Carotenoids standards preparation

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. 

It was stated that the method is relatively inexpensive, suitable for the preparation of carotenoid standards and provides a high sample throughput [44],... 

As pointed out above (see Basic Protocol 1), when preparing carotenoid standard solutions, complete dissolution of the solid material is essential however, it is always advisable to filter the solution through a solvent compatible filter. 

For normal-phase separations, solutions should be prepared using hexane. Crystalline carotenoid standards are available from Sigma, Indofine Chemical, Atomergic Chemetals, Huka Chemical, Kemin Industries, Wako, and others. 

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). 

The time taken to carry out the procedures outlined above will depend on the knowledge and experience of the analyst and their familiarity with the equipment however, once familiar with the procedure, the preparation and calibration of a standard solution of a carotenoid (see Basic Protocol 1) should not take >1 hr. 

Several other carotenoids have been synthesized by standard routes involving condensation of Wittig salts with the Cio and C20 dials (46) and (47). Thus the Wittig salts (48), (49), and (50) were used to prepare the acyclic 1,2-dihydrocarotenes... 

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 retinaldehydes (484) and (485), which carry a trifluoromethyl group at C-9, were obtained from the C15 acid (483) by a standard procedure (Asato et aL, 1982). The 9 acid (483) (carotenoid numbering scheme) was formed when the pyrone (482) was subjected to a stereospecific ring-opening reaction. Tyrone (482) was prepared from p-cyclocitral (480) by a Reformatsky reaction, using the C5 unit (481). 

Separation of carotenoids on magnesia layers may serve as a useful supplement to the more commonly used silica gel systems. Used alone, however, magnesia layers can easily lead to confusion, especially in the case of the xanthophylls. Relatively sharp bands are obtained, but the Ry-values may vary and the tabulated values should therefore be used only as a rough guideline. It is advisable to prepare standards from established sources with a more consistent system before testing the properties of the magnesia layers. 

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. ... 

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