6. AOACSPIFANMethods-2018Awards

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H ostetler : J ournal of AOAC I nternational V ol . 100, N o . 3, 2017  1

INFANT FORMULA AND ADULT NUTRITIONALS

Determination of Lutein and β-Carotene in Infant Formula and Adult Nutritionals by Ultra-High-Performance Liquid Chromatography: Single-Laboratory Validation, First Action 2016.13 G regory L. H ostetler Perrigo Nutritionals, Analytical Research and Development, 147 Industrial Park Rd, Georgia, VT 05468

An ultra-HPLC method for the determination of lutein and β-carotene in infant formula and adult nutritionals was validated using both unfortified and fortified samples provided by the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN). 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 isomerization during sample preparation. Linearity of the calibration solutions correlated to approximately 0.8–45 μg/100 g (reconstituted basis) for samples prepared for the lowest sample concentrations. With dilutions specified in the method, the range can be extended to approximately 2250 μg/100 g. The LOD for both lutein and β-carotene was 0.08 μg/100 g, and the LOQ for both was 0.27 μg/100 g. For all measurements in the range of 1–100 μg/100 g, repeatability RSD was ≤5.8% for lutein and ≤5.1% for β-carotene. For measurements >100 μg/100 g, repeatability RSD was ≤1.1% for lutein and ≤1.7% for β-carotene. Accuracy was determined by recovery from spiked samples and ranged from 92.3 to 105.5% for lutein and from 100.1 to 107.5% for β-carotene. The data provided show that the method meets the criteria specified in the Standard Method Performance Requirements for carotenoids (SMPR 2014.014). T he carotenoids present in human milk include α-carotene, β-carotene, β-cryptoxanthin, lutein, zeaxanthin, and lycopene (1–3). Of these, lutein and β-carotene are most commonly added to infant formula and adult nutritionals. Whereas β-carotene has provitamin A activity (4), lutein may Received November 16, 2016. Accepted by SG January 20, 2017. This method was approved by the AOAC Expert Review Panel for SPIFAN Nutrient Methods as First Action. The Expert Review Panel for SPIFAN Nutrient Methods invites method users to provide feedback on the First Action methods. Feedback from method users will help verify that the methods are fit-for-purpose and are critical for gaining global recognition and acceptance of the methods. Comments can be sent directly to the corresponding author or methodfeedback@aoac.org. Corresponding author’s e-mail: gregory.hostetler@perrigo.com DOI: 10.5740/jaoacint.16-0386

play a role in vision and cognitive function (5). Both lutein and β-carotene can occur as all- trans and cis isomers, and there is interest in separating these because of differences in absorption and biological activity. In addition to having twice the vitamin A activity of cis isomers, all- trans -β-carotene is preferentially absorbed over 9- cis -β-carotene (6). All- trans -lutein is the most common isomer found in human retinas (7) and infant brains (8). Because there were no official methods for the determination of lutein or β-carotene in infant formula and adult nutritionals, the current method was developed based on existing extraction and chromatographic procedures from various carotenoid methods. The saponification procedure was adapted from Granado et al. (9), the extraction solvents from Craft (10), the use of apocarotenal as an internal standard from Marx et al. (11), and the use of 10 mM α-tocopherol as an antioxidant from Scita (12). Chromatographic separation of lutein and β-carotene isomers with C30 columns and a methanol–methyl tert-butyl ether (MTBE) mobile phase has been demonstrated in several reports (11, 13–16), and the current method adapted these procedures for optimum resolution, sensitivity, and run time. Calculations for standard concentrations were based on purity from Müller et al. (17) using extinction coefficients from Craft and Soares (18). Response factors for cis isomers of β-carotene relative to the all- trans form were taken from Schierle et al. (13) and align with AOAC Official Method SM 2005.07 (19) and the United States Pharmacopeia (USP) monograph for β-carotene (20). AOAC Official Method 2016.13 Lutein and β-Carotene in Infant Formula and Adult Nutritionals Reversed-Phase Ultra-High-Performance Liquid Chromatography First Action 2016 (Applicable to the determination of all- trans -lutein, cis isomers of lutein, all- trans -β-carotene, and cis isomers of β-carotene in infant formula and adult nutritionals from 1 to 1300 μg/100 g reconstituted basis. 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 Material 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 (RTF)