ARTICLE IN PRESS

JOURNAL OF FOOD COMPOSITION AND ANALYSIS

Journal of Food Composition and Analysis 19 (2006) 97111 www.elsevier.com/locate/jfca

Study Review

Analysis of carotenoids in vegetable and plasma samples: A review

Ana Rodr guez-Bernaldo de Quiros, Helena S. Costa
de Centro de Seguranca Alimentar e Nutrica Instituto Nacional de Sau Dr. Ricardo Jorge, Av. Padre Cruz, 1649-016 Lisboa, Portugal - o, Received 29 June 2004; received in revised form 11 April 2005; accepted 15 April 2005

Abstract Some carotenoids, besides provitamin A activity, have antioxidant capacity. These properties together with epidemiological studies that establish an association between a high vegetable intake and a lower risk of chronic degenerative diseases, such as certain types of cancer or cardiovascular diseases have increased the interest on the analysis of carotenoids in vegetable samples as well as in human plasma and serum samples. The present paper is an updated review on the analysis of carotenoids in vegetable, plasma and serum samples. Traditional liquidliquid extraction, as well as supercritical uid extraction (SFE) is reviewed. General aspects of chromatographic analysis are commented on, and examples of carotenoids separation in different samples are shown. r 2005 Elsevier Inc. All rights reserved.
Keywords: Carotenoids; Analysis; Vegetables; Plasma; Review

1. Introduction Carotenoids are fat soluble compounds that are associated with the lipidic fractions. From a chemical point of view, carotenoids are polyisoprenoid compounds and can be divided into two main groups: (a) carotenes or hydrocarbon carotenoids only composed of carbon and hydrogen atoms and (b) xanthophylls that are oxygenated hydrocarbon derivatives that contain at least one oxygen function such as hydroxy, keto, epoxy, methoxy or carboxylic acid groups. Their structural characteristic is a conjugated double bond

Abbreviations: ACN, acetonitrile; APCI, atmospheric pressure ionization interfaces; BHA, butylated hydroxyanisole; BHT, butylated hydroxytoluene; DAD, photodiode array detector; DCM, dichloromethane; ESI, electrospray ionization interfaces; EtOH, ethanol; LC-MS, liquid chromatography-mass spectrometry; MeOH, methanol; MTBE, methyl-tert-butyl ether; HPLC, high-performance liquid chromatography; RP, reversed-phase; SFE, supercritical uid extraction; TEA, triethylamine; THF, tetrahydrofuran; UV-Vis ultraviolet-visible Corresponding author. Tel.: +351 21 751 9267; fax: +351 21 752 6400. E-mail address: helena.costa@insa.min-saude.pt (H.S. Costa). 0889-1575/$ - see front matter r 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.jfca.2005.04.004

system, which inuences their chemical, biochemical and physical properties. This class of natural pigments occurs widely in Nature. Carotenoids are synthesized by plants and many microorganisms, so animals have to obtain them from food. Up to now, more than 600 carotenoids have been isolated from natural sources (Pfander, 1987). They are responsible for the beautiful colors of many birds, insects and marine animals, as well as the colors of many owers and fruits (Carotenature, 2000). This attribute is of great importance in foods, since color is often a criterion of quality and is typically modied by food processing (Chen et al., 1995). In addition, carotenoid content in fruits and vegetables depends on several factors such as, genetic variety, maturity, postharvest storage, processing and preparation. However, the great interest in studying these compounds is due to their physiological and biological functions which have been extensively and in detail revised by van den Berg et al. (2000). In addition to the provitamin A activity of some carotenoids, they also have other functions, such as antioxidants and enhancers of the immune response. Furthermore, some of them are involved in the cell communication and

ARTICLE IN PRESS
98 s, A. Rodrguez-Bernaldo de Quiro H.S. Costa / Journal of Food Composition and Analysis 19 (2006) 97111

