The health benefits of the carotenoids have encouraged research on the potential effects of the individual carotenoids on human health. Due to their extremely high natural occurrence as well as their susceptibility to some reactions of oxidation and isomerization under certain conditions (light, heat, acids, oxygen), carotenoid analysis is a tricky task. Consequently, the analytic procedure and the separation in particular, must be adequate to the type of sample investigated must be thoughtfully developed, in order to avoid the degradation and transformation of the initial carotenoid content. For determination of carotenoids in Capsicum annuum L. the preliminary extraction with a tri-solvent mixture based on methanol/ethyl acetate/petroleum ether turned out to be the best choice recovering both non-polar and polar carotenoids; in particular within the polar carotenoids the xanthophylls bearing the keto groups have been shown for the first time.
The developed NP-LC×RP-LC system, due to the combination of two orthogonal separation systems in the dimensions allowed to successfully up identify carotenoids belonging to ten different chemical classes thanks to the complementary information afforded by the two detection techniques.

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