Xanthophylls HPLC

FIGURE 7.1 (a) Structures of the five most common xanthophylls. (b) HPLC separation profile of the... 

The horizontal distribution (Figure 13.4, top) of the macular xanthophylls across the retina has been studied in detail by measuring concentrations in postmortem eyes via HPLC (Bone et al. 1997). The macular xanthophylls are detectable across the entire retina but have their highest concentration in the center of the macula. The local zeaxanthin to lutein ratio depends on the distance from the fovea and decreases from about 2 1 at its center to a low of near 1 2 in the peripheral retina. The variation in the zeaxanthin/lutein ratio across the retina suggests that the chemical and biochemical influences operating on the xanthophylls in the peripheral retina are different from those in the central macula. This is an area about which not much is known and would constitute an interesting field of research. 

The HPLC analysis of milkweed, the food-plant source for Monarch butterflies, demonstrates that it contains a complex mixture of carotenoids including lutein, several other xanthophylls, xanthophyll epoxides, and (3-carotene, Figure 25.3b. There is a component in the leaf extract that is observed to elute near 8min, which has a typical carotenoid spectrum but is not identical to that of the lutein metabolite observed at near the same retention time in the extracts from larval tissue. 

The composition and amount of pigments in marine environments have also been vigorously investigated. Thus, on RP-HPLC method has been developed for the study of the effect of variable irradiance on the xanthophyll cycle of the seagrass Zostera marina. Extraction of pigments from seagrass was carried out by grinding the samples with acid-washed sand in the presence of 1 ml of 90 per cent acetone and the liquid phase was... 

Lyophilized and dry samples should be reconstituted with water prior to extraction. They may require saponification if the samples contain chlorophylls or xanthophyll esters. Beadlet materials should be suspended in hot water, saponified, or enzymatically hydrolyzed before extraction. Follow the manufacturer s recommendations before analyzing by HPLC. 

In general, RP-HPLC has been applied successfully to the qualitative and quantitative estimation of carotenes (64), xanthophylls, cis- and trans-carotenoids (54,60), and carotenoid fatty acid esters (53,65). Figure 5 shows an HPLC separation of (A) paprika extract, containing carotenoid esters, and (B) saponified paprika extract, containing the corresponding carotenoids (74a). 

Van Berkel and Zhou first tested (3-carotene with ESI positive in 1994 (van Berkel and Zhou, 1994). In this study, a doubly charged molecular ion of (3-carotene was observed as the primary species when triflur-oacetic acid was present in the solution. Van Breemen was the first to utilize ESI as an interface between HPLC and MS to analyze carotenoids (van Breemen, 1995). In this study, ESI operated in negative mode ionized xanthophylls (astaxanthin, (3-cryptoxanthin, and lutein), but did not ionize hydrocarbon carotenes (lycopene and (3-carotene). In contrast, ESI positive produced only [M" ] for all carotenoids in this study, and the addition of halogenated solvents to the post-column effluent greatly enhanced signal intensity (van Breemen, 1995). A later study by Guarantini et al. demonstrated the ability of ESI positive to produce both [M" "] and [M + H]" " for a number of xanthophylls, and these authors attributed the production of the two species to solvent system... 

All lutl alleles are recessive mutations (Fig. 3 A) that accumulate an additional carotenoid with an HPLC retention time and UV/visible absorption spectrum characteristic of a monohydroxy / , e-carotenoid (an a-carotene derived xanthophyll). The identification of the accumulating compound as zeinoxanthin ()3,e-caroten-3-ol) was confirmed by mass spectrometry (Pogson et al., 1996). The only difference between lutein and zeinoxanthin is the presence of an hydroxyl group on carbon 3 of the -ring in lutein. The decrease in lutein and its partial replacement by its immediate precursor, zeinoxanthin, defines lutl as a mutation... 

Thayer SS and Bjorkman O (1990) Leaf xanthophyll content and composition in sun and shade determined by HPLC. Photosynth... 

The separation of photosynthetic pigments by HPLC was obtained by using the system described in material and methods which permitted the clear resolution of zeaxanthin and lutein (Fig. 1). This method allowed to study the changes produced in photosynthetic pigments, and in particular, those of the xanthophylls involved in the violaxanthin cycle. 

A developing area of analytical development in HPLC is the determination of several groups of minor food components, like flavonoids and polyphenols in general, xanthophylls, and other carotenoids. Most of them have important bioactivities and have stimulated much interest in food and nutrition studies. 

Dissolve 1-2 mg lutein, zeaxanthin, p-cryptoxanthin, and other xanthophylls directly in 100 ml reagent alcohol containing 30 ppm butylated hydroxytoluene (BHT). Dissolve l-2 mg lycopene, a-carotene, and p-carotene in 10 ml HPLC grade THF stabilized with 250 ppm BHT and then dilute to 100 ml with reagent... 

Moros EE, Damoko D, Cheryan M, Perkins EG, Jerrell J (2002) Analysis of xanthophylls in com by HPLC. J Agric Food Chem 50 5787... 

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