2018

the stock standard at 265 nm (1/cm); E 1 % 1cm = extinction coefficient for vitamin D 2 in ethanol (461 dL/g.cm); and 10 000 = concentration conversion factor (g/dL to µg/mL). (b) Concentration of SIL vitamin D 3 in SILD 3 SS. —

in the 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. —

0 : 01 25

CS1 Dconcn = NLWS Dconcn ×

SILD 3 SS abs ð l max Þ E 1% 1cm

SILD 3 SS D 3 concn =

× 10000

0 : 05 25

where SILD 3 SS D 3 concn = concentration of d6-vitamin D 3 in the stock standard (µg/mL); SILD 3 SS abs( l max) = UV absorbance of the stock standard at 265 nm (1/cm); E 1 % 1cm = extinction coefficient for vitamin D 3 in ethanol (485 dL/g.cm); and 10 000 =

CS2 Dconcn = NLWS Dconcn ×

0 : 25 25

concentration conversion factor (g/dL to µg/mL). (c) Concentration of SIL vitamin D 2 in SILIS. —

CS3 Dconcn = NLWS Dconcn ×

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1 : 0 10

× 1000

SILIS D 2 concn = SILD 2 SS D 2 concn ×

0 : 5 25

CS4 Dconcn = NLWS Dconcn ×

where SILIS D 2 concn = concentration of d6-vitamin D 2 in the internal standard (ng/mL); SILD 2 SS D 2 concn = concentration of d6-vitamin D 2 in the stock standard (µg/mL); and 1000 =

1 : 25 25

CS5 Dconcn = NLWS Dconcn ×

concentration conversion factor (µg/mL to ng/mL). (d) Concentration of SIL vitamin D 3 in SILIS. —

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. —

1 : 0 10

× 1000

SILIS D 3 concn = SILD 3 SS D 3 concn ×

where SILIS D 3 concn = concentration of d6-vitamin D 3 in the internal standard (ng/mL); SILD 3 SS D 3 concn = 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. —

0 : 25 25

CS1 − 5 Dconcn = SILIS Dconcn ×

where CS1 – CS5 Dconcn = concentration of d6-vitamin D 2 or d6- vitamin D 3 in the CSs (ng/mL); 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. —

NLD 2 PS abs ð l max Þ E 1% 1cm

NLD 2 PS D 2 concn =

× 10000

where NLD 2 PS D 2 concn = concentration of vitamin D 2 in the purity standard (µg/mL); NLD 2 PS abs( l max) = UV absorbance of the purity standard at 265 nm (1/cm); E 1% 1cm = extinction coefficient for vitamin D 2 in ethanol (461 dL/g.cm); and 10 000 = concentration conversion factor (g/dL to µg/mL). (f) Concentration of nonlabeled vitamin D 3 in NLD 3 PS. —

D mass ð D mass +W mass Þ

× A mass

S mass =

where S mass = mass of powder in the slurried sample (g); D mass = mass of the dry powder used tomake 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; SILIS Dconcn = 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 NLD PA SILD × SILIS Dconcn L × SILIS alqt S mass × 100 1000

NLD 3 PS abs ð l max Þ E 1% 1cm

NLD 3 PS D 3 concn =

× 10 000

where NLD 3 PS D 3 concn = concentration of vitamin D 3 in the purity standard (µg/mL); NLD 3 PS abs( l max) = UV absorbance of the purity standard at 265 nm (1/cm); E 1% 1cm = 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. —

1 : 0 10

× 1000

NLWS D 2 concn = NLD 2 PS D 2 concn ×

where NLWS D 2 concn = concentration of vitamin D 2 in the working standard (ng/mL); NLD 2 PS D 2 concn = 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. —

1 : 0 10

NLWS D 3 concn = NLD 3 PS D 3 concn ×

× 1000

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