AOAC SPIFAN Nutrients ERP Review (March 14, 2019)

2016.13 (SEPTEMBER 2017) CAROT-02 METHOD

FOR ERP USE ONLY DO NOT DISTRIBUTE

Lutein, β-Carotene, and Lycopene in Infant Formula and Adult Nutritionals Reversed-Phase Ultra-High-Performance Liquid Chromatography

(Applicable to the determination of all- trans -lutein, cis isomers of lutein, all- trans -β-carotene, cis isomers of β-carotene, and lycopene in infant formula and adult nutritionals. Materials tested must not contain measurable levels of β-apo-8′-carotenal.) Caution: Tetrahydrofuran (THF) can form peroxides and only THF stabilized with butylated hydroxytoluene (BHT) should be used. Refer to safety data sheets when using any reagent, and use appropriate personal protective equipment when performing analyses. Note : Throughout this method, estimated sample concentrations for standard and sample preparations are stated per 100 g on a reconstituted basis (as is for ready-to-feed liquids, 25 g sample + 200 g water for powder samples, or diluted 1:1 by weight for liquid concentrates) in accordance with Standard Method Performance Requirements (SMPRs ® ) 2017.006, 2017.007, and 2017.008 (21-23). Test sample concentrations calculated in H(f-h) are expressed per 100 g on an as is basis for all samples, allowing each laboratory to apply appropriate reconstitution factors. A. Principle Test samples (reconstituted powders, liquid ready-to-feed and liquid concentrates) are spiked with an internal standard and saponified with potassium hydroxide. Samples are then extracted with MTBE and THF, followed by hexane. The supernatants from the liquid-liquid extraction are dried under nitrogen and reconstituted in 2-propanol. Separation is performed by reversed-phase chromatography on a C30 column. All- trans lutein, β-carotene, and lycopene are separated from their major cis isomers, as well as from zeaxanthin and α-carotene.