Carotenoids HPLC

Figure 10.2 Carotenoid HPLC profile of pooled extract from liver of mice supplemented with multicarotenold mixture for 24 weeks HPLC conditions described in text.

More specific methods involve chromatographic separation of the retinoids and carotenoids followed by an appropriate detection method. This subject has been reviewed (57). Typically, hplc techniques are used and are coupled with detection by uv. For the retinoids, fluorescent detection is possible and picogram quantities of retinol in plasma have been measured (58—62). These techniques are particularly powerful for the separation of isomers. Owing to the thermal lability of these compounds, gc methods have also been used but to a lesser extent. Recently, the utiUty of cool-on-column injection methods for these materials has been demonstrated (63). 

HART J D and SCOTT K J (1995) Development and evaluation of an HPLC method for the analysis of carotenoids in foods and the measurement of carotenoid content of vegetables and fruits commonly consumed in the UK. Food Chem. 54(1) 101-111. 

Muller, H., Determination of the carotenoid content in selected vegetables and fruit by HPLC and photodiode array detection, Z. Lebensm. Enters. Forsch. A, 204, 88, 1997. 

Nyambaka, H. and Ryley, J., An isocratic reversed-phase HPLC separation of the stereoisomers of the provitamin A carotenoids (a- and (3-carotene) in dark green vegetables, Food Chem., 55, 63, 1996. 

Gama, J.J.T. and Sylos, C.M., Major carotenoid composition of Brazilian Valencia orange juice identification and quantification by HPLC, Food Res. Int, 38, 899, 2005. 

Guyomarc h, R, Binet, A., and Dufosse, L., Production of carotenoids by Brevibac-terium linens variation among strains, kinetic aspects and HPLC profiles, J. Ind. Microbiol. BiotechnoL, 24, 64, 2000. 

High performance liquid chromatography (HPLC) has been by far the most important method for separating chlorophylls. Open column chromatography and thin layer chromatography are still used for clean-up procedures to isolate and separate carotenoids and other lipids from chlorophylls and for preparative applications, but both are losing importance for analytical purposes due to their low resolution and have been replaced by more effective techniques like solid phase, supercritical fluid extraction and counter current chromatography. The whole analysis should be as brief as possible, since each additional step is a potential source of epimers and allomers. 

Many other authors, as reviewed extensively by Schwartz and Lorenzo, and by Eder improved the C18 RP-HPLC methods that have been largely applied using similar but not exactly identical systems to separate and to quantify complex mixtures of chlorophylls and carotenoids. 

Although saponification was found to be unnecessary for the separation and quantification of carotenoids from leafy vegetables by high performance liquid chromatography (HPLC) or open column chromatography (OCC), saponification is usually employed to clean the extract when subsequent purification steps are required such as for nuclear magnetic resonance (NMR) spectroscopy and production of standards from natural sources. 

A variety of mobile phases have been employed for carotenoid separation by reversed phase HPLC. Most are based on MeOH or acetonitrile, with the addition of CH2CI2, THF, methyl-tert-butyl ether (MTBE), acetone, or EtOAc. In general, recoveries of carotenoids are higher with methanol-based systems compared to acetonitrile-based ones." ... 

Although some normal phase methods have been used, the majority of carotenoid separations reported in the literature were carried out by reversed phase HPLC. Among the Cjg columns employed for determination of complete carotenoid compositions in foods, the polymeric Vydac brand is preferably used for separation of cis isomers. Several examples of different C,g columns and mobile phases are cited in the literature, but not aU carotenoids are baseline separated in most systems. Table 6.2.1 shows some examples employing different brands of Cjg columns." Acetonitrile did not improve selectivity toward separation of carotene isomers in a Vydac 201TP column and resolution was strongly dependent on the Vydac column lot. ... 

HPLC Systems Employing Reversed Phase C,8 Columns for Separation of Carotenoids... 

Temperature has an influence on the retention and consequently on the capacity factors of carotenoids in HPLC columns. Usually, as the column temperature increases, the retention decreases however, in a polymeric C30 column, after an initial decrease of the t values of cis isomers of carotenoids, the retention of cis isomers actually increases at temperatures above 35°C. This different behavior can be explained by the increased order and rigidity of the C30 stationary phase at lower temperatures that in turn induce preferential retention of long, narrow solutes as the trans isomer and partial exclusion of bent and bulky cis isomers. The greater chain mobihty and less rigid conformation of the C30 at higher temperatures may increase the contact area available for interaction with the cis isomers and also may lower... 

Capillary electrophoresis (CE) has several unique advantages compared to HPLC, snch as higher efficiency dne to non-parabolic fronting, shorter analytical time, prodnction of no or much smaller amounts of organic solvents, and lower cost for capillary zone electrophoresis (CZE) and fused-silica capillary techniques. However, in CZE, the most popular separation mode for CE, the analytes are separated on the basis of differences in charge and molecular sizes, and therefore neutral compounds snch as carotenoids do not migrate and all co-elute with the electro-osmotic flow. 

Solvent — The transition energy responsible for the main absorption band is dependent on the refractive index of the solvent, the transition energy being lower as the refractive index of the solvent increases. In other words, the values are similar in petroleum ether, hexane, and diethyl ether and much higher in benzene, toluene, and chlorinated solvents. Therefore, for comparison of the UV-Vis spectrum features, the same solvent should be used to obtain all carotenoid data. In addition, because of this solvent effect, special care should be taken when information about a chromophore is taken from a UV-Vis spectrum measured online by a PDA detector during HPLC analysis. 

For HPLC, it is necessary to establish the relationship between the detector signal, of which the most used is peak area, and the concentrations of the pigments. Calibration curves for external quantification should be constructed for each carotenoid. Internal calibration is also used for quantification of carotenoids, using as internal standards all-trfln5 -p-apo-8-carotenal, ° Sudan 1, and decapreno-P-carotene. ... 

The accuracy and precision of carotenoid quantification by HPLC depend on the standard purity and measurement of the peak areas thus quantification of overlapping peaks can cause high variation of peak areas. In addition, preparation and dilution of standard and sample solutions are among the main causes of error in quantitative analysis. For example, the absorbance levels at of lutein in concentrations up to 10 mM have a linear relationship between concentration and absorbance in hexane and MeOH on the other hand, the absorbance of P-carotene in hexane increased linearly with increasing concentration, whereas in MeOH, its absorbance increased linearly up to 5 mM but non-linearly at increasingly higher concentrations. In other words, when a stock solution of carotenoids is prepared, care should be taken to ensure that the compounds are fuUy soluble at the desired concentrations in a particular solvent. 

Breithanpt, D.E., Simultaneons HPLC determination of carotenoids nsed as food coloring additives applicability of accelerated solvent extraction. Food Chem., 86, 449, 2004. 

Mercadante A.Z., Rodriguez-Amaya, D.B., and Britton, G., HPLC and mass spec-trometric analysis of carotenoids from mango, J. Agric. Food Chem., 45, 120, 1997. 

Khachik, F. and Beecher, G.R., Separation and identification of carotenoids and carotenol fatty acid esters in some squash products by liquid chromatography. 1. Quantification of carotenoids and related esters by HPLC, J. Agric. Food Chem., 36, 929, 1988. 

Scott, K. et al.. Interlaboratory studies of HPLC procedures for the analysis of carotenoids in foods. Food Chem., 57, 85, 1996. 

Minguez-Mosquera, M.I. and FIomero-Mendez, D., Separation and quantification of the carotenoid pigments in red peppers, paprika and oleoresin by reversed phase HPLC, J. Agric. Food Chem., 41, 1616, 1993. 

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