AOAC SPIFAN Nutrients ERP Review (March 14, 2019)

2016.13 (SEPTEMBER 2017) CAROT-02 VALIDATION REPORT FOR ERP USE ONLY DO NOT DISTRIBUTE

Summary

This is an updated report for a method for carotenoids that was origianlly submitted to the SPIFAN Expert Review Panel in August 2016. The orginal method included data for lutein and β-carotene and was approved by the ERP and codified as AOAC First Action Method 2016.13. This report includes the original data and extends the validation to lycopene. A UHPLC method for determination of lutein, β-carotene, and lycopene in infant formula and adult nutritionals was validated using the SPIFAN sample kit, including both unfortified and fortified samples. The data provided show that the method meets the requirements of the SMPRs.  Linearity Linearity of the calibration solutions correlates to approximately 0.8-45 μg lutein or β-carotene per 100 g as fed for samples prepared for the lowest sample concentrations. The lycopene linear range correlates to approximately 0.4-23 μg/100 as fed. With dilutions specified in the method, the range can be extended to approximately 1500 μg/100 as fed for lutein or β-carotene and up to 800 μg/100 as fed for lycopene. This extends beyond the ranges specified in the SMPRs. Linearity of the internal standard was also demonstrated.  LOD/LOQ The LOD for lutein and β-carotene is 0.08 μg/100 g and for lycopene it is 0.09 μg/100 g. The LOQs for lutein (0.27), β-carotene (0.27), and lycopene (0.30) meet that of ≤1 μg/100 g specified in the SMPRs.  Specificity All experiments showed separation of all- trans -lutein and β-carotene from their major cis isomers, apocarotenal, α-carotene, lycopene, and zeaxanthin. Samples spiked with all- trans -lutein and β-carotene showed no significant isomerization during sample preparation.  Precision For all measurements in the range of 1-100 μg/100 g, repeatability was ≤5.8% RSD for lutein, ≤5.1% RSD for β-carotene, and ≤1.4% RSD for lycopene. For measurements >100 μg/100 g, repeatability was ≤1.1% RSD for lutein and ≤1.7% RSD for β-carotene. The method meets the precision requirements of the SMPRs.  Accuracy The SMPRs call for 90-110% recovery from spiked samples, and all data meets this requirement. Reference values for NIST SRM 1869 have not yet been certified.