Validation of an HPLC Method for the Simultaneous Determination of trans-Lutein, (3R,3′R)-trans-Zeaxanthin, and (3R,3′S)-trans-Zeaxanthin

trans-Lutein, (3R,3'R)-trans-zeaxanthin, and (3R,3'S,meso)-trans-zeaxanthin are carotenoid isomers found in the human eye. Trans-lutein and trans-zeaxanthin can be separated under NP chromatographic conditions; however, (3R,3'R)-trans-zeaxanthin and (3R,3'S)-trans-zeaxanthin coelute and require chiral separation techniques. The validation of an HPLC analytical method for the simultaneous determination of trans-lutein, (3R,3'R)-trans-zeaxanthin, and (3R,3'S)-trans-zeaxanthin is described. The method uses a hexanes and 2-propanol gradient on a Daicel AD-H chiral column to separate the carotenoids for detection at 446 and 450 nm. The method was found suitable for use in quantifying the three analytes over a wide range of concentrations with the limiting factor being the upper solubility limit of 14 mu g of (3R,3'S)-trans-zeaxanthin/mL of the injection solution of hexane-2-propanol (95+5, v/v). The precision of the method, expressed by the coefficient of variation, was <5% for the analytes when triplicate injections were used. The average recoveries ranged between 95 and 103% for standards that were 50 to 200% of the target analyte level. The LOQ in the injection solution was determined to be between 1 and 6 ng/mL for the analytes. The method is valid for the separation and quantification of trans-lutein, (3R,3'R)-trans-zeaxanthin, and (3R,3'S)-trans-zeaxanthin.