6. AOACSPIFANMethods-2018Awards

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264 S CHIMPF ET AL . : J OURNAL OF AOAC I NTERNATIONAL V OL . 101, N O . 1, 2018

INFANT FORMULA AND ADULT NUTRITIONALS

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

K AREN J. S CHIMPF , L INDA D. T HOMPSON , and S HANG -J ING P AN Abbott Nutrition, 3300 Stelzer Rd, Columbus, OH 43219

10 – 250 µg/L trans- lutein. with average calibration errors of <1%. For b -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, b -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. T he term “ carotenoids ” is used to describe a distinct class of naturally occurring yellow, orange, and red compounds containing extensive networks of light-absorbing conjugated double bonds. Carotenoids containing only hydrogen and carbon are called carotenes, whereas those containing hydrogen, carbon, and oxygen are known as xanthophylls. Although there are over 600 naturally occurring carotenoids, the number of carotenoids found in foods is much less (1). The most common carotenoids that may be present in human milk and infant and adult nutritionals include a -carotene, b -carotene, b -cryptoxanthin, lutein (plus zeaxanthin), and lycopene. In 2014, the AOAC Stakeholder Panel on Infant and Adult Nutritionals (SPIFAN) working group for carotenoids, made up of experts from government, industry, academia, and nongovernment organizations, agreed that applicable carotenoid methods should determine all trans - a -carotene and cis isomers of a -carotene; all trans - b -carotene and cis isomers of b -carotene; and all trans -lutein and cis isomers of lutein and lycopene and developed Standard Method Performance Requirements (SMPRs ® ) for these carotenoids. SPIFAN approved the carotenoid SMPR at the September 2014 AOAC INTERNATIONAL annual meeting and AOAC issued a call for methods. At this time there were no AOAC official methods for the determination of carotenoids in infant and adult nutritionals. The only AOAC official methods for the determination of carotenoids were Method 938.04 (2), a colorimetric method for the determination of carotenoids in macaroni products; Method 2005.07 (3), an HPLC method for the determination of b -carotene in dietary supplements and raw materials; and Method 2009.04 (4), an HPLC method for the determination of lycopene in dietary supplements and raw materials. In 2016, the AOAC Expert Review Panel approved Method 2016.13 (5), a reversed-phase ultra-HPLC method for the determination of cis- and trans -lutein and cis- and trans - b -carotene in infant and

This reversed-phase HPLC method uses C30 chromatography and UV-Vis spectroscopy to determine cis and trans isomers of lutein, b -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, b -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 b -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, b -carotene, and lycopene, respectively. The method also demonstrated good linearity. For lutein, r averaged 0.99991 over a standard range of approximately Received July 18, 2017. Accepted by SG August 11, 2017. This method was approved by the AOAC Expert Review Panel for Nutrient Methods as First Action. The Expert Review Panel for 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: karen.schimpf@abbott.com DOI: https://doi.org/10.5740/jaoacint.17-0287