Carotenoid analysis quantification

Alkaline hydrolysis (saponification) has been used to remove contaminating lipids from fat-rich samples (e.g., pahn oil) and hydrolyze chlorophyll (e.g., green vegetables) and carotenoid esters (e.g., fruits). Xanthophylls, both free and with different degrees of esterification with a mixture of different fatty acids, are typically found in fruits, and saponification allows easier chromatographic separation, identification, and quantification. For this reason, most methods for quantitative carotenoid analysis include a saponification step. 

The only technique that is of major interest for carotenoid analysis is LC. Selected applications to biological samples are presented in Table 2. These methods are mostly satisfactory in terms of selectivity and sensitivity. However, accurate quantification requires precautions because of relative lability of the analytes during sample preparation, their incomplete recovery from LC columns, and the limitations of the available internal standards. 

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. 

Mass spectrometry data is often paired with UV-Visible spectra, NMR, or HPLC retention time for carotenoid identification. In addition, HPLC coupled with MS as a detector has been reported to be 100 times more sensitive than PDA for detection and quantification of some carotenoids (van Breemen, 1995 van Breemen et ah, 1996). While mass spectrometry can be a powerful tool, it should be noted that the analysis of carotenoids (which are nonvolatile, thermally labile, and inherently unstable) presents a special challenge to the mass spectrometry analyst. An overview of common MS components and techniques is provided in Chapter 2 (techniques not previously mentioned are briefly described below). 

The detection and quantification of tocopherols, carotenoids, and chlorophylls in vegetable oil were effectively used for authentication pnrposes. The presence of tocopherols, carotenoids, and chlorophylls influence the oxidative stability of vegetable oils and their potential health benefits. Puspitasari-Nienaber et demonstrated the application of a rapid and reliable analysis method of direct injection of C-30 RP-NPLC with electrochemical detection for the simultaneous analysis of the above mentioned substances. Aliquots of vegetable oils were dissolved in appropriate solvents and injected directly without saponification, thus preventing sample loss or component degradation. Thus the effective separation of tocopherols, carotenoids, and chlorophylls was achieved. 

The continuous advances in instrumental techniques for organic compound analysis enable us to be rigorous in the analysis of carotenoid pigments. The following sections describe the main stages in the procedures of extraction, isolation, identification, and quantification of carotenoid pigments in foods of plant and animal origin. 

To avoid quantification errors caused by the multiple manipulations of the sample during the various steps of extraction and preparation, the use of an internal standard (IS) in combination with the external calibration is advisable. The IS must be chosen carefully, as it has to meet a series of minimum requirements. It must be a carotenoid pigment not present in the sample to be analyzed, it must be chromato-graphically separable from the others under the analytical conditions used, it must have a A ax absorption as close as possible to the A of detection employed, and it must be as stable as possible. Various ISs have been proposed (3-apo-8 -carotenal and canthaxanthin are commonly used in the analysis of vegetable foods.The use of artificial colorants, such as Congo red and Sudan 1, and of synthetic carotenoids not present in natural samples, such as C45-(3-carotene, has also been proposed. ... 

Detection is normally by absorption or fluorescence spectrophotometry. The high coefficients of extinction of the chlorophyll Soret band enable sensitive detection between 380 and 445 nm. This region of the spectmm also includes the carotenoids, which accompany the chlorophylls in plant pigment extracts and whose analysis and quantification are also usually of interest (see Chapter 6). When these compounds are not of interest, and their possible interference must be excluded, a selective detection of chlorophylls and derivatives can be carried out at 654 [136] or 667 nm [230], where there is no absorption of these pigments. Detection... 

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