Carotenoids absorbance detection

Fig. 11 HPLC of carotenoids solvent-extracted from (A) raw and (B) thermally processed carrots. Column, 5-/um polymeric C1(J (250 X 4.6-mm ID) mobile phase, methyl tert-butyl ether/methanol (11 89), 1 ml/min absorbance detection, 453 nm. Tentative peak identifications (1) all-trans-lutein (2) 13-cis-a-carotene (3) a cis-a-carotene isomer (4) 13 -cA-a-carotene (5) 15-cis-/3-carotene (6) 13-cis-/3-carotene (7 and 8) cis-fi-carotene isomers (9) all-frans-a-carotene (10) 9-cis-a-carotene (11) all-frans-/3-carotene (12) 9-ci. -/3-carotene. (Reprinted with permission from Ref. 192. Copyright 1996, American Chemical Society.)...

For accurate determination of carotenoids, LC is the preferred technique and many, LC procedures have been published. Generally the sample is extracted into a solvent before being analyzed by reversed-phase LC with UV absorbance detection. These methods have the advantage of being able to detect and quantify individual carotenoids, allowing for more accurate calculation of vitamin A activity. 

The highest sensitivity and selectivity in vitamin E LC assays are obtained by using fluorescence or electrochemical detection. In the former, excitation at the low wavelength (205 nm) leads to improved detection limits but at the expense of selectivity, compared with the use of 295 nm. Electrochemical detection in the oxidation mode (amperometry or coulometry) is another factor 20 times more sensitive. In routine practice, however, most vitamin E assays employ the less sensitive absorbance detection at 292-295 nm (variable wavelength instrument) or 280 nm (fixed wavelength detectors). If retinol and carotenoids are included, a programmable multichannel detector, preferably a diode array instrument, is needed. As noted previously, combined LC assays for vitamins A, E, and carotenoids are now in common use for clinical chemistry and can measure about a dozen components within a 10 min run. The NIST and UK EQAS external quality assurance schemes permit interlaboratory comparisons of performance for these assays. 

The recognition of the importance of MP in maintaining the health of the retina has led to the development of a number of methods for determining its concentration in situ. These methods, necessarily noninvasive, are routinely employed in dietary supplementation studies with lutein or zeaxanthin to monitor the uptake of the carotenoids into the retina. Every method exploits the optical properties of lutein and zeaxanthin, specifically their absorbance at visible wavelengths. The detection of a light signal, modified by the carotenoids, is accomplished either by the retinal photoreceptors themselves (psychophysical methods) or by a physical detector such as a photomultiplier,... 

Fig. 2.23. Reversed-phase gradient HPLC profiles of carotenoids in human plasma. A human volunteer was given an oral dose of 5,6-epoxy-/l-carotene (9.1 /imol). Plasma was analysed for carotenoids before (a) and 6h after (b) the oral dose. Peak identification 1, bilirubin 2, lutein 3, zeaxanthin 4, /1-cryptoxanthin 5, 5,6-epoxy-/l-carotene 6, lycopene 7, /1-carotene. The detection wavelength was 445 nm. AU, absorbance unit. Reprinted with permission from A. B. Barua [50],...

P-carotene is only one of many antioxidants, which can be detected in the skin. Other carotenoids, for example, lutein and zeaxanthine, are preferentially found in the macula lutea, the so-called yellow spot in the eye. Here, carotenoids are subject to a metabolism typical for that tissue, which cannot be found in other tissues (e.g., formation of meso-zeaxanthine). In addition, they can specifically be absorbed into the macula. In the macula, they protect the retinal pigment epithelial cells against oxidative damage from UV light. Indeed, these two carotenoids can be protective against age-dependent macula degeneration. 

Radiation absorption monitoring of the column effluent at an appropriate wavelength provides the most versatile means of detection for the fat-soluble vitamins. Vitamins A, D, E, and K exhibit characteristic absorption spectra in the UV region, whereas the carotenoid pigments absorb light in the visible region. 

In the last century, it was already known from light microscopical studies that colored secondary products are located in distinct compartments of living cells, e.g., anthocyanins and anthraquinones in vacuoles and carotenoids in chromo-plasts. Since then the use of UV fluorescence microscopy or electron microscopy has led to the localization of many other secondary products (A 3). For the detection of compounds which do not directly absorb UV or electrons the following procedures have been applied ... 

More than 700 carotenoids have been described in nature, but it is estimated that we only have access to about 40 carotenoids that can be absorbed, metabolized, and/or used in our bodies. That number is reduced to 6 if we consider the carotenoid profile that is usually detected in human blood plasma a- and p-carotene, lycopene, p-cryptoxanthin, zeaxanthin, and lutein [21]. 

Lessin WJ, Catigani GL, Schwartz SJ (1997) Quantification of cis-trans isomers of provitamin A carotenoids in fresh and processed fruits and vegetables. J Agric Food Chem 45 3728 MacCrehan WA, Schonberger E (1987) Determination of retinol, a-tocopherol, and a-carotene in serum by liquid chromatography with absorbance and electrochemical detection. Clin Chem 33 1585... 

The eluted compounds can be detected either by photometric or fluorimetric detectors. In the former case, chlorophylls and carotenoids can be distinguished by a band in the region > 600 nm wavelength, where carotenoids do not absorb. However, the a band absorp-... 

Figure 12 Separation of carotenoids from Pelroselinum crispum. Solvent system light petroleum (40-.60 Q-r-butanol (80 20). Stationary phase silica gel 60 (0.25 nm, Merck). Developing distance 8.5 cm. Detection absorbance at 445 nm. Peak identities (1) p-carotene (2) lutein (3) violaxanthin (4) neoxanthin.

Newly absorbed carotenoid is most easily detected in the chylomicron or TRL plasma fraction, even in the absence of a whole-plasma response (Faulks etal. 1997) partly because this fraction is small and devoid of carotenoid in fasting volunteers, it is the most responsive pool and does not contain carotenoids sequestered and re-exported by the liver. The only disadvantage in this method is the necessity of obtaining fairly large plasma samples (5 ml) and the time taken to density adjust the plasma, ultracentrifugation and the quantitative recovery of the chylomicron fraction. A shorter and less demanding protocol for the isolation of chylomicrons has now been described (Borel et al. 1998). 

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