xanthophylls have shown to be effective as free radical scavengers (Krinskey, 1994). Recently, epidemiological studies have indicated an association between high vegetable intake and a lower risk of chronic degenerative diseases such as certain types of cancer, cardiovascular diseases (Machlin, 1995) and age-related macular degeneration (Bone et al., 2000). In this study, associations between dietary intake of lutein and zeaxanthin and their concentration in serum, and macular pigment density were found. These results are in accordance with the major risk of age-related macular degeneration and low concentrations of macular pigment. Regarding carotenoid analysis, originally, the separation was carried out by open column chromatography at atmospheric pressure (Almeida and Penteado, 1988), but this method required a large amount of sample (Mercadante, 1999; Su et al., 2002). However, Rodriguez-Amaya (1996) pointed out that with this technique, in spite of the disadvantages, a good separation could be achieved for food samples with complex carotenoids composition. Almeida-Muradian et al. (1997) have found similar results when HPLC methodology was compared with open-column chromatography for the determination of carotenoids with provitamin A activity in beet leaves, as well as in green leafy vegetables and other food samples (AlmeidaMuradian et al., 1998; Rodriguez-Amaya et al., 1988). With the development of high-performance liquid chromatography (HPLC), numerous methods, both normal- and reversed-phase (RP) have been described to separate xanthophylls and carotenes. In HPLC analysis of carotenoids, the most widely employed detector is ultraviolet-visible (UV-Vis) and, more recently the photodiode array detector (DAD), which allows a continuous collection of spectrophotometric data during the analysis has been used (Huck et al., 2000). When high sensitivity is required, electrochemical array detection is a good alternative (Ferruzzi et al., 1998; Rozzi et al., 2002). In complex matrixes, when DAD is not sufcient for identication because of spectral interferences, mass spectrometry coupled to liquid chromatography has been successfully used.

2. Extraction 2.1. Liquid liquid extraction 2.1.1. Extraction of carotenoids from vegetable and fruit samples Due to their complex structure and because of the wide variety of these compounds present in vegetables and fruits, there is not a reference method to analyze them. Numerous organic solvents such as acetone (AzevedoMeleiro and Rodriguez-Amaya, 2004; Englberger et al.,

2003a b; Heinonen et al., 1989; Otles and Atli, 2000; Hagg et al., 1994; Edelenbos et al., 2001; Mouly et al., 1999; Hegazi et al., 1998; Rodrigues et al., 1998), tetrahydrofuran (THF) (Hulshof et al., 1997; Granado et al., 1992; Khachik et al., 1986, 1992a), n-hexane (Gandul-Rojas et al., 1999; Ferreira de Franc a et al., 1999), pentane (Marsili and Callahan, 1993), ethanol (EtOH) (Howard et al., 1999; Moros et al., 2002), methanol (MeOH) (Melendez-Mart nez et al., 2003; Gokmen et al., 2002), chloroform (Rozzi et al., 2002), as well as solvent mixtures such as DCM:MeOH (6:1, v/v) (Markus et al., 1999), acetone:petroleum ether (50:50, v/v) (Hsieh and Karel, 1983), THF:MeOH (1:1, v/v) (Huck et al., 2000; Sharpless et al., 1999; Scott and Hart, 1993; Murkovic et al., 2002; Hentschel et al., 2002; Hart and Scott, 1995), n-hexane:toluene (5:4, v/v) (Kurilich et al., 2003), n-hexane:acetone (6:4, v/v) (Barth et al., 1995), 2-propanol:DCM (2:1, v/v) (Barua and Olson, 1998), n-hexane:ethyl acetate (85:15, v/v) (Gimeno et al., 2000), n hexane:acetone:EtOH (50:25:25, v/v/v) (Lee, 2001; Lee et al., 2001) have been widely used. Thus, Taungbodhitham et al. (1998) evaluated six different solvent combinations: acetone:hexane (4:6, v/v), EtOH: hexane (4:3, v/v), chloroform:MeOH (2:1, v/v), DCM:MeOH (2:1, v/v), hexane:isopropanol (3:2, v/v) and acetone:petroleum ether (50:50, v/v) to extract lycopene and a- and b-carotene from canned tomato juice, and the best recoveries were obtained with the EtOH:hexane mixture. Similar results were found by Lin and Chen (2003), that used ve solvent systems, EtOH:hexane (4:3, v/v), acetone:hexane (3:5, v/v), EtOH:acetone:hexane (2:1:3, v/v/v), ethyl acetate:hexane (1:1, v/v) and ethyl acetate (100%), to compare the extraction efciency of carotenoids in tomato juice. They concluded that the best extraction efciency was achieved with EtOH:hexane (4:3, v/v). Deli et al. (2001) used MeOH followed by diethyl ether to extract carotenoids from fruits of pepper (Capsicum annuum var. lycopersiciforme rubrum). Gandul-Rojas et al. (1999) extracted chlorophylls and free and monoesteried xanthophylls with dimethyl formamide and carotenoids with hexane from olive fruits. A method for determining lutein and zeaxanthin isomers from marigold ower (Tagetes erecta) extract using hexane and ethyl ether, as extracting solvent, has been reported by Hadden et al. (1999). Other authors (Lee, 2001; Lee et al., 2001) have used a more complex mixture, hexane:acetone:EtOH to extract the major carotenoids from juice of red Navel orange, New Sweet orange and Earlygold. A mixture of THF:MeOH was chosen by Murkovic et al. (2002) to isolate a- and b-carotene and lutein from pumpkins (Cucurbita pepo, C. maxima and C. moschata). Rozzi et al. (2002) reported a method to determine lycopene, a- and b-carotene, a-, g- and d-tocopherol

