Authentic Carotenoids

Application of NMR spectroscopy (777) to the carotenoid field was first reported in 1970 by Roberts and co-workers 103) using proton-noise decoupling and off-resonance partial proton decoupling. 

In a symposium paper Moss (135) published data for a series of methyl p-apocarotenoates and some fifteen C40-carotenoids. Long range i3c 3ip coupling was noted in allylic phosphonium salts. 

NMR assignments for (35,3 S)-astaxanthin (28) have been confirmed by Yb (dpm)3-induced shifts (69). Data for the 15-m isomer agreed with those previously obtained for 15-m-P,P-carotene (68, 135). Recently the NMR signals of azafrin (6) methyl ester and of 

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It is important to correctly identify the provitamin A peak(s) of interest in the chromatogram. A tentative identification can be made by a combination of retention time and spectral characteristics, using a photodiode array detector. Identification is aided by comparisons with authentic carotenoid standards in more than one chromatographic mode. Because of the ease of cis-trans isomerization when solutions of carotenoids are exposed to heat, light, oxygen, etc., it is difficult to ascertain whether a cis isomer occurs in nature or whether it is formed during its isolation. 

We thank Drs. D. H. Repeta and J. Ertel for providing authentic carotenoid standards and for helpful discussions during the course of this work. This research has been supported by the Oceanic Chemistry Program, Office of Naval Research, under contract N00014-85-C-001. Woods Hole Oceanographic Institution Contribution No. 6310. 

The structure of the C-5 monomethyl ether of azafrin (6) methyl ester was determined by partial analysis of the NMR spectrum (170), and a new isomer of phytoene (21) has been assigned the Z,E,Z or Z,E,E configuration from NMR evidence (18). Further application of NMR spectroscopy for the structural elucidation of carotenoids will be facilitated by the assigned spectra of authentic carotenoids gradually available. 

Kurz C, Carle R and Schieber A. 2008. HPLC-DAD-MSn characterization of carotenoids from apricots and pumpkins for the evaluation of fruit product authenticity. Food Chem 110 522-530. 

Pigments were separated on a normal (150 X 4 mm i.d., particle size 5 /tin) and on a microbore ODS column (150 X 2 mm i.d., particle size 4 jttm) using gradient elution. The steps of gradient elution are shown in Table 2.7. Carotenoids were detected at 440 nm. Columns were not thermostated and separations were performed at room temperature (20 2°C). The mean and the relative standard deviation of retention time and peak area were computed from three parallel measurements. The carotenoids capsanthin, zeaxanthin and j0-carotenein and the extracts were tentatively identified comparing their retention time with those of authentic standards. 

A similar study has been carried out in order to test the capacity of RP-HPLC for the authenticity test of chilli powders on the basis of pigment composition. Carotenoid pigments were extracted by shaking 3 g of chilli powder with 10 ml of acetone for 30 min. The supernatant was decanted and the procedure was repeated as the solid rest was nearly colourless. The collected organic phases were evaporated and redissolved in the mobile phase. Separations were performed on a narrow-bore ODS column (150 X 2 mm i.d., carbon loading, 9.5 per cent). Eluents A and B were methanol-ACN (80 20, v/v) and bidistilled water, respectively. Gradient elution was initiated by 15 per cent A increased to 80 per cent A in 25 min, held for 10 min, increased to 90 per cent A in 10 min, held for 10 min, increased to 97 per cent A in 3 min and held for 62 min. Each step of gradient elution was linear. Measurements were... 

Fig. 2.18. HPLC chromatogram of carotenoids in an authentic sample of orange juice I Sudan I, II lutein, III zeaxanthin, IV /J-cryploxanlhin. V a-carotene, VI / -carotene. For chromatographic conditions see text. Reprinted with permission from A. M. Pupin et al. [42].

LEVELS OF CAROTENOIDS (MG/LITRE) IN AUTHENTIC (HAND-SQUEEZED ORANGE JUICE, FROZEN CONCENTRATED ORANGE JUICE (FCOJ) AND IN FROZEN CONCENTRATED ORANGE PULP WASH (FCOPW), BOTH DILUTED TO 12° BRIX. (N = 5 FOR PERA RIO, N = 4 FOR NATAL, N = 3 FOR VALENCIA AND HAMLIN VARIETIES, AND N = 2 FOR FCOPW)... 

Hofsommer, H.J. (1994b) Analysis of carotenoids in fruit juices. Paper presented as part of the SGF symposium Progress in the Authenticity-Assurance for Fruit Juices, Parma, Italy, September 1994. 

The fatty/waxy products contained the lipophilic substances, including fatty oils, waxes, resins and colorants. Valuable pharmacological effects were proved for some minor constituents of these products (e.g. triterpenes, diterpenes, sterols and carotenoids). Thin layer chromatography and on-line UV-VIS spectroscopy were used for the quick identification and quantity determination of these compounds using authentic samples as standards. The SFE method proved favorable in terras of both extraction yield and speed of carotenoids. The CO2 extracts of the lavandin, clary sage and thyme have been enriched in triterpenic compounds (a-es P-amyrin, oleanic acid, ursolic acid, etc.) and phytosterols. Both free and esterified triterpenoids were present in the extracts of the different samples. Furthermore camosol and other diterpenes were detected in the SFE extract of Lamiaceae plants. The fatty acid composition was only slightly different for extracts obtained by SFE and conventional hexane extraction. 

