2.2. Analysis of carotenoids
Carotenoids were extracted using previously published methods with slight modifications (Riso and Porrini, 1997). Methanol(5 mL) was added to an accurately weighed food sample (1 g) in a 50-mL glass vial. The sample with the added methanol was then homogenized and then left to incubate overnight (16 h) in a refrigerator at 4 8C. After incubation, the sample was centrifuged at 800 g for 10 min. The methanol layer was transferred to a 25-mL volumetric flask.
After the addition of 5 mL tetrahydrofuran (THF), the vial was vortexed for 30 s, followed by a centrifugation at 800 g for 5 min. The THF layer was transferred into the methanol containing volumetric flask. The sample was extracted three more times using the same procedures and the THF layers were combined into the volumetric flask.
THF was added to make the final volume 25 mL. Then 10 mL (40% of the total) of extract was dried under nitrogen. For the spinach sample 5 mL was dried because of the relatively higher xanthophyll concentration. The extract was resuspended in 500 mL of ethanol. If the suspension appeared cloudy, oily or non-homogeneous (in the case of foods containing a relatively high fat), the extraction was repeated and ethanol:methyl-tert-butyl ether (MTBE) (2:1, v/v) or MTBE:methanol (2:1, v/v) was selected for the suspension solvent. After resuspension, the extract was vortexed for 30 s. Twenty-microliter samples were injected into the HPLC system for carotenoid analysis.