Determination of astaxanthin and astaxanthin esters in the microalgae Haematococcus pluvialis by LC-(APCI)MS and characterization of predominant carotenoid isomers by NMR spectroscopy

Abstract

The oily product ZANTHIN consists of natural astaxanthin, which is manufactured from the microalgae Haematococcus pluvialis by supercritical CO(2) extraction. An HPLC method was developed to separate all of the components of the complex astaxanthin extract using a C(30) column. The separation resulted in different isomers of astaxanthin accompanied by two other carotenoids. The main component consisted of astaxanthin singly esterified with several different fatty acids. C18:3, C18:2, C18:1 and C16:0 were identified as the most commonly occurring fatty acids. Doubly esterified astaxanthin was also found, although in lower concentrations compared to singly esterified astaxanthin. After performing a detailed fatty acid analysis by GC-MS, the peaks from the extract were assigned via HPLC-MS. A trans to cis transmutation of the all-trans compound was performed by thermal treatment in order to obtain an enrichment of cis isomers as the basis for unambiguous identification via NMR experiments. The all-trans as well as the 9- and 13-cis isomers of astaxanthin were characterized in detail by UV/Vis, (1)H, and (1)H,(1)H COSY NMR spectroscopy.

Tentative Identification of Torulene Cis/trans Geometrical Isomers Isolated from Sporidiobolus pararoseus by High-Performance Liquid Chromatography-Diode Array Detection-Mass Spectrometry and Preparation by Column Chromatography

“…In the UV region, an absorption peak referred to as the cis peak is apparent for cis isomers, with the intensity of this peak varying with the position and number of cis double bonds. 20,36 The cis peaks of Z-torulene isomers appeared at around 375 -380 nm (Table 3), being similar to those reported in the literature. 37 A distinction between mono-Z and di-Z carotenoid isomers can be made by considering the shift in their absorption maxima relative to those of the all-E isomers, which is normally around 2 -12 nm in the case of the former.…”

Astaxanthin in microalgae: pathways, functions and biotechnological implications

“…All the antioxidative enzymes studied showed a transient up-regulation pattern and then reverted to the basal or below basal level, suggesting that the enzyme defense system is an early response mechanism to oxidative stress, and as stress persists, the cells will adopt astaxanthin biosynthesis and accumulation as a long-term defense mechanism (Wang et al 2004a). the major fatty acid species that is conjugated to astaxanthin molecules (Holtin et al 2009). A linear correlation between the cellular fatty acid content and astaxanthin content in H. pluvialis under stress conditions has been reported in several independent studies (Zhekisheva et al 2002, Chen 2007, leading to a hypothesis that the biosynthesis of astaxanthin and fatty acids are coupled in this organism.…”

Carotenoids and Their Fatty Acid Esters in the Petals of Adonis aestivalis

“…The esterified fatty acids found in astaxanthin esters extracted from crustaceans were mainly C18:0, C18:1, C16:0, C16:1, C22:6, C20:5, and C20:4 10, 13 16 . In green algae H. pluvialisby , C18:3, C18:2, C18:1, and C16:0 were found to be major fatty acids esterifi ed with astaxanthin 17,18 . On the other hand, saturated and monounsaturated fatty acids were identifi ed as esterifi ed fatty acids with carotenoids in A. aestivalis.…”

“…The compositions of individual carotenoid esters expressed as a percentage of the total fraction of astaxanthin diesters, astaxanthin monoester, and adonirubun monoester are shown in Tables 2, 3, and 4, respectively Astaxanthin esters have been reported in several crustaceans 10, 13 16 and in green algae Haematococcus pluvialisby 17, 18 using GC 13 15 , LC-combined FD-MS 16 , LC/AP-CI-MS 17,18 , and FAB MS 10 methods. The esterified fatty acids found in astaxanthin esters extracted from crustaceans were mainly C18:0, C18:1, C16:0, C16:1, C22:6, C20:5, and C20:4 10, 13 16 .…”