Separation and identification of fatty acid esters of algal carotenoid metabolites in the freshwater mussel Dreissena bugensis, by liquid chromatography with ultraviolet/visible wavelength and mass spectrometric detectors in series

LC with photodiode array and APCI-ion trap mass spectrometry has made it possible to tentatively identify 76 carotenyl fatty acid esters (cFAEs) in solvent extracts from Dreissena bugensis, collected from Lake Erie: 16 mono- and 33 diFAEs of fucoxanthinol (FOH), and 27 diFAEs of mactraxanthin (MX). FOH and MX, previously identified in cFAE hydrolysates, were confirmed as parent carotenoids of the cFAEs, and as primary metabolites of fucoxanthin and violaxanthin, respectively, derived from diatoms and chiorophytes in the dreissenids' diet. The most abundant fatty acid substituents of cFAEs were 16:0 and 16:1; abundant fatty acid biomarkers were 16:1 and 20:5, from diatoms, and 17:0, from bacteria. Cleanup of solvent extracts by solid phase extraction (Florisil) was necessary to reduce neutral background lipids, which interfered with detection of MX-diFAEs by APCI(+), and detection of FOH-diFAEs by APCI(+/-). The FOH-monoFAEs, MX-diFAEs and FOH-diFAEs were found to elute in a well-defined chromatographic order, by two regression models for retention times increasing as a function of: i) increasing number of carbons but decreasing number of double bonds in the fatty acid and decreasing number of non-esterified OH-groups on the parent carotenoids; ii) increasing dispersive but decreasing polar and hydrogen-bonding interactions, described by solubility parameters calculated for each cFAE. The best separations of the dreissenid cFAEs, with free OH-groups decreasing from four to one, were achieved between 20 and 68 min, using a C18-column and moderately polar mobile phase (acetone, water), to obtain a reverse-phase gradient with a 56% decrease in hydrogen-bonding interactions. (C) 2017 Elsevier B.V. All rights reserved.