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Reliability of Carotenoid Analyses: A Review


Ladislav, Feltl* ,a , Vera, Packova , Karel, Stulka and Karel, Volkab
aDepartment bDepartment

of Analytical Chemistry, Charles University, Albertov 2030, 128 40 Prague 2, Czech Republic

of Analytical Chemistry, Institute of Chemical Technology, Technick 5, 166 28 Prague 6, Czech Republic
Abstract: This review critically discusses the most important properties of techniques applied to analyses for carotenoids and their impact on the reliability of the analytical results. The emphasis is placed on modern methods and the most recent references. Examples of analytical procedures illustrate the characteristics of experimental approaches to carotenoid analyses but no systematic survey of applications is given. Carotenoids are characterized chemically and their occurrence and functions in organisms are outlined. The principal implications of the (bio)chemical properties of carotenoids for the selection of an appropriate analytical procedure and for optimisation of the experimental conditions are dealt with. Various aspects of pretreatment and preconcentration of analytical samples are discussed, emphasizing modern approaches, such as microwave-assisted extraction or supercritical fluid extraction; the problems connected with chromatographic purification of carotenoids and with the obtaining of reliable standard materials are also dealt with. The approaches to determination of carotenoids involving high-performance separations are evaluated, emphasizing the importance of high performance liquid chromatography (HPLC) and capillary electrochromatography (CEC); of the detection techniques, spectrophotometry in ultraviolet and visible region (UV/VIS spectrophotometry), electrochemistry, thermal lens spectrometry (TLC), mass spectrometry (MS), nuclear magnetic resonance (NMR), and some aspects of vibrational spectrometry are primarily discussed.

1. INTRODUCTION IMPORTANCE AND BASIC PROPERTIES OF CAROTENOIDS Carotenoids belong in the group of yellow or red pigments that occur widely in plants, animals and humans. They are synthesized in plants and in some microorganisms and are only introduced with diet into human and animal organisms, which are incapable of their de novo synthesis but sometimes capable of their structural modification. They are highly physiologically important and fulfil many tasks. Primarily, they always accompany chlorophyll and assist photosynthesis and phototaxis as auxiliary light absorbers or, on the other hand, protect plants and microorganisms against excessive irradiation. Further, they strongly interact with reactive oxygen species and thus act in plant and animal organisms as potent free radical quenchers, singlet oxygen scavengers and lipid antioxidants (some of them are vitamin A precursors). In addition to this physiological and medical importance, changes in the contents and structure of carotenoids can also act as markers of environmental damage. Consequently, carotenoids have been intensely studied by organic chemists, food chemists, biologists, physiologists, medical doctors and recently also by environmentalists and great demands have been placed on their identification and determination. For the sake of illustration: the number of known natural carotenoids was about 80 in 1960 [1], around 500 in 1980 [2] and now is well over 700; since 1980, around 7500 papers have been
*Address correspondence to this author at the Department of Analytical Chemistry, Charles University, Albertov 2030, 128 40 Prague 2, Czech Republic; Tel: +420224913538; Fax: +420224913538; E-mail: pacakova@natur.cuni.cz 1573-4110/05 $50.00+.00

published about carotenoids and about 1100 papers about carotenoid analysis. Carotenoid research in the field of food chemistry is a very extensive area; for a detailed treatment see e.g., [3]. The high reactivity of carotenoids follows from their structure and places great demands on the methods of their analysis. Carotenoids are long, aliphatic, conjugated double bond systems, i.e., polyenes. A part of them are hydrocarbons, are usually composed of eight isoprene units and have the molecular formula C40H56. The central portion of the molecule contains four isoprene units, the centre two of which are joined tail-to-tail and open chain or ring structures form the ends of this chain. These hydrocarbons are called carotenes. A great majority of natural carotenes have double bonds in the all-trans position, where R is an open-chain structure or a ring system. Only a few natural carotenes exhibit a cis-trans configuration.
R

Another part of the carotenoid group are oxygenated derivatives of carotenes with various combinations of, e.g., hydroxy-, epoxy-, alcohol-, aldehyde-, keto-, lactone-, carboxylic acid-, ester- or phenolic functions. These compounds are called xanthophylls. The oxygen-containing functional groups are located at the chain ends, not within the multiconjugated system. No heteroatom other than oxygen has so far been known to be present in natural carotenoids. About one half of natural carotenoids are chiral, usually containing one to six chiral centres. Physico-chemical attacks (light, temperature, oxidants, substituents, etc.) at carotenoid molecules have profound effects on the structure
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and configuration of the products and thus also on their physico-chemical properties. Trans-cis shifts have especially strong effects on the overall shape of the molecule and thus also on its properties. In general, all-trans isomers have lower energy and are more stable than cis-trans and cis isomers.(For additional information, see, e.g., [1-5]). 2. SOME IMPLICATIONS ANALYSES FOR CAROTENOID

