2.2. Chemicals and analytical method
All reagents were at least analytical grade; organic solvents
used for HPLC analysis were of HPLC grade. All carotenoid determinations
in the different samples were done in duplicate.
The analytical method used for carotenoid determination is
described elsewhere (Dias, Camões, & Oliveira, 2009). In brief,
the samples were extracted four times with a methanol:tetrahydrofurane
mixture (1:1) after addition of basic magnesium carbonate
and an internal standard (b-apo-80-carotenal for fruits,
echinenone for vegetables). The solvent was evaporated (35 C
in a rotary evaporator) and the solid residue dissolved in 0.5
or 5 ml dichloromethane (accordingly to predicted carotenoid
mass fraction). The volume was made up to 5 or 50 ml, respectively,
with solvents to obtain a final composition near to the
mobile phase (see below). These extracts were injected in the
HPLC system. Fruit extracts (2 ml) were saponified with 5%
potassium hydroxide for 1 h at room temperature, under nitrogen
in the dark, in a pyrogallol antioxidant medium. After
extraction with petroleum ether and washing with water, the
solvent was evaporated and the residue dissolved in mobile
phase for analysis.