AOAC SPIFAN Nutrients ERP Review (March 14, 2019)

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

Study Title Determination of Lutein, β-Carotene, and Lycopene in Infant Formula and Adult Nutritionals by Ultra-High-Performance Liquid Chromatography: Multi Laboratory Testing Study Author Gregory Hostetler Perrigo Nutritionals 147 Industrial Park Road PO Box 2109 Georgia, Vermont 05468 USA Phone: +1 802 528 8736 Email: gregory.hostetler@perrigo.com Study Objective The objective of this study was to validate the AOAC First Action Method 2016.13 by determining its precision (repeatability and reproducibility) with multi laboratory testing (MLT). A full method description is given in Appendix C. The repeatability and reproducibility were compared with the criteria set out in the standard method performance requirements (SMPRs) for lutein (SMPR 2017.007), β-carotene (SMPR 2017.006), and lycopene (SMPR 2017.008). Method Principle Test samples (reconstituted powders, liquid ready-to-feed and liquid concentrates) are spiked with an internal standard and treated 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 with UV-visible detection. All- trans lutein, β-carotene, and lycopene are separated from their major cis isomers, as well as from zeaxanthin and α-carotene. Study Design A total of 13 laboratories agreed to participate in the study, including infant formula manufacturers, contract laboratories, government agencies, and a carotenoid supplier. Each participant received a set of 18 samples (9 samples in blind duplicate) and a practice sample (see Appendix A). Five of the samples were from the SPIFAN kit and were tested for homogeneity in 2015. One was a reference material (SRM 1869). The remaining three samples were locally sourced and tested for homogeneity by the study director. Before analyzing the full sample set, each participant analyzed the practice sample and resolved any issues with sample preparation or chromatography before proceeding with the full sample set. The MLT samples were analyzed on two separate days, with each sample assigned a specific day by the study director and duplicates assigned the same day. All results were entered in the

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