The general task of such an analysis is twofold: 1) To establish the identity of a compound that has been isolated or synthesized and to characterize its structure (and possibly also its configuration and conformation); 2) To determine the contents of required compounds in samples of biological materials (microbes, plant, animal and human tissues, physiological fluids). In view of the brief account of the carotenoid properties given above, the analyst must consider many factors and take appropriate measures, to obtain reliable results. Identification and characterization of an isolated or synthesized substance usually does not involve a special analytical separation step and the main problem is the selection of the measuring method (usually UV/VIS spectrometry, infrared spectroscopy (IR), Raman spectroscopy, NMR and MS, but sometimes also electrochemistry). However, it is necessary to ensure that the experimental conditions are sufficiently mild to avoid any changes in the analyte structure. On the other hand, when analysing biological samples with complex matrices and low analyte contents, direct measurement is rarely possible and a high-performance separation followed by a sensitive detection is usually required. Here it is often not so important whether the analyte character is affected during the analysis, provided that it can be separated from the sample matrix, unambiguously identified and reliably quantified. Of course, in some cases it is necessary to take special care, e.g. in chiral analyses. The crude sample preparation (sample decomposition, analyte preconcentration, etc.) is essential in all these analyses and may have the decisive effect on the reliability of the overall analysis. The properties of carotenoids briefly outlined above indicate a number of points to be considered when designing a carotenoid analysis: 1) Carotenoids are lipophilic pigments and thus are sometimes denoted as lipochromes or chromolipids [4]. Their hydrophobicity and fat-solubility have two primary consequences: a) they are transported by lipoproteins within organisms and thus their distribution is linked to the lipid profile [6]. This has a strong effect on the approach to the sample collection and pre-treatment; b) their insolubility in water and poor solubility in many organic liquids place considerable demands on proper selection of extraction and preconcentration agents and impose severe limitations on the composition of HPLC mobile phases. 2) Carotenoids exhibit pronounced photo- and thermal sensitivity. Irradiation and an increased temperature lead mainly to trans-cis isomerization which causes bending of the originally rod-like molecule and may also cause cleavage

of the molecule, especially in the presence of air and catalysts (iodine is the best known catalyst that is extensively used in organic chemistry of carotenoids, but many other paramagnetic substances, e.g., nitrogen oxide and dioxide, bromine, molecular oxygen, etc. have similar effects) [1,5]. These changes strongly influence spectral and chromatographic properties of carotenoids and also alter their optical activity if a chiral centre is present in the molecule, and thus may adversely affect the reliability of analytical measurements; on the other hand, they may serve as markers of external, e.g. environmental, effects on the studied organism. 3) Due to the presence of a long system of conjugated double bonds, carotenoids are intensely coloured and thus strongly absorb in the visible region, between 400 and 500 nm, usually exhibiting three absorption bands or two bands plus a shoulder, with vibrational fine structure, the middle band having the highest intensity [1,4]. The spectroscopic properties of carotenoid molecules depend on the highly complex mechanism and dynamics of energy transfers among various excited states; this is a very active research field because of the importance of carotenoids in photosynthesis (for a review see, e.g., ref. [7]). The absorption properties provide simple and cheap possibilities for direct analytical determinations and for chromatographic detection. Unfortunately, the position of the absorption bands strongly depends on the solvent used [4],which further complicates the selection of the experimental conditions. Of course, the effects discussed in point 2) above may complicate the measurement but may also serve as a means for studying chemical changes to the substances of interest. In general: bending of the molecule due to trans-cis conversion leads to a decrease in the absorbance and in the absorption wavelengths; moreover, a new band, the cis-peak, appears in the near UV region, at 320 to 380 nm. The longer the molecule, the stronger is the light absorption and the longer are the absorption wavelengths. Cooling of the studied system leads to enhanced absorption, narrowing of the absorption bands and their shift to lower wavelengths [1]. 4) Spectroscopic techniques, especially Raman spectroscopy, are extremely important for direct analyses and especially for carotenoid characterization. Molecules of carotenoids belong among the most efficient Raman scatterers and so Raman spectroscopy is usually the method of first choice for study of carotenoids. Resonance Raman spectroscopy produces strong lines even at concentration as low as 10-5 mol L-1 . The strongest lines are observed within the range of C=C and C-C stretchings of the conjugated backbone (1550-1500 and 1200-900 cm-1 ) when excited with a wavelength corresponding to the main electronic transition (1B u(S2) 1Ag(S0) (-*) [8] but an intensity enhancement can also be attained outside this resonance condition. As shown in detail (see ref. [9]) for carotene, its Raman spectrum is independent of the choice of wavelength from the visible to the near infrared (this statement holds for the fundamentals only, the intensities of overtones and combinations vary strongly with the excitation wavelength). A mechanism based on electron/phonon coupling seems to explain the observed intensity enhancement [10]. Near infrared excitation (usually

