AOAC Final Action Methods in 2019

Figure 2016.13F. Chromatogram of SRM 1869 sample. Lut = Lutein, Zea = zeaxanthin, Apo = apocarotenal, AC = α-carotene, BC = β-carotene, Lyc = lycopene.

( f ) Calculate the concentration of each carotenoid analyte (e.g., lutein, C Lut ) in the all- trans form, in μg/100 mL, in each calibration solution, E ( f ): C Lut = C LutInterm × (V MC /V Total ) where C LutIntermed = concentration of lutein in the mixed carotenoid intermediate solution calculated in H ( e ); V MC = volume of mixed carotenoid intermediate solution, E ( e ), used; V Total = dilution volume. ( g ) Calculate the concentration of the apocarotenal ISTD (C A ), in μg/100 mL, in each calibration solution, E ( f ): C Apo = C ApoInterm × (V ApoIntermed /V Total ) where C ApoIntermed = concentration of the apocarotenal intermediate solution calculated in H ( d ); V ApoIntermed = volume of apocarotenal intermediate solution, E ( d ), used; V Total = dilution volume. ( h ) For each calibration solution calculate ( 1 ) peak area ratio for each analyte: [Peak area (all- trans lutein, β-carotene, or lycopene)] / [peak area (ISTD)] and ( 2 ) concentration ratio: [Concentration (all- trans lutein, β-carotene, or lycopene)] / [concentration (ISTD)] Build a 5-point calibration curve with ISTD by plotting peak area ratios against concentration ratios, with relative concentration on the x -axis. Accuracy on calibration points should be 100 ± 10% for calibration solutions C1–C4, and the coefficient of determination (R 2 ) should be >0.995. The calibration and calculation may be achieved through data processing within the instrument software or off-line. ( i ) Calculate the mass of apocarotenal (M A ), in μg, added to the test samples: M A = (C A /100) × V A × (4/50) where C A = concentration of apocarotenal in the intermediate or working solution used in the ISTD solution (μg/100 mL); 100 = conversion from μg/100 mL to μg/mL; V A = volume of ISTD solution added to each sample, in mL; 4 = volume of apocarotenal

intermediate or working solution used in the ISTD solution, in mL; 50 = total volume of ISTD solution made, in mL. ( j ) Calculate the contents of all- trans lutein, cis isomers of lutein, and total lutein on an as-is basis in the test samples. For peak identification, refer to relative retention times of peaks in Figures  2016.13A , C , D , and F . Lut trans = (M A /M S ) × [(A Lut /A A ) – I Lut ] × (100/RF Lut ) where Lut trans = all- trans lutein in the sample (μg/100 g); M A = mass of apocarotenal added to the test sample, in μg; M S = sample weight, in g; A Lut = peak area of all- trans lutein in the sample chromatogram, in arbitrary units; A A = peak area of apocarotenal in the sample chromatogram, in arbitrary units; I Lut = y -intercept of the calibration curve for all- trans lutein; RF Lut = slope of the calibration curve for all- trans lutein. Lut cis = (M A /M S ) × ([([A 13cisLut × 1.4] + [A 13′cisLut × 1.4] + A 9cisLut + A 9′cisLut )/A A ] – I Lut ) × (100/RF Lut ) where Lut cis = cis isomers of lutein in the sample (μg/100 g); M A = mass of apocarotenal added to the test sample, in μg; M S = sample weight, in g; A 13cisLut = peak area of 13- cis lutein in the sample chromatogram, in arbitrary units; A 13′cisLut = peak area of 13′- cis lutein in the sample chromatogram, in arbitrary units; A 9cisLut = peak area of 9- cis lutein in the sample chromatogram, in arbitrary units; A 9′cisLut = peak area of 9′- cis lutein in the sample chromatogram, in arbitrary units; A A = peak area of apocarotenal in the sample chromatogram, in arbitrary units; I Lut = y -intercept of the calibration curve for all- trans lutein; RF Lut = slope of the calibration curve for all- trans lutein. Lut Total = Lut trans + Lut cis ( k ) Calculate the contents of all- trans β-carotene, cis isomers of β-carotene, and total β-carotene on an as-is basis in the test samples. For peak identification, refer to relative retention times of peaks in Figures 2016.13B – E . BC trans = (M A /M S ) × [(A BC /A A ) – I BC ] × (100/RF BC ) where BC trans = all- trans β-carotene in the sample (μg/100 g); M A = mass of apocarotenal added to the test sample, in μg; M S = sample weight, in g; A BC = peak area of all- trans β-carotene in the sample chromatogram, in arbitrary units; A A = peak area of apocarotenal

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