AOAC SPIFAN Nutrients ERP Review (March 14, 2019)
C
= W
× 2 × CP A
× 1000 × (3/50) × (V AI
/V
)
Lut
= (M
/M
S ) × )/A
A
A
Total
cis
A
{[(A
+ A
+ A
+ A
] – I
} × (100/RF
)
where W A = weight (mg) of apocarotenal used to make the stock = chromatographic purity of apocarotenal calculated in H(a)( 2 ); 1000 = conversion of milligrams to micrograms; (3/50) = dilution of stock solution to apocarotenal intermediate solution; V AI = volume of apocarotenal intermediate solution, E(d) , used; and V Total = dilution volume. ( d ) For each calibration solution in E(f) , calculate ( 1 ) the peak area ratio for each analyte: (peak area of all- trans lutein, β-carotene, or lycopene)/(peak area of internal standard); and ( 2 ) the concentration ratio: (concentration of all- trans lutein, β-carotene, or lycopene)/(concentration of internal standard). Build a five- point calibration curve with internal standard by plotting peak area ratios against concentration ratios, with relative concentration on the x -axis. The accuracy on calibration points should be 100 ± 10% for calibration solution C1–C4, and the coefficient of determination (R 2 ) should be greater than 0.995. The calibration and calculation may be achieved through data solution; 2 = conversion of 50 mL to 100 mL; CP A
13cisLut
13’cisLut
9cisLut
9’cisLut
A
Lut
Lut
where Lut
cis = cis isomers of lutein in the sample (μg/100 g); M A =
mass (μg) of apocarotenal added to the test sample; M S
= sample
weight (g); A
= peak area (AU) of 13- cis -lutein in the sample = peak area (AU) of 13′- cis -lutein in the
13cisLut
chromatogram; A
13′cisLut
sample chromatogram; A the sample chromatogram; A in the sample chromatogram; A A in the sample chromatogram; I Lut curve for all- trans -lutein; and RF Lut 9cisLut 9′cisLut
= peak area (AU) of 9- cis -lutein in = peak area (AU) of 9′- cis -lutein = peak area (AU) of apocarotenal = y -intercept of the calibration = slope of the calibration curve
for all- trans -lutein.
Lut
= Lut
+ Lut
Total
trans
cis
( g ) Calculate the contents of all- trans -β-carotene, cis isomers of β-carotene, and total β-carotene in the test samples. For peak identification, refer to relative retention times of peaks in Figures 2016.13B – D .
processing within the instrument software or off-line. ( e ) Calculate the mass (μg) of apocarotenal (M A
BC
= (M
/M
) × [(A
/A
) – I
] × (100/RF
)
) added to the
trans
A
S
BC
A
BC
BC
test samples:
where BC
trans = all- trans -β-carotene in the sample (μg/100 g); M A
= mass (μg) of apocarotenal added to the test sample; M S
= sample
M
= (C
× V
) × (4/50)
weight (g); A
BC = peak area (AU) of all- trans -β-carotene in the = peak area (AU) of apocarotenal in the
A
A
A
sample chromatogram; A A sample chromatogram; I BC all- trans -β-carotene; and RF BC
where C A = concentration (μg/100 mL) of apocarotenal in the intermediate or working solution used in the ISTD solution; V A = volume (mL) of ISTD solution added to each sample; 4 = volume (mL) of apocarotenal intermediate or working solution used in the ISTD solution; and 50 = total volume (mL) of ISTD solution made. ( f ) Calculate the contents of all- trans -lutein, cis isomers of lutein, and total lutein in the test samples. For peak identification, refer to relative retention times of peaks in Figures 2016.13A , 2016.13C , and 2016.13D .
= y -intercept of the calibration curve for = slope of the calibration curve for
all- trans -β-carotene. BC cis = (M A /M S
) × {{[(A
× 1.4) + (A
× 1.2)
15cisBC
13cisBC
+ A
+ A
]/A
} – I
} × (100/RF
)
9cisBC
XcisBC
A
BC
BC
where BC cis = cis isomers of β-carotene in the sample (μg/100 g); A = mass (μg) of apocarotenal added to the test sample; M S = sample weight (g); A 15cisBC = peak area (AU) of 15- cis -β-carotene in the sample chromatogram; A 13cisBC = peak area (AU) of 13- cis - β-carotene in the sample chromatogram; A 9cisBC = peak area (AU) of 9- cis -β-carotene in the sample chromatogram; A XcisBC = peak area (AU) of unidentified cis isomers of β-carotene in the sample chromatogram; A A = peak area (AU) of apocarotenal in the sample chromatogram; I BC = y -intercept of the calibration curve for all- trans -β-carotene; and RF BC = slope of the calibration curve for all- trans -β-carotene. BC Total = BC trans + BC cis M
Lut
= (M
/M
) × [(A
/A
) – I
] × (100/RF
)
trans
A
S
Lut
A
Lut
Lut
where Lut
trans = all- trans -lutein in the sample (μg/100 g); M A =
mass (μg) of apocarotenal added to the test sample; M S
= sample
weight (g); A
Lut = peak area (AU) of all- trans -lutein in the sample = peak area (AU) of apocarotenal in the sample Lut = y -intercept of the calibration curve for all- Lut = slope of the calibration curve for all- trans -
chromatogram; A A chromatogram; I trans -lutein; and RF
lutein.
Figure 2016.13F. Chromatogram of SRM 1869 sample.
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