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by Nd:YAG laser at 1064 nm) allows to overcome the serious problems with strong fluorescence of the biological samples observed with excitation in the visible range of spectrum. Carotenoid molecules, although constituting a very broad family of diverse structures, exhibit remarkably similar resonance Raman spectra, on the other hand there are many subtle changes in the spectra allowing to follow conformation changes [11]. Raman spectroscopy is the key method in the study of processes in photosystem II [12]. Identification and structure elucidation are based on combined approaches involving various spectroscopic methods, such as UV/visible, infrared, resonance Raman, NMR spectroscopy, circular dichroism and mass spectrometry [13]. Basic spectral characteristics of carotenoids are collected, e.g., in ref. [14]. 5) The conjugated double-bond system of carotenoids causes not only their spectral activity, but also their electroactivity. This provides a further analytical possibility, especially for chromatographic detection. The parent compounds are oxidized, with formation of cation radicals and dications; dications have tendency to decay by loss of a proton and undergo a comproportionation reaction with the parent carotenoid to form two cation radicals. The substituents at the terminal positions strongly affect the oxidation potentials: electron donors enhance the oxidation whereas electron acceptors hinder it. Electron acceptors stabilize the cation radicals and destabilize the dications [15]. A study of -carotenes with chain lengths between 5 and 23 double bonds using cyclic voltammetry and photoelectron spectroscopy [6] has demonstrated that carotenoids undergo redox reactions at low potentials (below 0.5 V, SCE). With increasing chain length ( 11 double bonds), even tetra-cations and tetra-anions are generated. The potentials converge to limiting values with increasing number of double bonds. The potential difference between the first oxidation and first reduction potential is a linear function of the reciprocal chain length and the radical cation formation is in principle determined by the chain length. It is obvious that, in view of the complexity of carotenoid chemistry, the reliability of carotenoid analyses needs rigorous checking. An example of a NIST check of measuring performance in this field over an interval of 15 years is given in ref. [16]. 3. PRETREATMENT AND PRECONCENTRATION OF ANALYTICAL SAMPLES As pointed out above, carotenoids are insoluble in water and even their solubility in methanol and acetonitrile is limited e.g., the solubility of -carotene in acetonitrile is only 10 mg.L-1 ; however, its solubility in hexane is much better, 800 mg.L-1 . Great demands are placed on solvent selection and its purity. The best solvents for carotenoids are chloroform, dichloromethane and tetrahydrofuran in which the solubility may attain values of 1000 to 10000 mg.L-1 [17]. However, these solvents may be contaminated by traces of hydrogen chloride and hydroperoxides, which chemically interact with carotenoids and cause their decomposition [18].

