AOAC SPIFAN Nutrients ERP Review (March 14, 2019)

2016.13 (Jan. 2019) Carot-02 MLT Report FOR ERP USE ONLY DO NOT DISTRIBUTE

(a) Chromatographic conditions.— Set up the UHPLC system according to the specifications in Table C . Follow the manufacturer’s instructions for column installation, cleaning, and storage. (b) System suitability checks.— (1) Resolution between lutein cis and trans isomers.— Inject the lutein system suitability solution, E(h) , and determine the resolution between the two major cis isomers and all- trans -lutein. Resolution should be ≥1.4 between 13- cis and 13′- cis lutein and ≥2.2 between 13′- cis and all- trans -lutein using the half-width method. See Figure A . (2) Resolution between all-trans-lutein, zeaxanthin, and apocarotenal .—From the chromatogram of the lutein system suitability solution, determine the resolution between all- trans -lutein, zeaxanthin, and apocarotenal. Resolution should be ≥3.7 between all- trans -lutein and zeaxanthin and ≥2.5 between zeaxanthin and apocarotenal. See Figure A . (3) Resolution between β-carotene cis and trans isomers and α-carotene .—Inject the β- carotene system suitability solution, E(g) , and determine the resolution between the two major cis isomers of β-carotene, all- trans -β-carotene, and α-carotene. Resolution should be ≥1.4 between 13- cis -β-carotene and cis/trans α-carotene and ≥2.6 between all- trans - β-carotene and 9- cis - β-carotene. See Figure B. (4) Inject the calibration solutions before and after each set of sample injections (up to 12 samples in each set). Calculate the slope relative to the internal standard as shown in H (d) . The coefficient of determination (R 2 ) for each curve should be >0.995. The slopes from the two curves should not differ by more than 2% for lutein or β-carotene

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