Identification of Isolutein (Lutein Epoxide) as cis-Antheraxanthin in Orange Juice ANTONIO J. MELE Ä NDEZ-MARTı ´NEZ, † GEORGE BRITTON, ‡ ISABEL M. VICARIO, † AND FRANCISCO J. HEREDIA* ,† Laboratory of Food Color & Quality, Departament of Nutrition and Food Science, Faculty of Pharmacy, University of Seville, 41012 Seville, Spain, and School of Biological Sciences, University of Liverpool, L69 7ZB Liverpool, United Kingdom The carotenoid profile of orange juice is very complex, a common characteristic for citrus products in general. This fact, along with the inherent acidity of the product, which promotes the isomerization of some carotenoids, makes the correct identification of some of these pigments quite difficult. Thus, one of the carotenoids occurring in orange juice has been traditionally identified as isolutein, a term used to refer to lutein epoxide, although enough evidence to support that identification has not been given. In this study, the carotenoid previously identified as isolutein/lutein epoxide in orange juice has been isolated and identified as a 9 or 9′-cis isomer of antheraxanthin as a result of different tests. To support this identification, a mixture of geometrical isomers of lutein epoxide isolated from petals of dandelions was analyzed under the same conditions used for orange juice carotenoids to check that neither their retention times nor their spectroscopic features matched with those of the orange juice carotenoid now identified as a cis isomer of antheraxanthin. KEYWORDS: Antheraxanthin; C30; carotenoids; isolutein; lutein epoxide; orange juice INTRODUCTION Orange juice is probably the most globally preferred fruit juice. The importance of carotenoids in orange juice color (1- 3) along with the growing interest in these compounds owing to their likely health benefits (4-6) have stimulated the development of a wide variety of analytical methods for their determination in orange juice (1, 7-10). Because there is a wide choice of methods for the analysis of orange juice carotenoids, research should focus on their accurate identification. The correct identification of these pigments in citrus fruits in general is more complicated, due to their complex carotenoid profile. On the other hand, their acidity is enough to promote rear- rangements of 5,6-epoxides to 5,8-epoxides. In addition, the use of C 30 columns, which are currently being used to study the carotenoid profile of different kinds of orange juices (2, 8, 11, 12), leads to complex chromatograms, since different isomers of the same carotenoid can be separated (13-15). Thus, around 40 peaks corresponding to orange juice carotenoids have been obtained in some studies (2, 7). Some of them remain unidenti- fied, whereas many others have been identified in a tentative way by comparison of the spectroscopic data obtained with those reported in the literature. The aim of this study was to provide new data concerning the identity of one of these carotenoids, which had been previously identified as isolutein. MATERIALS AND METHODS Pigment Extraction from Orange Juice and Saponification. The extract of carotenoids was obtained from 300 mL of ultrafrozen orange juice, kindly provided by Zumos Vitafresh (Almonte, Spain), which was thawed at room temperature. Extraction was carried out in a separatory funnel, using 500 mL of a mixture of ethanol:hexane (1:1). After the mixture was filtered, the colored extract was saponified overnight under an atmosphere of nitrogen. For that purpose, 500 mL of 10% ethanolic KOH was used. The saponified extract was washed four times with water (4 × 300 mL) to remove any trace of alkali and subsequently taken to dryness in a rotary evaporator at a temperature below 35 °C. Thin-Layer Chromatography (TLC). For a preliminary study of the carotenoid profile of orange juice, an aliquot of the extract was chromatographed on silica gel TLC aluminum sheets (Merck, Darm- stadt, Germany) using diethyl ether as the mobile phase. Lutein standard, obtained from spinach leaves according to standard procedures (16), was cochromatographed to determine the location of the band corre- sponding to dihydroxycarotenoids. After 30 min of development, it was seen that there were three bands below the one corresponding to dihydroxycarotenoids. The TLC aluminum sheet was left to dry exposed to air, and a few minutes later, it was clearly observed that two of those bands turned greenish, which revealed the presence of mono- epoxycarotenoids, whereas the band at the bottom turned blueish, which revealed the presence of diepoxycarotenoids (17, 18). The rest of the extract was chromatographed on laboratory-made 60GF 254 (Merck) silica gel plates (20 cm × 20 cm, 0.5 mm thickness) using the same solvent system. To determine the location of the dihydroxycarotenoid fraction, a small amount of lutein standard was cochromatographed on every plate. After 1 h, the bands located between the ones corresponding to dihydroxycarotenoids and hydroxycarotenoids * To whom correspondence should be addressed. Tel: ++34 954556761. Fax: ++34 95455 7017. E-mail: heredia@us.es. † University of Seville. ‡ University of Liverpool. J. Agric. Food Chem. 2005, 53, 9369-9373 9369 10.1021/jf051722i CCC: $30.25 © 2005 American Chemical Society Published on Web 11/05/2005