This general problem of chemical interactions, primarily the trans-cis conversion (see above), is encountered both during the sample pre-treatment and during the sample injection into a HPLC column. Carotenoids in their natural environment, when they are incorporated in lipoproteins or membranes, are relatively well protected. However, if they are isolated and transferred into stock solutions or dissolved in extractants, they readily undergo isomerization catalysed by light, acids and bases, oxygen, heat, traces of metal ions, etc. Special attention should be devoted to the sampling procedure. The content of carotenoids is known to be controlled by many factors and may be changed not only in living organisms, but also during their senescence and death related alterations. Removing, e.g., a leaf from a plant does not stop the photochemical processes immediately and thus the changes in the carotenoid contents are still controlled by many factors, including light, oxygen and water content [19]. It has been suggested that handling of the samples influences the ratio of the cis and trans isomers of carotene. As emphasized in the previous paragraph, such isomerization is less probable for carotenoids embedded in biological matrices. Therefore, a number of measures must be taken during the sample pre-treatment, primarily, antioxidants must be used, the laboratory operations carried out in dimmed, yellow or red light, evaporations should be carried out under a protective nitrogen or argon atmosphere at temperatures below ca. 40 oC and the samples should be stored in darkness, under nitrogen or argon, at temperatures around -20 oC [20-23]. The often used antioxidants are ascorbic acid, sodium ascorbate, pyrogallol and ethoxyquinine, but the most common one is butylated hydroxytoluene (BHT) at concentrations of 0.01 or 0.1% in extracted solutions [24,25]. Samples of tissues are homogenized prior to extraction, preferentially at a low temperature (in ice or in liquid nitrogen) and in the presence of an antioxidant [18]. There is no standard procedure for carotenoid extraction because of a wide spectrum of analysed materials (foodstuffs, plants, animal and human samples) and a wide range of carotenoids present. Samples of animal tissues or fluids are deproteinated prior to extraction by adding ethanol or other alcohol, e.g., propanol [26]. An internal standard is usually added to the sample, together with an agent precipitating the proteins present. A mixture of perchloric acid with ethanol is sometimes used for deproteination, even if oxidizing acids may decompose the analytes and induce their isomerization [18,27-32]. The second step of sample pre-treatment usually consists of extraction for which hexane is most often used; dichloromethane is sometimes added to improve the extraction efficiency which, however, increases the risk of altering the sample composition. A butanol ethyl acetate mixture is also a frequent extraction agent. Halogenated solvents are often used in mixtures with methanol in analyses of lipophilic antioxidants in subcellular fractions, such as mitochondria or microsomes [18,28,30,32-37]. As the extraction separates hydrophobic carotenoids from hydrophilic medium of foodstuffs, plants or animal materials, it is suitable to extract samples with minimum moisture content by a mixture of weakly polar and nonpolar

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solvents. To isolate carotenes, the sample should be extracted first with a polar solvent (e.g., methanol), which removes water and more hydrophilic xanthophylls, followed by extraction with a suitable solvent of low polarity. The literature contains many methods using acetone, chloroform, tetrachloromethane, petroleum ether, diethyl ether, tetrahydrofuran, methanol, ethanol, 2-propanol, hexane, dichloromethane, ethyl acetate, N,N-dimethylformamide and various mixtures thereof [21,38-66]. With materials containing much water, an organic solvent miscible with water is suitable, to enhance denaturation of carotenoidprotein complexes and to prevent formation of emulsions; acetone and tetrahydrofuran are used most often [48]. The sampled material matrix also affects the extraction efficiency and thus great differences can be found in the literature for carotenoid contents in fruit and vegetables (e.g., values of 1830 to 14700 and 490 to 20000 mg per 100g for -carotene in carrots and gourd, respectively). Consequently, a proper selection of the solvent for a particular carotenoid extraction is of prime importance. Additions of sodium, magnesium or calcium carbonate (0.10 g per gram of sample) and of an antioxidant (BHT) are recommended, to neutralize acids contained in the tissues sampled and to prevent oxidation [67-69]. Soxhlet extraction, sonication and other extraction techniques are very often replaced by microwave-assisted extraction [70], which shortens the extraction time and improves the yield of the extraction. Compared to traditional extraction procedures, microwave-assisted extraction is simple, is not limited by the solvent selectivity, a preconcentration (evaporation) step is avoided and the danger of contamination by solvent impurities suppressed. Mixtures of water with various contents of an organic modifier (acetone, dioxan, ethanol, methanol, tetrahydrofuran) have been used for extraction. The time of extraction and the electric input have been optimized, especially from the point of view of the temperature attained which should not exceed 40 oC to minimize chemical changes in the carotenoids. Solid-phase extraction with the C-18 phase, followed by reversed-phase ion-pair HPLC, has also been applied to carotenoid isolation [50]. A mild and quick extraction technique, matrix solid-phase dispersion, has been

