Enrichment procedure based on graphitized carbon black and liquid chromatography-high resolution mass spectrometry for elucidating sulfolipids composition of microalgae.

Microalgae have recently become a popular functional food due to their health benefits. Sulfolipids, a class of substances abundant in this matrix, have been reported to have interesting bioactivities, such as anti-carcinogenic activity. However, despite the potential interest in sulfolipids, a dedicated analytical method for their characterization is currently lacking but would significantly increase the coverage of sulfolipids with respect to the direct lipidomic analysis. To achieve this goal, in this work a procedure, based on graphitized carbon black solid phase extraction, was developed for clean-up and enrichment of sulfolipids (sulfoquinovosyldiacylglycerols and sulfoquinovosylmonoacylglycerols) and it was applied to spirulina (Arthrospira platensis) microalgae. A careful study of the solid phase extraction conditions was performed, first to maximize the recovery of reference standards, then to increase the total number of identified sulfolipids from the spirulina lipid extract. All samples were analysed by ultra-high performance liquid chromatography coupled to high resolution mass spectrometry and lipids were tentatively identified by Lipostar, for a reliable lipid structure assignment. The developed method was compared to the direct lipidomic analysis without enrichment, to establish the enrichment efficiency in terms of number of identifications. From the comparison, the enrichment procedure proved better and allowed the tentative identification of 199 sulfolipids, which is the largest number reported so far for the Arthrospira platensis species. The described method was validated in terms of precision, accuracy, recovery, limit of quantitation and detection for two sulfolipids. Finally, a relative lipid quantitation based on peak area was carried out on the microalgae sample, which indicated nine abundant sulfolipids as representing ca. 60% of sulfolipids in spirulina microalgae.