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). 

Unsaponifiable matter. Oils and fats contain variable amounts of sterols, hydrocarbons, tocopherols, carotenoids, and other compounds, collectively referred to as unsaponifiable matter because they do not produce soaps upon hydrolysis (Table 6). The sterol and tocopherol composition of commodity oils is discussed in another chapter. Some of these minor components are removed during refining, and the resulting concentrates may be useful byproducts, for example, tocopherol antioxidants. Characteristic fingerprints of minor components, particularly phytosterols and tocopherols, are also used to authenticate oils and detect adulteration (18). 

Katayama has identified aldehydes, monoterpenes, and alcohols in the steam-distillate of some dried Laminaria sp. However, they were not detected in fresh kelps, except for the secondary alcohol (8). With essential oils of the wet and undecomposed edible kelps, L. angustata, L. japonica, Kjellmaniella crassifolia, Costaria costata, Ecklonia crassifolia, E. cava and U. pinnantifida along the Sea of Japan, fifty three compounds including alcohols, aldehydes, esters, ketones, hydrocarbons, and carboxylic acids were identified by comparison of Kovats indices and MS data with those of authentic compounds (9,10). The nor-carotenoids such as P-cyclocitral, P-homocyclocitral, P-ionone, and dihydroactinidiolide, which have been... 

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. 

Gandul-Rojas, B., Roca-Lopez Cepero, M., and Minguez-Mosquera, M.I. Use of chlorophyll and carotenoid pigment composition to determine authenticity of virgin olive oU. Journal of American Oil Chemists Society, 77(8), 853-858. 2000. 

The mode of action of file phenylthiazolines has been investigated by looking for file accumulation of metabolic intermediates in chlorotic cress leaves. Treated plants showed an increase in levels of a compound that can not be observed in controls, identified by UV spectroscopy and HPLC cochromatography with an authentic phytoene. The latter has been produced by the application of file known carotenoid synthesis inhibitors such as flurtamon and identified independently by UV and mass spectroscopy. From these results, it can be assumed that chlorosis is caused by inhibition of carotenoid synthesis at the phytoene desaturase step. 

It should be emphasized that the VOO authentication regulatory standards, compiled in the European Commission Regulation (EC Reg No 2568/1991 and its later amendments EC Reg No 1989/2003) [19], the Codex Alimentarius Norm (Codex Alimentarius Commission Draft, 2013) [49], and International Olive Oil Council (lOOC) Trade standards (IOOC/T.15/NC n° 3/Rev.4, 2011) [50], give a particular importance to chromatographic techniques for the analysis of VOO composition in order to detect some fraudulent mixtures. Therefore, for a long time, several HPLC methods have been developed to detect the illegal addition of other oils, including the use of tocopherols, carotenoids, and chlorophylls in various research works to detect adulteration of VOO [51,52],... 

Spectrophotometric examination of isolated pigments is extremely valuable in determining the purity of a carotenoid. The absorption spectrum of a carotenoid is a function of its structure and, if pure, there is good band resolution. Comparison of the absorbance spectrum to that of an authentic sample in the same solvent or to the speqtrum reported in the literature, indicates the identity and purity of a carotenoid. Further evidence of purity is obtained from molar extinction coefficients. 

Spectra for a number of carotenoids can be found in 2 chmeister (1%2) and in Vetter et al. (1971). In addition, Davies (1976) has compiled an extensive list of the main absorption maxima of carotenoids in various solvents. Since the solvent also affects the absorption spectrum of a carotenoid, it is important to report the solvent in which the spectrum was determined. To identify a carotenoid by its absorption spectrum, the spectrum of the unknown carotenoid is compared to the spectrum of an authentic compound in the same solvent, or the positions of the absorbance maxima of the unknown are compared to those reported in the literature. 

The dried carotenoid samples in kiwifruits were dissolved in 0.8% butylated hydroxytoluene (BHT)/acetone. Their concentrations of carotenoids were determined by reversed-phase high performance liquid chromatography (HPLC). Their HPLC chromatographic peaks were identified by the comparison of their retention times and UVA is spectra with authentic standards of a-carotene (1), lutein (6), violaxanthin (8), antheraxanthin (15) and zeaxanthin (9). 

Di-cw-phytofluene (117 R = Me2C=CHCH2) is the first authentic poly-cw-polyene to be identified in carotenoid biosynthesis. The compound (117 R = H), which displays the same chromophore as the natural product, has now been synthesized. Careful choice of conditions for the Homer reaction between the phosphine oxide (114), derived from trans-(E),trans- E)-farnesyl chloride, and the trienal (115) led to a 3 4 mixture of the erythro- and threo- -hyAtoxy-phosphine oxides (116). These products were then separated by chromatography, and the erythro-vsomtx on treatment with base gave (117) in 35% yield (Scheme 22). Spectral data for (117 R = H) were found to be very similar to those for the natural product from Tangerine tomato fruits. 

O. 2 to 0.4% of dry biomass. Ten carotenoids were identified on the basis of their chromatographic properties (TLC, HPLC on nitrile and silica columns), visible, H-NMR and CD spectra and comparison with authentic compounds 54). Both races produce 6(R)-p,a-carotene (47),... 

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