Identification of the Composition of Isomeric Canthaxanthin Sample by NMR, HPLC, and Mass Spectrometry

Canthaxanthin crystal and three isomeric samples generated by different processes (solvent dissolving, iodine catalysis, and melting) were selected to study the chemical changes during different isomerization processing. No sharp difference could be found when the nuclear magnetic resonance spectra of canthaxanthin crystal and canthaxanthin obtained by solvent dissolving and iodine catalysis were compared, suggesting that only isomerization occurred in those isomeric samples. However, the content of unsaturated hydrogen decreased during the melting of canthaxanthin, and the measurements of gel permeation chromatography and UV/Vis spectroscopy showed that the canthaxanthin melt contained few compositions of polymer and degradation products. A high-performance liquid chromatography (HPLC) analysis system consisting of a C30 column and an elution system of methanol/methyl tert-butyl ether/water/dichloromethane (60:25:12:3, v/v/v/v) was employed to identify the compositions detected in HPLC. Using the combination of HPLC–diode array detector and HPLC–atmospheric pressure chemical ionization–mass spectrometry, ten isomers and four epoxides of canthaxanthin were successfully isolated. The isomer compositions were identified as 9,13′-di-cis-, 13,15-di-cis-, 9,15-di-cis-, 15-cis-, 13-cis-, 9,13-di-cis-, all-trans-, 11-cis-, 7-cis-, and 9-cis-canthaxanthin. The chromatographic purity and content of canthaxanthin sample also can be determined based on the C30-HPLC method.