| Summary: | <i>Haematococcus pluvialis</i>, a unicellular green microalga that produces a secondary metabolite under stress conditions, bears one of the most potent antioxidants, namely xanthophyll astaxanthin. The aim of our study was to determine the content of astaxanthin and its esterified forms using three different solvents—methyl <i>tert</i>-butyl ether (MTBE), hexane isopropanol (HEX -IPA) and acetone (ACE)—and to identify them by using high performance liquid chromatography coupled with diode array detection and the quadrupole time-of-flight mass spectrometry (HPLC-DAD and LC-QTOF-MS) technique. We identified eleven astaxanthin monoesters, which accounted for 78.8% of the total astaxanthin pool, six astaxanthin diesters (20.5% of total), while free astaxanthin represented the smallest fraction (0.7%). Astaxanthin monoesters (C16:2, C16:1, C16:0), which were the major bioactive compounds in the <i>H. pluvialis</i> samples studied, ranged from 10.2 to 11.8 mg g<sup>−1</sup> DW. Astaxanthin diesters (C18:4/C18:3, C18:1/C18:3) were detected in the range between 2.3 and 2.6 mg g<sup>−1</sup> DW. All three solvents were found to be effective for extraction, but MTBE and hexane-isopropanol extracted the greatest amount of free bioactive astaxanthin. Furthermore, MTBE extracted more low-chain astaxanthin monoesters (C16), and hexane-isopropanol extracted more long-chain monoesters (C18 and above) and more diesters. We can conclude that MTBE is the solvent of choice for the extraction of monoesters and hexane-isopropanol for diesters.
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