Development of a simple and economical analytical method for investigating the distribution of astaxanthin E/Z-isomers in foods and cosmetics

Astaxanthin has many theoretical E/Z-isomers, making separation and accurate analysis challenging, especially because of their structural similarities and polarities. The present study developed a simple and cost-effective reversed-phase high-performance liquid chromatography (HPLC) method to separate major astaxanthin isomers efficiently (i.e., the all-E-, 9Z-, 13Z-, and 15Z-isomers) and assess their distribution in various foods, processed products, edible insects, and cosmetics. Various HPLC columns (C18, C30, and cholesteryl-bonded silica) and mobile phase mixtures at different column oven temperatures were examined. Our developed method, using a C18 column (C18-HPLC), successfully separated the major isomers of astaxanthin at 30 °C in isocratic mode with methanol/H₂O (92.5:7.5, v/v) within 20 min. This method also minimizes the chromatographic interference from other dietary carotenoids. Additionally, although the C30 column allowed the efficient separation of astaxanthin isomers, it required the use of highly toxic and volatile mobile phase solvents, such as methyl tert-butyl ether and dichloromethane, as well as longer run times. The optimized C18-HPLC method provided an accurate assessment of astaxanthin isomers in commercially available foods, revealing higher isomer ratios in cooked samples than in raw samples. In conclusion, the developed C18-HPLC method is a simple, efficient, cost-effective, and rapid tool for astaxanthin isomer analysis.