Zeaxanthin HPLC analysis

In the egg yolk, zeaxanthin and lutein appear to be the primary carotenoids responsible for yellow color (Smith and Perdue, 1966 Schiedt et al, 1985 Schaeffer et al, 1988). Schaeffer et al. (1988) utilized HPLC analysis to reveal over 20 carotenoid species in the yolks of hens fed typical layer diets. Hamilton et al (1990) recently reported that the oleoresin of red pepper could be supplemented to laying hens to yield egg yolks with increased redness and yellowness. The three major pigments isolated were trans-lutein, trans-zeaxanthin and trans-capsanthin. The authors noted that the incorporation of small amounts of reddish capsanthin is advantageous for intensifying the yellow color of yolks. 

Aman, R. et al.. Application of HPLC coupled with DAD, APcI-MS and NMR to the analysis of lutein and zeaxanthin stereoisomers in thermally processed vegetables. Food Chem., 92 753, 2005. 

Both MS and NMR coupling to HPLC have been employed for the analysis of p-carotene isomers and determination of lutein and zeaxanthin isomers in spinach, sweet com, and in retina. Capillary high performance hquid chromatography with stop flow connected to NMR (600 MHz) was used for stracture elucidation of all-trans deoxylutein 11 and its isomers.Efforts are in progress to eliminate the remaining major drawbacks such as obligatory use of deuterated solvents in the mobile phase, poor sensitivity, and low throughput of HPLC-NMR analyses. 

Another study employed both TLC and HPLC for the analysis of carotenoids of Calendula officinalis L. TLC separation of all E(trans)-a,3-carotene, cryptoxanthin, zeaxanthin and lutein was performed on a silica layer using petroleum ether-j-propanol-CIICI, (90 10 70 v/v). The same carotenoid pigments were separated by RP-HPLC using an ODS column (250X4 mm, i.d.). The organic modifiers were methanol, THF and ethyl octane. The flow rate was 1 ml/min, pigments were detected at 440 nm [20],... 

Pigments with very similar structures might be difficult to separate using classic RP-C18 HPLC. This is the case of zeaxanthin and lutein. To overcome this difficulty, a particular HPLC method should be applied (Darko et al., 2000). Alternatively a capillary electrophoresis column can be used. This last method was successfully applied to the separation of zeaxanthin and lutein from eye humor (Karlsen et al., 2003). The good separation and the fast elution of the pigments obtained by these authors suggest that capillary electrophoresis is suitable for routine analysis of pigments contained in tiny samples. 

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