Extraction technique carotenoids analysis

On the other hand, quantitative extraction requires complete and exhaustive extraction and no material can be lost. To assure complete extraction when a food is analyzed for the first time in a laboratory, it is useful to carry out two or three extractions, pool the solvents, and keep separate the next extracts to verify the presence of carotenoids. Usually four to six extractions are enough to remove the carotenoids completely from a sample. The extraction can be carried out in a blender, vortex, or with a mortar and pestle. Accelerated solvent extraction (ASE), an important extraction technique in residue analysis, currently attracts interest due to its short duration, low level of solvent use, and high extraction yield. The average recoveries for all carotenoids with the exception of norbixin ranged from 88.7 to 103.3% using manual extraction and from 91.0 to 99.6% by ASE (70 bar and temperature of 40°C) both extractions were carried out with a mixture of MeOH, EtOAc, and petroleum ether (1 1 1). ... 

Hyphenation of Modern Extraction Techniques to LC-NMR for the Analysis of Geometrical Carotenoid Isomers in Functional Food and Biological Tissues... 

Matrix solid-phase dispersion (MSPD) is the extraction method of choice for the analysis of solid samples, such as plant material, foodstuffs or tissue samples [26]. This method has been developed especially for solid or viscous matrices. MSPD is preferable to other extraction techniques, because the solid or viscous sample can be directly mixed with the sorbent material of the stationary phase [27]. As the carotenoid stereoisomers stay bound in their biological matrix until the elution step, they are protected against isomerisation and oxidation [28]. The extraction scheme of MSPD is shown in Figure 5.2.1. 

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preparative applications, but both are losing importance for analytical purposes due to their low resolution and have been replaced by more effective techniques like solid phase, supercritical fluid extraction and counter current chromatography.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. 

The first comprehensive 2D system was developed in the late 1970s by Erni and Frei, who applied IEX x RPC to the analysis of senna glycosides from plant extracts.61 In the subsequent decades, comprehensive MD-HPLC methods have been further developed, mainly for peptides and proteins,62 3 but also for separation of various natural products such as phenolic and flavone antioxidants64 and carotenoids.65 The theoretical aspects of MD-HPLC techniques have also been further developed.66-68... 

The solvents used for the extraction and analysis of carotenoids should be carefully purified before use to remove impurities. In most cases this means redistillation of the solvent. In the case of diethyl ether, the removal of peroxides is also important. Petroleum ether used for the extraction and chromatography of carotenoids should be passed through silica gel to remove sulfur compounds and unsaturated hydrocarbons. The methodology used in the isolation and identification of carotenoids has been covered in a recent review (Davies, (1976). A brief introduction to these techniques is presented below. 

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). 

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. 

Since the carotenoids display similar absorption spectra it is recommended to identify these compounds after HPLC separation using a mass spectrometer or a NMR detector. Structural and geometrical isomers or epoxides were distinguished by this technique (Aman et al., 2005 Matsubara et al., 2012). An accelerated solvent extraction step in hexane-based solvent mixtures was successfully used prior to LC-ESl MS analysis of lutein and P-carotene from orange carrot (Saha et al., 2015). 

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