Lutein standard

For the analysis of lutein in egg yolks, 12 eggs, or six per group, were used. The yolk samples were prepared according to the Leeson and Caston's method (2004). Lutein in egg yolks was determined using the Shimadzu HPLC system by weighing 0.5 g of egg yolk in a test tube, overflowing it with 5 ml of acetone and vigorously stirring on a vortex mixer for 30 seconds. The samples were left to stand in the dark for one hour. Subsequent to a rest and filtration through a 0.45 μm membrane filter CHROMAFIL ® Xtra CA-45/25 (MACHEREY-NAGEL GmbH&Co. KG, Düren, Germany), 1 ml of acetone extract was transferred to the HPLC vials and gently evaporated by heating. The residue in the vial was dissolved by the addition of 1 ml of hexane / ethyl acetate solution (65:35, v/v) and mixed on the vortex. The sample thus prepared was analyzed on a Viva C18 column (5 μm, 250x4.6 mm; RESTEK Corporation, Bellefonte, PA, USA). The mobile phase consisted of a mixture of methanol and tetrahydrofuran (THF) 9:1 (v/v). The flow rate was 1 ml/min, the analysis time was 20 minutes, and the measurement wavelength was 450 nm. The injection volume was 20 μl. The standard lutein curve was prepared using the lutein standard purchased from ChromaDex (Irvine, CA, USA).