An improved method for the separation of carotenoids and carotenoid isomers by liquid chromatography-mass spectrometry

Carotenoids consist of a series of conjugated isoprene units that are characteristically highly conjugated through double bonds, leading to the formation of many isomers that are susceptible to oxidation and other chemical modifications. Extreme hydrophobicity and high complexity make carotenoids difficult to identify and quantify. We implemented the use of a common Syncronis C18 column with strong eluting solvent, here isopropanol, to successfully separate a mixture of 23 carotenoids standards with different structural properties. In addition, the method differentiated between three groups of isomeric carotenoids (lycopene/delta-carotene/gamma-carotene/epsilon-carotene/epsilon-carotene/beta-carotene, alpha-cryptoxanthin/beta-cryptoxanthin, and zeaxanthin/lutein) by optimizing the gradient profile and using liquid chrmatography-mass spectrometry. The LOD ranged from 0.05 to 551 ng/mL, and the recovery of carotenoids in Mytilus coruscus was from 63.54 to 93.25%, with standard deviations <10%. Twenty-five carotenoids were detected with a total content of 857 +/- 55.1 mg/kg, and three isomeric carotenoids were identified: epsilon-carotene, alpha-carotene, and beta-carotene. Our results show that this methodology is a significant improvement over other alternatives for analyzing carotenoids because of its compatibility with carotenoids of different categories, and most importantly, its ability to resolve isomeric carotenes, which is significant not only for assessing carotenoid species, but also for the tracing of metabolic pathways of carotenoids.

Automated summary

Carotenoids are complex compounds composed of highly conjugated isoprene units that are difficult to identify and quantify due to their extreme hydrophobicity. A new method using a C18 column and isopropanol as eluent successfully separated 23 carotenoid standards and differentiated three groups of isomeric carotenoids. The methodology showed improved compatibility with different categories of carotenoids and the ability to resolve isomeric carotenes, crucial for both assessing carotenoid species and tracing their metabolic pathways.