recommended for the analysis of carotenoid stereoisomers [71]. Supercritical fluid extraction (SFE) is a rapid and selective method that is readily automated [40,58,72,73]. The extraction selectivity can easily be varied by varying the carbon dioxide density (i.e., by varying the temperature and pressure), because the dissolving power of CO2 increases with its increasing density (see (Fig. 1) [5]); e.g., the selectivity of carotenoid extraction from palm oil deteriorates at higher pressures because triacylglycerols begin to be dissolved [74]. The same situation is encountered in extractions from other materials (paprika, leaves) a lower temperature and/or pressure lead to a higher selectivity. Using the Hildebrand solubility parameter for -carotene (8.71 cal1/2cm3/2) [75], the temperatures and pressures have been calculated from the Giddings equation [76] for which the CO2 density (0.98 or 1.04 g.mL-1 ) permitted the attaining of this solubility parameter value, i.e., the attaining of the highest possible solubility. SFE methods have been described for extraction of -carotene from leaves and vegetables, employing pure carbon dioxide and combining static and dynamic extraction, attaining yields similar to or higher than those obtained in liquid extraction [77-80]. The extraction selectivity of SFE can also be influenced by adding a polar modifier at concentrations around 1% (methanol, ethanol, 2-propanol) [81-84]. The small modifier molecules hasten the desorption process by displacing the analyte from the binding sites and by disintegrating the matrix of the extracted material; e.g., an addition of ethanol increases the yield in the -carotene from carrots and tomato residues [74]. However, modifications that improve the dissolution power of carbon dioxide usually lead to a decrease in the extraction selectivity. An advantage of SFE with pure carbon dioxide lies in the possibility of obtaining carotenoids from natural materials in the absence of organic solvents and with minimum changes caused by thermal oxidation. A certain danger comes from a loss of the extracted carotenoid through its oxidation by oxygen present in the carbon dioxide; therefore, it is advisable to add an antioxidant (usually BHT) even in extractions with supercritical CO2 [85].

Fig. (1). Solubility of -carotene in supercritical carbon dioxide [5].

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Polar xanthophylls, which contain hydroxyl groups, may form esters with fatty acids. Alkaline saponification must thus be included in the analytical procedures for xanthophylls, usually using aqueous-ethanolic or methanolic solutions of potassium hydroxide [22]; however, the saponification process also causes degradation of carotenoids [40,44,57,86-88]. One of the great problems of carotenoid analyses lies in the unavailability of standard compounds caused by natural instability of carotenoids. Some carotenoids, e.g., carotene, can be obtained commercially but their purity is insufficient for their use as chromatographic standards and thus they must be purified under spectrophotometric control. The literature contains spectral data of many carotenoids in various solvents [19,23,89]. However, the purity of standard solutions must also be verified chromatographically (HPLC). The importance of certified materials in analyses for carotenoids in vegetables has been emphasized in an interlaboratory study [90]. Carotenoids can be purified using classical column chromatography on alumina, silica gel, magnesium oxide or carbonate, calcium hydroxide or carbonate, Cellite and further adsorbents, with various solvent systems [23]. Possible problems with catalytic activity of these adsorbents have not been reported in the literature. Preparative TLC can be used [24,91] but preparative HPLC is most common [22,92,93]. Degradation and isomerization of carotenoids must be prevented during the preparation of standards, by preparing and storing stock solutions in darkness, at -20 oC, under a protective atmosphere (nitrogen, argon) and using solvents containing antioxidants. An interesting procedure, based on recrystallization and fractional dissolution, has been used for the obtaining of pure all-trans -carotene [94]. This paper also describes the spectral characteristics for the pure and a partially degraded product and points out a high reactivity with atmospheric and dissolved oxygen. The procedure is based on recrystallization and fractional dissolution. 4. DETERMINATION OF CAROTENOIDS INVOLVING HIGH-PERFORMANCE SEPARATIONS 4.1. Separation Classical column chromatography and TLC were originally used for determination of carotenoids. However, these methods are time-consuming, need large sample amounts, their separation efficiency is not particularly high, and they suffer from a poor reproducibility of results and low recoveries of the analytes. Among the high-performance separation methods, gas chromatography (GC) is unsuitable, primarily because of low volatility and thermolability of carotenoids (however, the volatility of carotenoids can be increased by reducing the double bonds). Capillary zone electrophoresis (CZE) is inapplicable because of the absence of charge on the carotenoid molecules. Therefore, the most common method used in the analysis of carotenoids is HPLC employing various detection techniques. Both normal- and reversed-phase systems are used, either in isocratic or gradient elution modes. Reversed-phase systems are generally preferred because normal-phase HPLC has

