Carotenoids analytical techniques

These types of technique have been employed to identify a number of carotenoids in multiple types of samples. Recently GC-MS and authentic standards were used to identify volatile carotenoid metabolites from plant tissues (Vogel et ah, 2008) and numerous studies have identified P-carotene metabolites in animals and humans using a variety of analytical techniques (Hu et ah, 2006 Ho et ah, 2007). These techniques have also been used to identify lycopene metabolites in both foods and biological samples (Khachik et ah, 1997 Bouvier et ah, 2003 Kopec et ah, 2010) and the metabolism of lutein, zeaxanthin, and P-cryptox-anthin (Bernstein et ah, 2001 Prasain et ah, 2005 Mein et ah, 2011). 

A practical way of extracting, isolating, and purifying carotenoids from plant materials is described in this chapter. The method is based mainly on the natural form in which carotenoids are found (esterified or free) and to some extent on their polarity and/or solubility in the solvents used. Common and readily available solvents and laboratory equipment are suggested. Preparation and determination of carotenoids are described. Moreover, an advanced extraction and analytical technique of carotenoids is also explained. 

CO2 extraction has been prevalent for the isolation of essential oils and other natural lipophilic pigments like carotenoids. Hot water and superheated water extraction methods are used for analytical preparation of polar pigments. The technique is commonly referred to as subcritical water extraction because the practitioners of this approach come from SEE backgrounds. 

High performance liquid chromatography (HPLC) has been by far the most important method for separating chlorophylls. Open column chromatography and thin layer chromatography are still used for clean-up procedures to isolate and separate carotenoids and other lipids from chlorophylls and for preparative applications, but both are losing importance for analytical purposes due to their low resolution and have been replaced by more effective techniques like solid phase, supercritical fluid extraction and counter current chromatography. The whole analysis should be as brief as possible, since each additional step is a potential source of epimers and allomers. 

Capillary electrophoresis (CE) has several unique advantages compared to HPLC, snch as higher efficiency dne to non-parabolic fronting, shorter analytical time, prodnction of no or much smaller amounts of organic solvents, and lower cost for capillary zone electrophoresis (CZE) and fused-silica capillary techniques. However, in CZE, the most popular separation mode for CE, the analytes are separated on the basis of differences in charge and molecular sizes, and therefore neutral compounds snch as carotenoids do not migrate and all co-elute with the electro-osmotic flow. 

Eor the majority of foods, especially those containing high levels of chlorophyll, carotenoids, waxes or fats, a cleanup technique is usually used to minimize contamination of the analytical instruments, especially for GC. There are a number of cleanup techniques that can be employed based on partition, adsorption, ion exchange and size exclusion. 

Natural colours include annatto, anthocyanins, beetroot red (betalaines), caramel, carotenoids, cochineal and lac pigments, flavanoids, chlorophylls and tumeric. There is a trend towards encapsulating natural colours for food use, but this is not yet reflected in the extraction techniques described in the published analytical methods. Lancaster and Lawrence (1996) described the extraction and... 

Earlier methods of ionization applied to carotenoids, including electron impact (El), chemical ionization (Cl), a particle beam interface with El or Cl, and continuous-flow fast atom bombardment (CF-FAB), have been comprehensively reviewed elsewhere (van Breemen, 1996, 1997 Pajkovic and van Breemen, 2005). These techniques have generally been replaced by softer ionization techniques like electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), and more recently atmospheric pressure photoionization (APPI). It should be noted that ESI, APCI, and APPI can be used as ionization methods with a direct infusion of an analyte in solution (i.e. not interfaced with an HPLC system), or as the interface between the HPEC and the MS. In contrast, matrix-assisted laser desorption ionization (MALDI) cannot be used directly with HPEC. 

In the APPI method of ionization, the solvent is vaporized in a heated nebulizer and the gaseous analytes are then ionized with photons from a lamp (Rivera et ah, 2011). It has been observed that certain solvents, called dopants, enhance the ionization of analytes via this technique. To date only one study has been published with carotenoids and APPI (Rivera et ah, 2011). APPI was compared to ESI and APCI as ionization techniques, and the authors observed that APPI positive produced approximately a 2- to 4-fold greater total ion signal for lycopene and (3-carotene as compared to APCI positive and ESI positive. In contrast, APCI positive outperformed APPI positive for a number of xantho-phylls and phytoene and phytofluene. 

Craft, N.E. 2001. Chromatographic techniques for carotenoid separation. In Current Protocols in Food Analytical Chemistry (Wrolstad, R.E., Ed.). John Wiley Sons Inc., New York, pp. F2.3.1-F2.3.12. 

SEE has so far been the technique most frequently used to validate DPSE methods such as those for the extraction of dioxins from high- and low-carbon fly ash [48], triazolo-pyrimidine sulphonanilide herbicides, trichloropyridinol and PCBs from soil [150,152, 168], and carotenoids and tocopherol from spice red pepper [177]. As noted earlier, neither technique can be said to be better than the other it depends on the characteristics of the analytes to be extracted (e.g. on their polarity and high-temperature stability). Thus, in the extraction of cloransulam-methyl from soil, while the use of subcritical water provided higher recoveries than SEE, the analyte was not hydrolytically stable above 150°C, which entailed using a lower temperature and hence an increased extraction time [152]. In the extraction of PAHs from bituminous coal fly ash [180], extraction with supercritical CO, yielded better recoveries than DPHSE using toluene and methylene... 

Time-resobed resonance Raman spectrometry is a technique that allows collection of Raman spectra of excited state molecules. It has heen used lo study in-lermediaies in enzyme reactions, the spectra of carotenoid excited slates, ultrafasi electron transfer steps, and a variety of olher biological and bioinoiganic processes, I inie discrimination methods have been used to overcome a major limitation of resonance Raman spectroscope, namelv. fluorescence interference either by the analyte itself or by other species present in the sample. 

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. 

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