LC-PDA/APCIitMS identification of algal carotenoid and oxysterol precursors to fatty acid esters in hydrolyzed extracts of the freshwater mussel Dreissena bugensis.

Carotenyl fatty acid esters (carotenyl-FAEs) were extracted in acetone from freeze-dried Dreissena bugensis (Lakes Erie and Ontario) and hydrolyzed to identify the carotenoid precursors. Analysis by liquid chromatography (LC) with photodiode array (PDA) and atmospheric pressure chemical ionization-ion trap mass spectrometry (APCIitMS) revealed the major hydrolysis products: fucoxanthinol (FOH) from fucoxanthin (FX, diatoms); mactraxanthin (MX) from violaxanthin (VX, chlorophytes); 4-fold higher levels of an unknown, tentatively identified as an adduct of two closely eluting C27H46O3 and C27H48O3 steryl triols. Enzymatic hydrolysis (Candida rugosa) of dreissenid extracts yielded FOH and MX, but residual carotenyl-FAEs remained. Alkaline hydrolysis yielded isoFOH, MX, and steryl triols, without residual carotenyl-FAEs: isoFOH decreased, but two FOH hemiketal by-products increased, when the dose of potassium hydroxide in methanol was too high. The PDA detector profiled carotenyl-FAEs and products of enzymatic and alkaline hydrolysis, without interference. The APCIitMS detector revealed carotenoid and oxysterol products of alkaline hydrolysis but was adversely affected by background from bile salts used for enzymatic hydrolysis. LC retention times and elution order were correlated to solubility parameters, calculated from the analyte structure, to cross-check MS interpretations. A multiple linear regression of LC retention times on solubility parameters for 12 carotenoid standards included FOH, isoFOH, and MX (r 2 0.97). The model revealed the close similarity of polar carotenoid metabolites to C27-steryl triols tentatively identified by APCIitMS, suggesting that further LC-MS analyses would be beneficial, to explicitly link oxysterols and the polar carotenoids, as metabolites of algal precursors in the dreissenid diet. Graphical abstract Methods of analysis and major neutral products of hydrolysis from fatty acid esters in D. bugensis.

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 chlorophytes 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 68min, 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.