several disadvantages, namely, a lower column stability, a poorer reproducibility of the retention times and a longer time required for column equilibration. In reversed-phase systems, non-aqueous mobile phases are recommended, in view of the pronounced hydrophobicity of carotenoids that makes their separations in mobile phases containing water difficult or impossible. Various mixtures of solvents, mostly of methanol, acetonitrile and tetrahydrofuran, have been successfully applied to the purpose (see, e.g., review by Su et al. [48]). Antioxidants, such as BHT, are added to the mobile phase, and the temperature of the HPLC column should be maintained low and constant (around 20 0C), to prevent decomposition of carotenoid samples during the HPLC analysis and improve the reproducibility of quantitative analysis [95]. An example is the analysis of carotenoids in orange juice [96] ( and carotenes, lutein, zeaxanthin, -cryptoxanthin) on a C-18 reversed phase, using a ternary mobile phase consisting of a mixture of acetonitrile - methanol - dichloromethane (60:35:5), with additions of the BHT antioxidant (0.1 %), triethylamine (0.1 %), and ammonium acetate (0.05 mol.L-1 solution in methanol). Triethylamine and ammonium acetate minimize the effects of acidity generated by the free silanol groups present on the silica support. So far the best separations of various carotenoids have been attained on a C-30 chemically-bonded phase (e.g., ref. [97]). This stationary phase has the highest separation selectivity. Applications of the C-30 stationary phase to food analyses have recently been reviewed [98]. An example of the analysis of carotenoids in a blood serum extract on the C-30 stationary phase in combination with a coulometric array detector is given in Fig. 2 [43]. A very promising method for carotenoid analysis is capillary electrochromatography (CEC). Due to a flat elution profile, CEC offers a high separation efficiency, comparable to capillary electrophoresis (CE) and gas chromatography (GC). Stationary phases similar to those of HPLC are used, as demonstrated on an analysis of carotenoids in baby food and Dunaliella algae. The analysis was carried out on a polymeric C-30 stationary phase in combination with an acetone - 1 mmol.L -1 borate buffer (99:1) mobile phase [99]. In natural environments, carotenoids are usually present as esters. The degree of esterification differs for individual carotenoids and is related to the number of hydroxyl groups present in the molecule. Identification of peaks in chromatograms is thus complicated. To simplify interpretation of experimental results, esters are saponified prior to analysis. However, significant degradation and losses of carotenoids have been observed due to saponification. Simultaneous analysis of free and esterified carotenoids has been suggested, thus avoiding saponification [22]. 4.2. Detection The success of the determination depends equally on the separation and the detection steps. Whereas the selection of applicable separation techniques is rather limited by the chemical character of carotenoids and their sensitivity to experimental conditions, these very properties open wide possibilities for spectroscopic and electrochemical detection.

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Fig. (2). HPLC of blood serum extract with electrochemical detection [43]. Stationary phase: C-30, mobile phase methanol-methy tert. butyl ether (11:89), coulometric electrochemical array detector. 1, lutein, 2, zeaxanthin, 3, -cryphoxanthin, 4, 13-cis--carotene, 5, trans--carotene, 6, trans--carotene, 7, 9-cis--carotene.

4.2.1. UV/VIS Photometry This detection is by far the most common because carotenoids absorb strongly in the visible region between 400 and 500 nm (all-trans isomers), while cis- and cis/transisomers also exhibit absorption in near UV region, around 320 nm (see also Sect. 1), the detectors are simple and cheap and photometric detection is readily compatible with gradient elution. Diode array detection (DAD) provides certain possibilities for analyte identification, which, however, should not be overestimated, as DAD yields spectra without fine structure and the spectra of many carotenoids are very similar (e.g., those of -carotene and zeaxanthin) [100]. Nevertheless, DAD may provide very useful information on the chromatographic peak purity, i.e., on the separation efficiency and resolution. Similar detection limits and similar reproducibilities of results have been reported with DAD and with singlewavelength detectors that usually operate at 450 nm, which is a compromise among the absorption maxima of various carotenoids. Typical characteristics of quantitative determination of -carotene in a vegetable product, olive oil, can be found in ref. [52]: for a sample of 2.75 mg.kg-1 , a RSD equals 4.27 %, the calibration curve is linear within a range of 0.5 5 g.mL -1 with a correlation coefficient R 2 better than 0.999, the recoveries at three concentration levels (from 3.6 to 6 mg.kg-1 ) are around 107 %, DL amounts to 5.5 ng and QL to 31.0 ng..The time of analysis of -tocopherol and -carotene is 8 min. The quantitative analysis always suffers from instability of the standards used, which are usually less pure than expected (see also Sect. 3). The purity of standards has always to be checked, e.g., by DAD measurements. 4.2.2. Electrochemical Detection The level of -carotene in plasma is usually below the limit of detection reached by UV/VIS detection. Therefore,

