Carotenoids determination
Carotenoids (lutein, -carotene and -carotene) were
extracted with acetone in conditions of restricted lighting.
Samples were analyzed using an Ultra High Performance
Liquid Chromatographer Agilent 1290 (Agilent Technologies,
USA) with a Zorbax Eclipse Plus C18 (column
50 × 2.1mm; 1.8m particle size) coupled to a DAD detector
(measuring at 460nm). The gradient mobile phase
was a mix of acetonitrile/methanol 85:15 (solvent A) and
acetonitrile/methanol/ethyl acetate 60:20:20 (solvent B) containing
0.1% BHT (butylated hydroxytoluene) and 0.05% TEA
(Tetraethylammonium): initially of 100% solvent A for 4min,
then 5min with 100% solvent B and finally 100% solvent A
until the end of the run. Flow rate was 0.3mL/min and column
temperature was maintained at 28 ◦C using a column oven.
Carotenoids were identified and quantified by their retention
times using standard solutions. Results are expressed as mg
100 g−1 of sample weight.