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

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.

Phase optimized liquid chromatography as an instrument for steroid analysis.

A new method using phase optimized LC (POPLC) for the analysis of steroids is described. The retention factors and the theoretical plate numbers of different steroids were determined for four different stationary phases. Based on these values, an optimal stationary phase composition and the resultant chromatogram have been calculated by an optimization software and compared to the measured data. It is demonstrated that these predicted data show only little differences to the experimental results. Furthermore, it is shown that the overall selectivity of the optimal stationary phase composition is much better than the selectivity of any individual stationary phase.

Characterization of bixin by LC-MS and LC-NMR.

An overview upon modern analytical techniques for the isolation, separation, and structural identification of the essential bioactive carotenoid bixin is given. Isolation from biological matrices is performed by matrix solid phase dispersion (MSPD). The extract is separated with shape-selective C(30 )columns. Structural assignment of the separated compounds is done by online LC-MS and capillary HPLC-NMR.