Carotenoids sample preparation

In order to obtain reliable results, three steps are involved in the analysis sampling and sample preparation carotenoid extraction, separation, identification. 

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. 

This chapter reviews recent findings about the health benefits of phytochemicals present in fruits, vegetables, nuts, seeds, and herbs, including phenolics, carotenoids, sterols, and alkaloids. These phytochemicals are extracted using emerging technologies such as supercritical carbon dioxide (SC-CO2) extraction, PEF, MWE, HPP, UE, and OH. The impact of important parameters related to sample preparation (particle size and moisture content) and extraction process (temperature, pressure, solvent flow rate, extraction time, and the use of a cosolvent) on the efficiency of extraction and on the characteristics of the extracted products is evaluated based on an extensive review of recent literature. The future of extraction of phytochemicals is certainly bright with the... 

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). 

Describes sample preparation and matrix requirements for analyzing carotenoids using MALDI-TOF MS and discusses the use of postsource decay for obtaining structurally significant fragment ions of carotenoids during MALDI-TOF MS. 

Keywords Carotenoids analysis sample preparation UV/Visible spectroscopy geometrical isomers optical isomers HPLC mass spectrometry nuclear magnetic resonance metabolites. 

If history is any guide, then we foresee great potential for growth in the field of carotenoid analysis in the coming years. Sample preparation methods that quickly and effectively break down and remove sample matrix while preserving carotenoids intact will improve the accuracy of both parent carotenoid and carotenoid metabolite identification and quantitation. 

Finally, the field of metabolomics is still in its early stages of development. With advances in all the areas listed above (sample preparation, HPLC analysis, and MS analysis), the use of metabolomics to analyze metabolic pathways involving carotenoids may evolve from a simple targeted approach to a more sophisticated fingerprinting approach. 

Vitamin activity in foods and food products serves as an example where typical problems arise with traditional liquid solvent extraction and then where SFE has been used to address these concerns [30]. In addition to the routine assay of food products, there is a considerable amount of research being conducted on the role of carotenoids and xanthans as antioxidants in the human body. This antioxidant-role may address many health concerns such as aging and various diseases. The "friendly-extracting-environment" of SFE has some merit of consideration for such studies, particularly with regard to the lesser possibility of oxidation of the analytes during the sample preparation 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. 

Some natural complex matrices do not need sample preparation prior to GC analysis, for example, essential oils. The latter generally contain only volatile components, since their preparation is performed by SD. Citrus oils, extracted by cold-pressing machines, are an exception, containing more than 200 volatile and nonvolatile components. The volatile fraction represents 90-99% of the entire oil, and is represented by mono- and sesquiterpene hydrocarbons and their oxygenated derivatives, along with aliphatic aldehydes, alcohols, and esters the nonvolatile fraction, constituting 1-10% of the oil, is represented mainly by hydrocarbons, fatty acids, sterols, carotenoids, waxes, and oxygen heterocyclic compounds (coumarins, psoralens, and polymethoxylated flavones—PMFs) [92]. 

Inject 10-50 pi sample (see Sect. Sample Preparation ) dissolved in a solvent miscible with the mobile phase (e.g., ethanol, 90% ethanol/10% isopropanol). Note The complete separation from lutein to lycopene requires 40 min with an additional 15 min to equilibrate the colunm back to the initial mobile phase. Figure 111.7 illustrates the separation of carotenoid in the nuxed natural products extract using this LC system. The elution order using this method difers from many other methods lutein, zeaxanthin, (l-cryptoxanthin. 

The simultaneous determination of fat-soluble vitamins is possible, thus, simultaneous analysis of retinol, tocopherols, as well as other carotenoids present in milk, among other products, can be achieved by ultra-performance liquid chromatography (UPLC) with detection at 325, 292, and 450 nm, respectively. Another possibility is to select a compromise wavelength, in which the overall sensitivity is acceptable. For instance, 265 nm can be employed for the detection of vitamins E and D in foods using an RP method. In addition, the sample preparation method has to be carefully selected, given that several compounds are involved. In general, compromise conditions should be selected to have a maximum... 

The use of a mass spectrometer as chromatographic detector offers a great advantage in vitamin analysis the possibility of simplifying the extraction procedure. The selectivity of the LC—MS technique reduces problems due to intrusive peaks from matrix components, while its sensitivity (ng or pg injected for real samples) allows the direct injection of an extract, eliminating the concentration step and the exposure to heat (most water-soluble vitamins posses low thermal stability). Sample preparation time is reduced as well as the duration of exposition to air and light (most vitamins and carotenoids are susceptible to these factors). 

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