6. AOACSPIFANMethods-2018Awards

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H ostetler : J ournal of AOAC I nternational V ol . 100, N o . 3, 2017  7

(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 . ( ) ( ) ( ) = ×   − × BC M M A A I 100 RF trans A S BC A BC BC

above; 1000 = the conversion of milligrams to micrograms; (3/50) = the dilution of stock solution to apocarotenal intermediate solution; V AI = the volume of apocarotenal intermediate solution, E(d) , used; and V Total = the 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 or β-carotene)/(peak area of internal standard); and ( 2 ) the concentration ratio: (concentration of all- trans lutein or β-carotene)/(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%, and the coefficient of determination (R 2 ) should be greater than 0.995. The calibration and calculation may be achieved through data processing within the instrument software or off-line. (e)  Calculate the mass (μg) of apocarotenal (M A ) added to = the concentration (μg/100 mL) of apocarotenal in the intermediate or working solution used in the ISTD; V A = the volume (mL) of ISTD added to each sample; 4 = the volume (mL) of apocarotenal intermediate or working solution used in the ISTD; and 50 = the total volume (mL) of ISTD 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 . ( ) ( ) ( ) = ×   − × Lut M M A A I 100 RF trans A S Lut A Lut Lut = the peak area (AU) of apocarotenal in the sample chromatogram; I Lut = the y -intercept of the calibration curve for all- trans -lutein; and RF Lut = the slope of the calibration curve for all- trans - lutein. ( ) ( ) ( ) ( ) = × + + +   − × Lut M M A A A A A I 100 RF cis A S 13cisLut 13'cisLut 9cisLut 9'cisLut A Lut Lut where Lut cis = the concentration (μg/100 g) of cis isomers of lutein in the sample; M A = the mass (μg) of apocarotenal added to the test sample; M S = the sample weight (g); A 13cisLut = the peak area (AU) of 13- cis -lutein in the sample chromatogram; A 13′cisLut = the peak area (AU) of 13′- cis -lutein in the sample chromatogram;A 9cisLut = the peak area (AU) of 9- cis -lutein in the sample chromatogram; A 9′cisLut = the peak area (AU) of 9′- cis - lutein in the sample chromatogram; A A = the peak area (AU) of apocarotenal in the sample chromatogram; I Lut = the y -intercept of the calibration curve for all- trans -lutein; and RF Lut = the slope of the calibration curve for all- trans -lutein. = + Lut Lut Lut Total trans cis where Lut trans = the concentration (μg/100 g) of all- trans -lutein = the mass (μg) of apocarotenal added to the in the sample; M A test sample; M S = the sample weight (g); A Lut = the peak area (AU) of all- trans -lutein in the sample chromatogram; A A the test samples: ( ) ( ) = × × M C V 4 50 A A A where C A

= the concentration (μg/100 g) of all- trans -β-

where BC trans

= the mass (μg) of apocarotenal = the sample weight (g);

carotene in the sample; M A added to the test sample; M S

A BC = the peak area (AU) of all- trans -β-carotene in the sample chromatogram; A A = the peak area (AU) of apocarotenal in the sample chromatogram; I BC = the y -intercept of the calibration curve for all- trans -β-carotene; and RF BC = the slope of the calibration curve for all- trans -β-carotene. ( ) ( ( ) ) ( ) = × ×   +   ×   + +  − × BC M M A 1.4 A 1.2 A A A I 100 RF cis A S 15cisBC 13cisBC 9cisBC XcisBC A BC BC where BC cis = the concentration (μg/100 g) of cis isomers of β-carotene in the sample; M A = the mass (μg) of apocarotenal added to the test sample; M S = the sample weight (g); A 15cisBC = the peak area (AU) of 15- cis -β-carotene in the sample chromatogram; A 13cisBC = the peak area (AU) of 13- cis -β- carotene in the sample chromatogram; A 9cisBC = the peak area (AU) of 9- cis -β-carotene in the sample chromatogram; A XcisBC = the peak area (AU) of unidentified cis isomers of β-carotene in the sample chromatogram; A A = the peak area (AU) of apocarotenal in the sample chromatogram; I BC = the y -intercept of the calibration curve for all- trans -β-carotene; and RF BC = the slope of the calibration curve for all- trans -β-carotene. = + BC BC BC Total trans cis SMPR 2014.014 calls for the determination of all- trans and cis isomers of lutein and β-carotene, as well as the separation of lutein from zeaxanthin. Selectivity was evaluated with visual inspection of chromatograms and by measuring the resolution of system suitability standard mixtures. Because apocarotenal is used as an internal standard, samples were prepared without internal standard to ensure there were no interfering peaks. To identify major cis isomers of α-carotene, β-carotene, and lutein, standard mixtures were isomerized by heating at 80°C for 2 h. The separation of all- trans -lutein, cis isomers of lutein, zeaxanthin, and apocarotenal is shown in Figure 2016.13A , whereas the separation of geometric isomers of α-carotene and β-carotene is shown in Figure 2016.13B . Peak assignments were based on relative retention times from previous studies using C30 columns and methanol–MTBE as the mobile phase (11, 14–16). A chromatogram showing separation of lutein and β-carotene from lycopene is shown in Figure 1, and isomerized standard solutions showing major cis isomers of the carotenoids are shown in Figures 2 and 3. One of the minor cis isomers of β-carotene elutes before 15- cis -β- carotene, and this peak has a similar retention time to one Validation Selectivity