Carotenoids saponification

Other problems in identification are the formation of artifacts during saponification. Carotenoids in citrus occur mainly as esters. For ease of separation, they are saponified with a strong base to their alcohols. During this reaction, traces 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. 

Although saponification was found to be unnecessary for the separation and quantification of carotenoids from leafy vegetables by high performance liquid chromatography (HPLC) or open column chromatography (OCC), saponification is usually employed to clean the extract when subsequent purification steps are required such as for nuclear magnetic resonance (NMR) spectroscopy and production of standards from natural sources. 

A general procedure that our laboratory generally employs is the addition of an equal amount of methanolic 10% potassium hydroxyde (KOH) to an ethereal carotenoid extract. This solution is bubbled with N2 and allowed to stand overnight at room temperature. Other conditions that shorten time at room temperature have also been used, such as saponification of the dichloromethane (CH2CI2) extract with the same amount of 10% KOH in MeOH for 1 hr (peppers and fruits ) and ethereal extract treated with 30% methanolic KOH under N2 for 3 hr (green leaves, vegetables and fruits ). 

However, complete hydrolysis of carotenoid esters sometimes is not achieved in 1 to 3 hr. The saponification degree can be verified easily by the presence of carotenol ester peaks eluting later than the peaks of P-carotene on reversed phase columns. Retinol palmitate, added as an internal standard to orange juice, also serves to indicate whether saponification is complete, since it is converted to retinol which elutes at lower retention time. The mixture is subsequently washed with water until free of alkali in a separatory funnel. Other more polar solvents such as CH2CI2 or EtOAc, and diethyl ether alone or mixtured with petroleum ether can be used to increase the recovery of polar xanthophylls from the water phase. 

More severe conditions, 35 ml of 35% methanolic KOH added to 10 mL extract in EtOAc and shaken for 20 min at 50°C, are necessary for the total conversion of bixin, an ester of a carotenoid acid, to norbixin in snacks. Since saponification yields the norbixin salt (K or Na, depending on the alkali) that is soluble in the aqueous phase, the pH should be decreased to 3.5 or even lower to allow extraction of the protonated norbixin by EtOAc and diethyl ether. ... 

Carotenoids with ally lie hydroxy and keto groups such as the 3-hydroxy-4-keto group in astaxanthin which is widespread in marine animals, microorganisms, and algae undergo oxidation in the presence of alkali and air. Eor such samples, saponification is not recommended or must be carried out under anaerobic conditions. Eor this purpose, a special apparatus and procedure were developed by Schiedt et al. ... 

In recent years, the methods for carotenoid determination without saponification have increased. Independently of the mobile phase and food composition, there are similar patterns of chromatographic separation on reversed phase columns. A chromatograph can be divided roughly into four zones the first zone corresponds to free xanthophyUs, the second zone to monoesterified pigments, the third zone contains carotenes, and finally the fourth zone corresponds to diesterified carotenoids. - ... 

The effect of saponification on the concentration of carotenoids in fatty foods has also been investigated by RP-HPLC. Sausages containing 5.6 per cent powdered paprika were extracted exhaustively with chloroform-methanol (2 1, v/v). The extracting solvent contained 0.01 per cent butylated hydroxyanisole (BHA). An aliquot of the combined extracts was evaporated to dryness and saponified at 50°C for 5min with 10 per cent KOH in methanol in the presence of 0.01 per cent BHA. Free carotenoid pigments were extracted with diethyl ether, washed with water, dried over anhydrous NajSC and evaporated under... 

J. Oliver, A. Palou and A. Pons, Semi-quantification of carotenoids by high-performance liquid chromatography saponification-induced losses in fatty foods. J. Chromatogr.A 829 (1998) 393-399. 

Carotenoids A large number of solvents have been used for extraction of carotenoids from vegetables matrices, such as acetone, tetrahydrofuran, n-hexane, pentane, ethanol, methanol, chloroform [427-431], or solvent mixtures such as dichloromethane/methanol, tetrahydrofuran/methanol, -hexane/acetone, or toluene or ethyl acetate [424,432-435], SPE has been used as an additional purification step by some authors [422,426], Supercritical fluid extraction (SEE) has been widely used, as an alternative method, also adding CO2 modifiers (such as methanol, ethanol, -hexane, water, methylene chloride) to increase extraction efficiency [436-438], In addition, saponification can be carried out, but a loss of the total carotenoid content has been observed and, furthermore, direct solvent extraction has been proved to be a valid alternative [439],... 

Bixin is an oil soluble, highly stable coloring ingredient. The saponification of the methyl ester group to form the dicarboxylic acid yields the water-soluble form of bixin, sometimes called norbixin. Annatto colorants date back into antiquity. The colorant has been used for centuries in connection with various textiles, medicinals, cosmetics, and foods. Annatto colors have also been used to color cheese, butter, and other dairy products for over a century. See also Carotenoids. 

This protocol begins with the extraction of a dehydrated sample. It continues with a saponification scheme to initiate the isolation of the carotenoid mixture. During saponification, the esters are hydrolyzed and the free pigments released. Then, to continue the isolation, column chromatography is suggested as a simple and fast means of separating the three main groups of carotenoids based on their different polarities. 

Following extraction, an efficient way of initiating the isolation of carotenoids is to saponify the extract. This removes many of the unwanted lipids present in the sample as well as chlorophyll. The saponification by-products, which to a great extent are sodium or potassium salts, are easily separated by an aqueous solution of a highly polar salt. The addition of water also helps wash off excess alkali and other water-soluble and water-complexed compounds. This procedure hydrolyzes xantho-phyll esters to form the hydroxylated carotenoid. 

Samples containing esterified carotenoids, chlorophyll, or high levels of fat may require saponification (VNITF2.1). 

The problem relating to chlorophylls can be overcome to some extent by saponification of the sample, which will remove the chlorophylls however, care must be taken in the choice of conditions, as some carotenoids, particularly the xanthophylls, may be degraded (see UNITF2.1). On the other hand, it is possible to use an alternate wavelength for the carotenoids. For example most of the major carotenoids of interest in foods have an absorption peak around 480 nm, where any absorption of chlorophylls causes less interference however, it is then necessary to use alternate extinction coefficients (e.g., 2180 for (3-carotene). 

M Kimura, DB Rodriguez-Amaya, HT Godoy. Assessment of the saponification step in the quantitative determination of carotenoids and provitamins A. Food Chem 35 187-195, 1990. 

Saponification is a purification procedure to remove unwanted lipids and chlorophylls. It has to be omitted when alkali-labile carotenoids (e.g., astaxanthin, fucoxanthin) or carotenoid esters are to be analyzed. To prevent the formation of artifacts produced by aldol condensation between acetone and carotenals, all traces of acetone have to be removed prior to saponification (41). 

Orange juice 9 carotenoids Centrifugation, saponification with KOH, extraction with DCM C-18 ACN-MeOH- dcm-h2o HPLC/UV-vis (454 nm) 0.22 pmol for p-carotene 82... 

Plant extract 12 carotenoids Saponification, extraction with diethyl ether, SPE with silica C-18 Petroleum- MeOH-ACN TLC 57... 

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. 

Larsen, E. Christensen, L.P. 2005. Simple saponification method for the quantitative determination of carotenoids in green vegetables. J. Agric. Food Chem. 53 6598-6602. 

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