Abstract
A fast and high-resolution UPLC-MSE analysis was used to identify phytoplankton pigments in an ethanol extract of Porphyridium purpureum (Pp) devoid of phycobiliproteins. In a first step, 22 standard pigments were analyzed by UPLC-MSE to build a database including retention time and accurate masses of parent and fragment ions. Using this database, seven pigments or derivatives previously reported in Pp were unequivocally identified: β,β-carotene, chlorophyll a, zeaxanthin, chlorophyllide a, pheophorbide a, pheophytin a, and cryptoxanthin. Minor amounts of Divinyl chlorophyll a, a chemotaxonomic pigment marker for prochlorophytes, were also unequivocally identified using the database. Additional analysis of ionization and fragmentation patterns indicated the presence of ions that could correspond to hydroxylated derivatives of chlorophyll a and pheophytin a, produced during the ethanolic extraction, as well as previously described galactosyldiacylglycerols, the thylakoid coenzyme plastoquinone, and gracilamide B, a molecule previously reported in the red seaweed Gracillaria asiatica. These data point to UPLC-MSE as an efficient technique to identify phytoplankton pigments for which standards are available, and demonstrate its major interest as a complementary method for the structural elucidation of ionizable marine molecules.
Highlights
Phytoplankton species present a high genetic and metabolic diversity, and evolved a wide range of photoprotective and photosynthetic pigments capable of collectively harvesting most of the wavelengths of visible light available in underwater marine habitats [1,2,3,4]
Because of their very low abundance, these minor pigments usually remain unidentified, in spite of their possible interest as chemotaxonomic markers or for biotechnological or biomedical applications. They can correspond to molecules effectively present in living algal cells, to biosynthetic precursors and intermediates, or to artifact or natural derivatives produced by the alteration of chlorophylls or carotenoids in environmental conditions or during extraction and/or purification [12,19,23]
The selected UPLC conditions allowed for a separation of all pigments except for chlorophyll b and DV-chlorophyll b, which exhibited the same retention times (Rt) (4.71 min, Table 1) but could be discriminated by their accurate masses and fragmentation patterns (Table 1 and Supplementary Figure S1)
Summary
Phytoplankton species present a high genetic and metabolic diversity, and evolved a wide range of photoprotective and photosynthetic pigments capable of collectively harvesting most of the wavelengths of visible light available in underwater marine habitats [1,2,3,4]. Because of their very low abundance, these minor pigments usually remain unidentified, in spite of their possible interest as chemotaxonomic markers or for biotechnological or biomedical applications They can correspond to molecules effectively present in living algal cells, to biosynthetic precursors and intermediates, or to artifact or natural derivatives produced by the alteration of chlorophylls or carotenoids in environmental conditions or during extraction and/or purification [12,19,23]. A complex mix of metabolites can be analyzed in a single run, no precursor ion is selected for individual fragmentation, and fragment ions can be related to their precursors using a mass fragmentation software and high-resolution mass databanks This technique, coupled with UV analysis, was already applied with success to the identification and quantification of major carotenoids and chlorophylls in Dunaliella salina [30]. Ions whose accurate masses and fragmentation patterns did not match with the database were compared to mass data in the literature, to determine if they could correspond to previously reported metabolites, including pigments and pigment derivatives
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