Saponification carotenoid analysis

To simplify carotenoids analysis, saponification procedures are employed before LC to release all the carotenoid esters as their respective free forms. However, it has been demonstrated that this saponification may change the native carotenoid composition, producing a loss of... 

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. 

In the field of carotenoids, most TLC-MS utilizations to date have been made offline and have used TLC merely for the purpose of purification or isolation of these pigments. Mass spectrometry was introduced into carotenoid analysis in 1965 [16]. In the 1970s, separations by open column chromatography on aluminum oxide were often combined with TLC separations on silica gel and MgO/Kieselguhr to achieve sufficient purification degree of carotenoids from tomato. These were analyzed afterward by direct-insertion electron impact-MS (EI-MS) [17-19]. Such isolation procedures, applied reactions (acetylation, saponification, and reduction), Rf values, absorption, and MS spectra enabled identification of phytoene 1,2-oxide, and related compounds as the first naturally occurring epoxides of acyclic carotenoids [17]. 

Saponification is often used to extract xanthophylls as well as remove chlorophylls and lipids from samples prior to analysis, as these compounds can interfere with the chromatographic detection. Although saponification with methanol and potassium hydroxide is routinely used to facilitate carotenoid extraction, numerous studies indicate that saponification can also result in losses of carotenoids. For example, Khachik et al.60 demonstrated that saponification actually caused the loss of total carotenoids in samples. Alternatively, enzymatic saponification using lipase can be used to help prevent the loss and isomerization of some carotenoids. Fang et al.32 suggested that saponification of plasma samples should be avoided to prevent unnecessary lycopene degradation. 

Granado, F. Ohnedilla, B. Gil-Martinez, E. Blanco, 1.2001. A fast, reliable and low-cost saponification protocol for analysis of carotenoids in vegetables. J. Food Comp. Anal. 14 479 89. 

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. 

Recent developments have been directed toward the simultaneous determination of multiple vitamins in foods. Separation of vitamins A, E, D2, and D3 in lacteal matrices has been performed using RP-LC with methanohwater as the mobile phase and UV detection at 265 nm (or at the wavelength of maximum absorption of each individual form), after exhaustive saponification. Normal-phase LC has also been used to analyze multiple fat-soluble vitamins in seed oils after extraction or even direct injection of the sample prior to analysis. UV detection has also been used. Sometimes a combination of two detection systems such as UV-visible detection and fluorescence have been used to, for example, determine, respectively, the carotenoids and fat-soluble vitamins in foods. 

Saponification of the extracts is generally desirable to remove unwanted lipid materials. However, this step is omitted in the isolation of carotenol esters, since these are hydrolyzed by this procedure. It is also omitted in the isolation of carotenoids such as fucoxanthin and peridinin, which are alkali-labile. If acetone has been used in the initial extraction, it is essential that all traces be removed before saponification. The general procedure used involves dissolving the total lipid fraction in an alcoholic (ethanol or methanol) solution of potassium hydroxide. The mixture is then either heated for a short period of time while kept in the dark, or left in the dark at room temperature for 12-16 h. There has been considerable discussion of the merits of these two procedures. Which method is used is dependent on the nature of the samples being analyzed and the requirements of the analysis (Davies, 1976 Liaaen-Jensen, 1971). After saponification, water is added, and neutral lipids (the unsaponifiable fraction) are extracted with diethyl ether or hexane. Acidic carotenoids remain in the alkaline phase and are extracted with diethyl ether or hexane after acidification with acetic acid. The unsaponifiable fraction usually contains sterols as well as carotenoids. If desired, sterol contaminants can be removed by precipitation from cold (- 10°C) petroleum ether or by precipitation of these compounds as their digitonides. 

Food sample pretreatment may consist of either (a) saponification to quantify the free forms (retinol or xanthophylls may occur free or ester-ified in foods) [95,96] or (b) direct extraction to determine the unaltered A vitamers [84,88]. Alkaline hydrolysis is also an expedient to simplify the vitamin A analysis, since retinol is the only form to be quantified nevertheless, due to its sensitivity to light and oxygen, it is important to prevent photo-oxidation by inclusion of a antioxidant (ascorbic acid, hydroquinone, or pyrogallol). A drawback of hot saponification is the generation of artifacts, such as geometric isomers of retinol and carotenoids [97]. 

For the analysis of carotenoids in animal products, saponification is normally advisable to remove the fats, although this should be avoided in the case of astaxanthin, because of its lability to alkalis. The HPLC methods for this type of analysis are varied, and although those described above can be used in many cases, there are specific methods listed in the References section at the end of the chapter. Once the pigment extract has been prepared in each particular case, any HPLC method for the analysis of carotenoids in blood plasma can be used. The method employed by Khachik s group is ideal, with the separation of a wide range of carotenoids and their metabolites.The chromatographic separation is performed on a reversed-phase column (Rainin Microsorb 5 pm C18, 25X0.46 cm). 

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