Carotenoids are fat-soluble substances consisting of a backbone of 40 C atoms, formed through the condensation of eight isoprene units. They are classified into carotenes and xanthophylls, with the latter including end-groups with oxygen-based functionalities. Such end-groups may extend the conjugated central polyene chain of carotenoids, that is responsible for absorption of radiation in the visible region and characteristic orange pigmentation [1]. Due to their importance as bioactive secondary metabolites, a method based on reversed-phase liquid chromatography coupled to a diode array detector and to Fourier transform mass spectrometry by atmospheric pressure chemical ionization (RPLC-DAD-APCI-FTMS) has been recently developed in our laboratory for the characterization of carotenoids in Brassica innovative products, particularly microgreens, which are already known for their nutraceutical properties. In particular, β-carotene, β-cryptoxanthin, lutein, violaxanthin, and 9-cis-neoxanthin were identified and separated from further 22 isomeric species using a ternary elution gradient. In order to clarify the structure of these isomers, a combination of detection approaches was exploited after RPLC separation, initially considering standard carotenoids as model compounds. Specifically, UV–Vis absorption spectroscopy was employed to determine the position of cis double bonds and the number of epoxide groups. In-source fragmentation patterns revealed by APCI-FTMS spectra enabled the identification of terminal end groups through the recognition of the number of free hydroxyl groups. Electrospray ionization-ion mobility spectrometry following RPLC (RPLC-ESI-IMS) was finally used to assess the eventual occurrence of multiple mobilities for carotenoids ions. IMS data clearly revealed the presence of multiple protomers for single ions generated in the source and provided collision cross section (CCS) values not previously reported in databases. Individual protomers separated by ion mobility were also subjected to tandem mass spectrometry experiments (IMS-MS/MS), which showed distinct fragmentation patterns for each species. This analytical strategy enabled a five-dimensional characterization of each carotenoid, based on retention time, UV–Vis absorption, APCI-FTMS in-source fragmentation pattern, CCS values and MS/MS spectra of each protomer, providing useful analytical information both on major carotenoids and on their isomeric forms in innovative products of Brassicaceae plants.
Britton G, The FASEB Journal, (9.15) 1995, 1551-1558, DOI:10.1096/fasebj.9.15.8529834.