Zeaxanthin Trade Association   Home Contact Us

An Association-developed method to see the differences: Chiral Analysis
of 3R 3’R Zeaxanthin.

An accurate testing method for determination of Zeaxanthin identity is chiral chromatography. A typical high-pressure liquid chromatography (HPLC) cannot distinguish between dietary and non-dietary isomers; a chiral chromatogram clearly identifies all Zeaxanthin isomers.

The term chiral describes an object that is non-superposable on its mirror image. Human hands are an example of chirality: the left hand is a non-superposable mirror image of the right hand. The term chirality is derived from the Greek word for hand.

Chirality is a form of stereoisomerism where two or more isomers having the same chemical structure (the same linkages between atoms in a molecule) but different configurations (orientation of atoms in space). A carbon atom linked to four different substituents is called chiral. Zeaxanthin has two chiral centers – at position 3 (on the left ring) and 3’ (on the right ring). The presence of chiral atoms in Zeaxanthin introduces chirality to the molecule.

Zeaxanthin has three isomers: 3R, 3’R Zeaxanthin; 3R,3’S Zeaxanthin; and 3S,3’S Zeaxanthin. 3R,3’S Zeaxanthin is also called meso-Zeaxanthin. The natural form of Zeaxanthin is 3R,3’R Zeaxanthin. The R / S system is a nomenclature system for denoting isomers. It labels each chiral center R or S according to a system by which its substituents are each assigned a priority, according to the Cahn Ingold Prelog priority rules (CIP), based on atomic number. If the center is oriented so that the lowest-priority of the four is pointed away from a viewer, the viewer will then see two possibilities: If the priority of the remaining three substituents decreases in clockwise direction, it is labeled R (for Rectus), if it decreases in counterclockwise direction, it is S (for Sinister).

Molecular structure of Zeaxanthin Isomers

Due to the similarity in chemical structure, three stereoisomers of Zeaxanthin will not be separated on regular C8, C18 and C30 or any silica based columns in normal phase or reverse phase HPLC. However, these molecules will elute differentially on Chiral Stationary Phases (CSP). Chiral Chromatography is defined as a chromatographic technique employing Chiral Stationary Phases (CSP) to separate various chiral substances.

Non chiral and chiral chromatograph of Zeaxanthin

Non-Chiral HPLC chromatogram of Zeaxanthin containing meso-Zeaxanthin (Stereoisomers 3R,3’S Zeaxanthin and 3R,3’R Zeaxanthin not separated) on silica based C30 column.

Chiral HPLC chromatogram of meso-Zeaxanthin (3R,3’S Zeaxanthin) on a Chiral Stationary Phase. 3R,3’S Zeaxanthin and 3R,3’R Zeaxanthin are resolved.

Examples of the different non-dietary Zeaxanthin products available in the market analyzed via chiral chromatography


Difficulty in distinguishing natural 3R 3’R Zeaxanthin from the other non-dietary forms is a problem partially created by its manufacturers, which so far have not provided a complete picture of their products on the Certificates of Analysis. A typical Certificate of Analysis for Zeaxanthin these days only shows an HPLC analysis, when it fact it should also include a chiral analysis.

3R 3’R Zeaxanthin

The Zeaxanthin found in nature is the 3R 3’R isomer. This naturally occurring Zeaxanthin is the predominant form used in clinical research. Substantial safety and toxicity data has been compiled for the FDA for the NDI approval of 3R 3’R Zeaxanthin. Its chiral chromatogram looks quite different from the other isomers as you can see below.

Chiral chromatograph of 3R 3’R Zeaxanthin

Even the smallest structural change in a molecule can have a tremendous impact on how the molecule functions in the human body. Isomers that have not had a thorough health and safety validation have been known to cause severe problems. Insuring the identity of Zeaxanthin is important now that dietary supplements have to comply with Good Manufacturing Practices (GMP). GMPs “ensure the quality of dietary supplements so that consumers can be confident the products they purchase contain what is on the label.” Identity testing must be performed on every dietary ingredient used in each batch. This can be performed by the manufacturer or by the supplier, but the manufacturer is responsible for verifying the reliability of the supplier.

GMP requires “that the dietary supplement consistently meets the established specifications for identity, purity, strength, and composition and has been manufactured, packaged, labeled, and held under conditions to prevent adulteration under section 402(a) (1), (a) (2), (a) (3), and (a) (4) of the Federal Food, Drug, and Cosmetic Act.”

The only way to fully comply with GMP when it comes to the identity of the Zeaxanthin used is to perform a chiral analysis. Ask your Zeaxanthin supplier to routinely include a chiral chromatogram in their Certificate of Analysis.

Chiral Analysis Approved Method

The Zeaxanthin Trade Association has developed a method to carry out chiral analysis. This comprehensive method includes analysis of powders, beadlets, softgels and oil suspensions.

Click here to view method >>

  ©2012 Zeaxanthin Trade Association   |   622 Town Road, West Chicago, IL 60185-2698   |   USA   |   Phone: ???-000-000   |   Fax: ???-000-0000   Design by Image Perspective