other detection methods have been investigated. The electroactivity of carotenoids (see ref. [6,15] offers the use of electrochemical detectors. Electrochemical detection seems to be somewhat more sensitive than UV/VIS detection. When a detector with multiple working electrodes (electrode array) maintained at different potentials (an analogy of DAD) is used, then the peak purity can be checked, analyte identification aided and certain cis and trans isomers differentiated, but with rather serious limitations in practical application. For example, a coulometric detector with 8 electrodes placed in series whose potentials ranged from 100 to 520 mV in 60 mV increments was capable of distinguishing -carotene isomers (all-trans, 9-cis-, 13-cis and 15-cis) and other carotenoids (see Fig. 2). The day-today reproducibility better than 10 % was attained [43]. 4.2.3. Thermal Lens Spectrometry Promising results have been obtained by HPLC combined with a thermal lens spectrometric detector (TLS). TLS is based on generation of heat during the non-radiative relaxation of the absorbed optical radiation. The immediate consequence of such a process is an increase in the sample temperature, which results in changes of physical properties of the sample (the density and refractive index). TLS measures the absorbance indirectly through the changes of the temperature-dependent refractive index of the sample. The main advantages over conventional spectrometry are a higher sensitivity and a lower susceptibility to errors resulting from light scattering. Consequently, the sample volumes can be smaller and thus the effects of possible incompatibility between the solvent used for sample extraction and the mobile phase can be suppressed. TLS detection is suitable for compounds with low fluorescence quantum yields, such as most of carotenoids. In combination with favourable optothermal properties of solvents used as mobile phases in HPLC (a low thermal

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conductivity, a high temperature coefficient of the refractive index), a very sensitive detection can be obtained (a 100-fold improvement in sensitivity compared to UV detection, i.e. 100 pg.mL-1 ) [101]. Limits of detection for gradient separation with TLS are 1.3- to 10-fold lower than for the UV-Vis detection [102]. The validation of the method demonstrated a good reproducibility and accuracy. 4.2.4. Mass Spectrometry The main advantage of MS detection is in that it enables not only the analyte quantification, but also the elucidation of its structure, on the basis of the molecular mass and of fragmentation. Most of the methods applied to carotenoids use the API mode, due to the absence of protonation sites in carotenoids. Labelled or deuterated compounds are used to study the bioavailability and metabolism of carotenoids [103]. The advantage of using 13C labelled compounds is that their retention behaviour in reversed-phase chromatography is the same as that of the unlabelled compounds [104,105]. The sensitivity of the ESI-MS detection is two orders of magnitude higher than that of the UV/VIS detection (e.g., a detection limit of 0.7 pmol for -carotene). APCI in positive and negative modes gave detection limits 1-13 pmol, slightly higher than the ESI measurement. APCI has a broader linear dynamic range than ESI (over at least three orders of magnitude). The determination of lycopene in serum by HPLC-MS/MS with APCI in the negative mode yielded a standard deviation of less than 10 % over a sample range of 5-500 pmol (on-column injection), with a limit of detection of 11.2 fmol. [106]. Detection limits within a region of units to hundreds of fmol have been obtained also for the MALDI technique [107,108]. Another technique involves the use of a particle-beam interface with electroncapture negative ion chemical ionization (PB-NCI-MS). Detection limits between 0.2 and 3 ng have been obtained for carotenes using this technique [109]. A mild electrospray ionization technique has been developed especially for unsaturated compounds (sometimes it is called coordination ion-spray (CIS) MS). Silver ions are added to the analytes after separation. All carotenoids form [M +Ag+ ] ions and radical [M + ] ions are formed additionally. DL values as low as 300 fmol for -carotene have been reported [110]. The ultrasensitive AMS technique exhibiting low detection limits and wide linear dynamic range values is promising for C-14 nutrition studies [111]. 4.2.5. Nuclear Magnetic Resonance NMR is the best method for identification of unknown stereoisomers [71,112]. It can be used either off- or on-line. A prerequisite for reliable results in direct coupling of HPLC-NMR is a complete separation of all the isomers. The C-30 stationary phase is so far the best stationary phase for this purpose [112]. Measurement of NMR spectra requires pure components in higher amounts than UV/Vis and MS detection modes. All lutein stereoisomers were identified using HPLC-NMR coupling with a concentration in the upper nanogram range. The importance of combinations of HPLC-MS and HPLC-NMR for identification and determination of carotenoid stereoisomers has been emphasized [71,113].

