AOAC SPIFAN Nutrients ERP Review (March 14, 2019)

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

Figure 21. Comparison of total lycopene data for SRM 1869 compared to the reference value and uncertainty limits.

Collaborators’ Comments The following comments were received from collaborating labs during the course of the MLT: 1. Reinjected a few Level 1 working standards due to elevated trans-lycopene areas including those that were similar to standard areas observed on Day 1. The areas for trans beta-carotene and trans-lutein were similar to all other injections so it doesn't appear to be due to any variability in injection volumes. This was also observed on the first attempt at day 2 which led us to re-extract the batch. Notable difference in response between curves with observed decrease in area over time. We have seen lycopene behave in a similar manner in the past, Study director’s response: Lycopene analysis is challenging for many labs, and it probably relates to stability and solubility. Adding THF to the diluent would help with solubility, but would likely be detrimental to the separation of cis and trans lutein peaks which elute much earlier. Although the method specifies sonicating stock solutions when they are removed from the freezer, the method will be updated to specify sonicating all standard solutions after storage. 3. Nowhere in the method information about identifying the cis-Lycopene peaks is given. Now most of the peaks between all-t-BC and all-t-Lycopene have been named cis-lycopene. More (small) peaks do elute in this area, but are not quantified as cis-Lycopene because UV- spectrum deviates from all-trans-Lycopene. Study director’s response: When determining background lycopene levels in the MLT samples, the first cis lycopene peak has a relative retention time of approximately 0.87 compared to all- might be remedied with the addition of some THF in the diluent? 2. Poor peak shape for lycopene is calibration solutions C1 and C2.

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