2.3. Sample extraction
100 g of black and orange fresh carrots were peeled and finely
chopped, to avoid the traces of carotenes in the black carrots. These
samples were subjected to extraction with 100 mL EtOH:H2O (1:1,
v/v), containing 0.01% HCl (37% v/v) and left with stirring at room
temperature (25 8C) for a period of 2 h. Then, the extracts were
filtered through a 0.45 mm polytetrafluoroethylene (PTFE) membrane,
and the ethanol was evaporated under vacuum. The
resulting extracts were purified by liquid–liquid extraction with
chloroform (4 50 mL), pentane (4 50 mL) and cyclohexane (4
50 mL). The resulting aqueous fractions were concentrated in a
vacuum rotary evaporator until 2 mL at a temperature of 30 8C and
stored at 4 8C in the absence of light. The extraction was carried out
in triplicate for each sample, and the further analyses were
performed within 3 days.