Detection limits and reproducibility of measurements (relative standard deviations) for various detection modes used in HPLC of carotenoids are summarized in Table 1 [30,101,114-124]. 5. OTHER METHODS OF DETERMINATION OF CAROTENOIDS A separation step is needed in all cases where detailed information on the contents of individual carotenoids is needed. On the other hand, methods providing information on the total content of carotenoids are also useful. The conjugated polyene structures of carotenoids provide them with unique absorption spectra and high molar absorptivities in ultraviolet and visible spectral range, and hence outstandingly low limits of detection are attained. The total contents of carotenoids are determined spectrophotometrically in biological samples [125]. The accuracy of the method can be disputed, even if the dependence of the molar absorption coefficients of individual carotenoids and the resolution of the spectrometers are respected, but 155 citations of this method given on the Web of Science in April 2004 illustrates the popularity of this method. The current challenges in carotenoid analysis include the determination of these compounds at trace levels in biological tissues. Raman spectroscopy exhibits not only good detection limits, but also allows non-invasive analysis. Raman spectroscopy was used for detection of the carotenoids in human skin [126], in the human eye [127], in live corpus luteum cells [128] and for evaluation of general health or stress status of living plants and plant products [129]. The crucial problem lies in the finding an appropriate method of calibration. 6. CONCLUSIONS It can be seen that the pronounced physico-(bio)chemical properties of carotenoids have two-fold significance. On one hand, they yield many possibilities for their selective and sensitive detection, identification and characterization, provided that they are synthesized or isolated from a natural matrix. A number of spectroscopic methods, from UV/VIS photometry, to primarily Raman, NMR and MS, are available for the purpose. It seems that electrochemistry of carotenoids might and should play a more important role in this respect. On the other hand, these very properties of carotenoids seriously limit the selection of high-performance separation methods that are unavoidable in analyses of natural materials. In fact, there are only two generally applicable approaches, namely, HPLC and CEC. These techniques are, however, highly developed and can cope with great differences in the sets of carotenoids to be determined and the type and complexity of matrices of natural samples. The critical stage in analyses of natural materials is the sample preparation and preconcentration during which the sensitivity of carotenoids to light, temperature and oxidation has the greatest effect. Moreover, the techniques of sample pre-treatment and extraction of the analytes are empirical to a considerable extent and thus their selection and optimisation are difficult, specific for particular matrices and often

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Table 1.

Comparison of Detection Limits and Reproducibility of Measurements (Relative Standard Deviations) for Various Detection Modes Used in HPLC of -Carotene in Plasma and Serum
Detection limit mg.L-1

Detection mode UV/Vis, isocratic elution

Coefficient of variation %

Recovery %

Ref.

12 12 6.5 1.48 Gradient elution 7 9.4 1.5 Electrochemical 0.004-0.012 TLS 0.1 0.58 Mass spectrometric APCI-MS 0.015 0.003 ESI-MS ESI-MS, NMR 0.015 0.0065

9.3 7.6 6.4 7.6

101 77 -

114 115 116 117

3.4 5.2 5.6

100 95 99

118 119 30,120

3-14

102

121

5 4.1

101

101 122

3 5.4 6.5

102 -

123 124 110 71

- data not given

dubious. This is illustrated, e.g., in ref. [90] reporting an interlaboratory study of determination of carotenoids in foodstuffs: of the overall RSD of ca. 23%, the sample preparation contributed 13%. ACKNOWLEDGEMENTS Support of this research from the Ministry of Environmental Protection of the Czech Republic (project VaV 340/1/01) and from the Ministry of Education, Youths and Sports of the Czech Republic (project MSM 223400008) is gratefully acknowledged. LIST OF ACRONYMS APCI BHT CEC CZE C-30 DAD = = = = = = Atmospheric pressure chemical ionization Butylated hydroxytoluene Capillary electrochromatography Coordination ion-spray mass spectrometry Capillary zone electrophoresis Hydrocarbon triacontane Diode array detection

DL ESI GC HPLC IR MS NMR QL

= = = = = = = =

Detection limit Electrospray ionization Gas chromatography High-performance liquid chromatography Infrared spectroscopy Matrix assisted laser desorption ionization Mass spectrometry Nuclear magnetic resonance Quantification limit Particle-beam interface with electron-capture negative ion chemical ionization Correlation coefficient Relative standard deviation Saturated calomel electrode Supercritical fluid extraction Thin-layer chromatography Thermal lens spectrometry Spectrophotometry in ultraviolet and

MALDI =

PB-NCI- = MS R2 RSD SCE SFE TLC TLS = = = = = =

CIS-MS =

UV/VIS- =

Reliability of Carotenoid Analyses

Cuuent Analytical Chemistry ,2005, Vol. 1, No. 1 [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] [71] [72] [73] [74] [75] [76] [77] [78] [79] [80]

101

spectrophotometry

visible region

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Received: 21 Septermber, 2004

Accepted: 6 October, 2004

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