Standards carotenoid

The majority of carotenoids exhibit absorption in the visible region of the spectrum, between 400 and 500 nm. Because they obey the Beer-Lambert law (i.e., absorbance is linearly proportional to the concentration), absorbance measurements can be used to quantify the concentration of a pure (standard) carotenoid (see Basic Protocol 1) or to estimate the total carotenoid concentration in a mixture or extract of carotenoids in a sample (see Basic Protocol 2). Considerations for the preparation of carotenoid-containing samples are presented in Critical Parameters (see Sampling and Sample Preparation). 

In this protocol, commercially purchased carotenoid standards are dissolved in a suitable solvent and the absorbance measured at its maximum wavelength (A.max). Using published extinction coefficients and taking into consideration the dilution factor, the concentration of the standard carotenoid is calculated. The spectrum is also scanned in order to evaluate the fine structure (see Spectral Fine Structure in Background Information). The carotenoid solution should ideally be assayed by HPLC as described in unit F2.3 to establish chromatographic purity and thus correct the calculated concentration. 

In certain instances, degradation can be rapid, thus it is essential that the chances of it occurring be lessened or avoided completely. This is particularly important in the case of standard carotenoid solutions. It has been reported that lycopene in particular can be rapidly... 

In milk approximately 90% of the yellow color is because of the presence of -carotene, a fat-soluble carotenoid extracted from feed by cows. Summer milk is more yellow than winter milk because cows grazing on lush green pastures in the spring and summer months consume much higher levels of carotenoids than do cows ham-fed on hay and grain in the fall and winter. Various breeds of cows and even individual animals differ in the efficiency with which they extract -carotene from feed and in the degree to which they convert it into colorless vitamin A. The differences in the color of milk are more obvious in products made from milk fat, since here the yellow color is concentrated. Thus, unless standardized through the addition of colorant, products like butter and cheese show a wide variation in shade and in many cases appear unsatisfactory to the consumer. 

The ready availability of carotenoid oxidation products through chemical methods will facilitate their use as standard identification tools in complex media such as biological fluids, and enable in vitro investigation of their biological activity. Moreover, these studies can help reveal the mechanisms by which they can be chemically or biochemically cleaved in vivo. 

Traditionally, dried or powdered plant material is used and extracts can be obtained by mixing the material with food-grade solvents like dichloromethane or acetone followed by washing, concentration, and solvent removal. The result is an oily product that may contain variable amounts of pheophytins and other chlorophyll degradation compounds usually accompanied by lipid-soluble substances like carotenoids (mainly lutein), carotenes, fats, waxes, and phospholipids, depending on the raw material and extraction techniques employed. This product is usually marketed as pheophytin after standardization with vegetable oils. 

Lipid-soluble food grade copper chlorophyll is manufactured similarly by extraction of adequate plant material, followed by replacement of magnesium by copper, and purihcation steps to remove carotenoids, waxes, sterols, oils, and other minor components that are co-extracted. Commercial copper chlorophylls may vary physically, ranging from viscous resins to fluid dilutions in edible oils as well as granulated forms and emulsions standardized with edible vegetable oil. Colors may vary... 

The National Institute of Standards and Technology (NIST) released certified standard materials of a baby food composite (SRM 2383) and an infant formula (SRM 1846) containing carotenoids however, the relative uncertainties of certified values are considerably high, ranging Irom 20% for P-carotene (cis + trans) to 28% for lutein (including esters) and to a 47% for free lutein reference value in SRM 2383. ... 

An alternative for evaluating accuracy is spiking known amounts of standards to a food, as reported in several papers,although percent recoveries of spikes do not truly address the influence of the food matrix complexity on the extraction efficiency. Data evaluation procedures were developed as a manual system to assess the quality of analytical data for carotenoids in foods. ... 

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. 

However, complete hydrolysis of carotenoid esters sometimes is not achieved in 1 to 3 hr. The saponification degree can be verified easily by the presence of carotenol ester peaks eluting later than the peaks of P-carotene on reversed phase columns. Retinol palmitate, added as an internal standard to orange juice, also serves to indicate whether saponification is complete, since it is converted to retinol which elutes at lower retention time. The mixture is subsequently washed with water until free of alkali in a separatory funnel. Other more polar solvents such as CH2CI2 or EtOAc, and diethyl ether alone or mixtured with petroleum ether can be used to increase the recovery of polar xanthophylls from the water phase. 

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. ... 

One point of discussion is the number of necessary concentration points to construct calibration curves. Statistics demonstrated that it is not necessary to use more than six points. Because of the difficulty of handling carotenoid standards, a minimum coefhcient of correlation of 0.9 was suggested by Khachik et al.while Mantoura and Repeta recommended a coefficient above 0.95. The curves should intercept close to the zero value. 

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. 

Traditionally, carotenoid standards are prepared in each laboratory using the best sources of each individual carotenoid, for example, violaxanthin from spinach, antheraxanthin from potatoes, capsanthin and capsorubin from paprika, a- and P-carotene from carrots, and lycopene from tomatoes. 

Sharpless, K.E. et al., Valne assignment of retinol, retinyl palmitate, tocopherol, and carotenoid concentrations in standard reference material 2383 (baby food composite), J. AOAC Int., 82, 288, 1999. 

Detailed information about carotenoids found in food or extracted from food and evaluated for their potential as food colorants appeared in Sections 4.2 and 6.2. We would like to mention some new data about the utilization of pure carotenoid molecules or extracts as allowed food additives. Looking to the list of E-coded natural colorants (Table 7.2.1), we can identify standardized colorants E160a through f, E 161a, and E161b as natural or semi-synthetic derivatives of carotenoids provided from carrots, annatto, tomatoes, paprika, and marigold. In addition, the extracts (powders or oleoresins) of saffron, - paprika, and marigold are considered more economical variants in the United States and European Union. 

For many decades, the standard technique for measuring carotenoids has been high-pressure liquid chromatography (HPLC). This time consuming and expensive chemical method works well for the measurement of carotenoids in serum, but it is difficult to perform in human tissue since it requires biopsies of relatively large tissue volumes. Additionally, serum antioxidant measurements are more indicative of short-term dietary intakes of antioxidants rather than steady-state accumulations in body tissues exposed to external oxidative stress factors such as smoking and UV-light exposure. 

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