Optimized separation of astaxanthin stereoisomers from microbial sources using chiral HPLC

Astaxanthin (AST) is a high-value antioxidant, and its efficient isolation and utilization are challenging owing to the presence of different stereoisomers from various sources. In the present study, a semi-preparative HPLC method for the efficient separation of AST stereoisomers using a Chiralpak IC chiral column with good loading capacity and chiral recognition ability was successfully developed. The mobile phase was methanol-methyl <i>tert</i>-butyl ether (90 : 10, v/v), with a flow rate of 3.06 mL min^-1 and a maximum injection volume of 0.32 mg. The results indicated that the purity of all-trans AST was 97.9% for <i>Haematococcus pluvialis</i> and 97.5% for <i>Phaffia rhodozyma</i>. Additionally, molecular weights and fragmentation patterns analyzed using mass spectrometry were consistent with those of all-trans AST. Linearity validation and reproducibility experiments revealed that all calibration curves had coefficients of determination (<i>R</i>²) greater than 0.999 and a relative standard deviation (RSD) of <3.8%. This is because all-trans AST stereoisomers could undergo specific rotations or spins due to π-π interactions, hydrogen bonding, and inclusion interactions. This process allowed the successful separation of the three all-trans AST optical isomers and provides a theoretical basis for large-scale preparation of all-trans AST stereoisomers from different sources.