Contents lists available at ScienceDirect Talanta journal homepage: www.elsevier.com/locate/talanta Enrichment procedure based on graphitized carbon black and liquid chromatography-high resolution mass spectrometry for elucidating sulfolipids composition of microalgae Michela Antonelli a , Barbara Benedetti a , Chiara Cavaliere a,* , Andrea Cerrato a , Giorgia La Barbera a , Carmela Maria Montone a , Susy Piovesana a , Aldo Laganà a,b a Department of Chemistry, Università di Roma “La Sapienza”, Piazzale Aldo Moro 5, 00185, Rome, Italy b CNR NANOTEC, Campus Ecotekne, Università del Salento, Via Monteroni, 73100 Lecce, Italy ARTICLE INFO Keywords: Sulfolipids Spirulina microalgae Lipidomic analysis HPLC-HRMS Enrichment Graphitized carbon black ABSTRACT Microalgae have recently become a popular functional food due to their health benefts. Sulfolipids, a class of substances abundant in this matrix, have been reported to have interesting bioactivities, such as anti-carcino- genic activity. However, despite the potential interest in sulfolipids, a dedicated analytical method for their characterization is currently lacking but would signifcantly 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, frst to maximize the recovery of reference standards, then to increase the total number of identifed 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 identifed by Lipostar, for a reliable lipid structure assignment. The developed method was compared to the direct lipidomic analysis without enrichment, to establish the enrichment ef- ciency in terms of number of identifcations. From the comparison, the enrichment procedure proved better and allowed the tentative identifcation 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. 1. Introduction Sulfolipids are polar glycolipids where the sugar moiety possesses a sulfate substituent (Supplementary Material, Fig. S1). Sulfolipids are constituents of the thylakoid membrane in plant and algae chloroplasts [1]; these membranes are mainly composed of mono- and digalacto- syldiacylglycerols, with sulfolipids and phospholipids constituting the anionic fraction [2]. Identifcation of sulfolipids, in particular sulfoquinovosylmonoacylglycerols (SQMGs, Fig. S1, structures a1, a2) and sulfoquinovosyldiacylglycerols (SQDGs, Fig. S1, structure b), has been reported in plant leaves such as conifer leaves, parsley, daisy, green onion leaves and several green foods such as spinach and broccoli [3–5]. In the recent years, sulfolipids have been attracting the interest of the scientifc community due to their promising bioactive properties. In fact, a range of diferent actions, such as anti-infammatory, immunosuppressive, anti-viral and anti-carcinogenic activities, have been attributed to these https://doi.org/10.1016/j.talanta.2019.120162 Received 11 April 2019; Received in revised form 15 July 2019; Accepted 17 July 2019 Abbreviations: UHPLC-HRMS, Ultra-high performances liquid chromatography-high resolution mass spectrometry; GCB, graphitized carbon black; LC-MS, liquid chromatography-mass spectrometry; SQDG, sulfoquinovosyldiacylglycerol; SQMG, sulfoquinovosylmonoacylglycerols; SPE, solid phase extraction; SLE, solid-liquid extraction; MeOH, Methanol; DCM, dichloromethane; TMACl, tetramethylammonium chloride; HESI, heated electrospray ionization; HCD, higher-energy collisional dissociation; LOD, Limit of detection; LOQ, Limit of quantitation; RSD, relative standard deviation * Corresponding author. E-mail addresses: michela.antonelli@uniroma1.it (M. Antonelli), barbara.benedetti@uniroma1.it (B. Benedetti), chiara.cavaliere@uniroma1.it (C. Cavaliere), andrea.cerrato@uniroma1.it (A. Cerrato), giorgia.labarbera@uniroma1.it (G. La Barbera), carmelamaria.montone@uniroma1.it (C.M. Montone), susy.piovesana@uniroma1.it (S. Piovesana), aldo.lagana@uniroma1.it (A. Laganà). Talanta 205 (2019) 120162 Available online 18 July 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved. T