Determination of Carotenoids in Infant, Pediatric, and Adult Nutritionals by HPLC with UV-Visible Detection: Single-Laboratory Validation, First Action 2017.04

: This reversed-phase HPLC method uses C30 chromatography and UV-Vis spectroscopy to determine cis and trans isomers of lutein, β-carotene, and lycopene in infant, pediatric, and adult nutritionals. Samples are saponified with a mixture of potassium hydroxide, tetrahydrofuran, and methanol, and carotenoids are extracted from saponified samples with 75 + 25 hexane-methyl tertiary butyl ether (MtBE). After extraction, a portion of the organic layer is evaporated to dryness, and the residue is dissolved in 75 + 25 10% butylated hydroxytoluene in methanol-MtBE. Prepared samples are injected into a C30 HPLC column where cis and trans isomers of lutein, β-carotene, and lycopene are separated with a methanol-MtBE gradient and detected with UV-Vis spectroscopy at 445 nm. Total carotenoid concentrations are calculated by comparison of sample peak areas with the areas of trans carotenoid standards of known concentration. During a single-laboratory validation of this method, total lutein repeatability and intermediate precision ranged from 1.89 to 14.9 and 1.93 to 14.0%, respectively, in infant and adult nutritional matrixes with concentrations >1 μg/100 g. Total β-carotene repeatability and intermediate precision ranged from 1.81 to 6.77 and 3.07 to 16.2%, respectively, in infant and adult nutritional matrixes with concentrations >1 μg/100 g, and total lycopene repeatability and intermediate precision ranged from 3.01 to 6.37 and 4.29 to 10.3%, respectively, in infant and adult nutritional matrixes with concentrations >1 μg/100 g. Mean overspike recoveries ranged from 90.3 to 95.3, 89.3 to 108, and 97.3 to 109% for lutein, β-carotene, and lycopene, respectively. The method also demonstrated good linearity. For lutein, r averaged 0.99991 over a standard range of approximately 10-250 µg/L trans-lutein. with average calibration errors of <1%. For β-carotene and lycopene, r averaged 0.99993 and 0.9998 over standard ranges of approximately 25-500 and 5-100 µg/L with calibration errors of <1 and <1.5%, respectively. Lutein, β-carotene, and lycopene LOQs in ready-to-feed nutritionals were estimated to be 0.4, 0.1, and 0.3 µg/100 g, respectively. This method met AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals Standard Method Performance Requirements and was approved as a First Action Official Method at the AOAC INTERNATIONAL 2017 midyear meeting.