2.5.1. Carotenoids Analysis in Blood Samples

For plasma separation, blood samples were collected in tubes with EDTA K2 and centrifuged (500× g). Plasma samples were aliquoted and stored at −80 °C until analysis. The quantification of plasma carotenoids was performed by high-performance liquid chromatography with diode-array detection (HPLC-DAD) using a methodology adapted from the literature [16,17,18]. Briefly, plasma (100 µL) was extracted with ethanol/n-butanol (1:1, v/v, 200 µL) containing β-apo-8′-carotenal as an internal standard. After incubation at −20 °C for 30 min, the mixture was centrifuged (21,000× g for 3 min) and 20 µL of the clear supernatant was analyzed on a Waters^® HPLC Alliance using a LiChrospher^® 100 RP-185 μm (250 × 4.0 mm) column at 450 nm as previously described in detail [18]. Pure carotenoid standard mixtures were prepared and analyzed using the same analytical conditions as for the sample for quantification. Results are expressed in µg·L⁻¹ of plasma.