A new method for the identification and the structural characterisation of carotenoid esters in freshwater microorganisms by liquid chromatography/electrospray ionisation tandem mass spectrometry

Abstract

Liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS) has been employed to identify carotenoid esters present in raw organic extracts of pigmented freshwater microalgae and to gain structural information on these compounds. In particular, acyl carotenoid derivatives of Haematococcus pluvialis and Euglena sanguinea have been characterised by tandem mass spectrometry (MS/MS) in a quadrupole ion trap. ESI-MS/MS allows recognition of the presence of carotenoid esters in complicated mixtures without any initial chromatographic work-up and without the need to use UV-Vis photo-diode array (PDA) detectors. Product ion scans of the [M + Na]+ ion lead to known neutral losses of the C7H8 and C8H10 residues from the conjugated polyene moiety of the carotenoid unit, that permit the unambiguous identification of the carotenoid itself. These structurally relevant ions are not observed in positive or negative ion APCI (atmospheric pressure chemical ionisation) mass spectra. Moreover, the several product ions observed in positive and/or negative ion ESI-MS/MS not only are a diagnostic signature of the main structural features of the acyl chains such as length, position and unsaturation, but also display the nominal mass of the parent xanthophyll. Our methodology has been validated (i) by using esters of astaxanthin obtained from off-line purification of the H. pluvialis extracts and structurally elucidated through proton nuclear magnetic resonance (1H-NMR) spectroscopy and (ii) by product analysis of esters by alkaline hydrolysis. The characterisation of the unknown carotenoid esters of E. sanguinea is a demonstration of the capabilities of this methodology.

Sodiation as a tool for enhancing the diagnostic value of MALDI‐TOF/TOF‐MS spectra of complex astaxanthin ester mixtures from Haematococcus pluvialis

“…In contrast, [M + H] + parent ions do not form toluene or xylene fragments, at least not with ESI ion trap MS . When [M + Na] + adducts of xanthophyll esters were ion trap fragmented, loss of toluene and xylene was more pronounced, and fragmentation patterns showed less non‐diagnostic fragments than with [M + H] + adducts . Similar observations have been obtained for astaxanthin and canthaxanthin in ESI tandem quadrupole and for fucoxanthin‐type carotenoids in APCI ion trap .…”

“…Esterified astaxanthins exhibited a 1–2 nm hypsochromic shift compared to free astaxanthin as reported earlier . For identification of fatty acids esterified to asthaxanthin, MS 2 data were required.…”

“…A). PDA visible absorption, full MS and MS 2 spectra were used for peak assignment of free carotenoids and porphyrins (Table ) and astaxanthin esters (Table ) …”

“…In order to study the compatibility of esterified astaxanthins with food matrices, fractions enriched in natural astaxanthin esters are required. Preparative Flash chromatography is the method of choice to obtain such fractions . For correct pooling of the esters desired, characterization of the fractions obtained is necessary.…”

“…For instance, radical [M] +● parent ions of xanthophyll esters show a clear toluene loss in PSD fragmentation . In contrast, [M + H] + parent ions do not form toluene or xylene fragments, at least not with ESI ion trap MS . When [M + Na] + adducts of xanthophyll esters were ion trap fragmented, loss of toluene and xylene was more pronounced, and fragmentation patterns showed less non‐diagnostic fragments than with [M + H] + adducts .…”