ARTICLE IN PRESS
s, A. Rodrguez-Bernaldo de Quiro H.S. Costa / Journal of Food Composition and Analysis 19 (2006) 97111 99

from tomato skin and seeds using chloroform as extraction solvent. In spite of THF and ethyl ether being widely used because of the great solubility that carotenoids present in them, some researchers (Khachik et al., 1986; Marsili and Callahan, 1993) have pointed out that these solvents can develop peroxides that can rapidly degrade b-carotene, and may contribute to the production of artifacts. Therefore, some researchers have advised the addition of antioxidants such as butylated hydroxytoluene (BHT) to the solvent. On the contrary, Quackenbush and Smallidge (1986) have pointed out that the use of strong antioxidants can promote autooxidation of b-carotene. The extraction of carotenoids must be carried out very quickly, avoiding exposure to light, oxygen, high temperatures and to prooxidant metals, such as iron or copper, in order to minimize autooxidation and cis trans isomerization (van den Berg et al., 2000; Marsili and Callahan, 1993). Moreover, to prevent carotenoid losses during the extraction procedure, the addition of antioxidants, such as ascorbic acid, pyrogallol has been recommended (Sharpless et al., 1999; Nierenberg and Nann, 1992; Hagg et al., 1994; Granelli and Helmersson, 1996). It is also advisable to add magnesium or calcium carbonate to the sampleextracting solvent mixture, in order to neutralize trace levels of organic acids that are usually present in the samples (Khachik, et al., 1986, 1992a; Hart and Scott, 1995; Granado et al. 1992). The next step in the extraction procedure would be to homogenize the mixture and ltrate the resulting carotenoid extract. This process must be repeated until the ltrate is colorless. The combined ltrate is concentrated, and the carotenoids are partitioned into an appropriate organic solvent and water. The organic phase is removed and evaporated; during evaporation, it is advisable not to reach temperatures above 40 1C, to avoid degradation, and nally the residue is dissolved in a suitable amount of solvent (Sharpless et al., 1999; Hulshof et al., 1997; Khachik, et al., 1986, 1992a; Granado et al., 1992; Lin and Chen, 2003). In some studies, solid-phase extraction technique has been used prior to analysis, in order to provide a better purication of the analyte (Fisher and Rouseff, 1986; Iwase, 2002). 2.1.2. Extraction of carotenoids from plasma and serum samples In general, the extraction of carotenoids from human serum and plasma is carried out with an organic and immiscible solvent. Prior to the extraction, the samples are usually treated with EtOH to precipitate the proteins. Proteins can also be denatured by exposure to perchloric acid (Lee et al., 1992). Gueguen et al. (2002) pointed out that the addition of ultrapure water

to EtOH for deproteinizing the serum samples improved considerably the recoveries of carotenoids. They assayed different EtOH:water ratios in the range 1:4 to 1:1 (v/v), and best results were achieved with the proportion 1:1 (v/v). The most common solvent employed in the extraction step is n-hexane (Gueguen et al., 2002; Olmedilla et al., 1997; Lyan et al., 2001; Epler et al., 1993; Gimeno et al., 2001; Olmedilla et al., 1990, 1992; Talwar et al., 1998; Nierenberg and Nann, 1992). Other organic solvents are also used: butanol:ethyl acetate (1:1, v/v) (Lee et al., 1992), 2-propanol:dichlorometane (2:1 v/v) (Barua and Olson, 1998), diethyl ether (Khachik et al., 1992b) and ethyl acetate (Barua et al., 1993). Tzouganaki et al. (2002) proposed two extraction procedures: liquidliquid extraction using n-hexane as extracting solvent and solid-phase extraction using an ISOLUTE C18 cartridge for isolating lycopene from plasma samples; a relatively low recovery (62%) was achieved when solid-phase extraction was applied, probably due to the loss of lycopene during the washing and elution steps. On the contrary, good recoveries (95%) were obtained with the liquidliquid extraction method. According to Epler et al. (1993), the critical point in the analysis of carotenoids from serum samples is the dissolution of the serum extracts, because the concentrations of carotenoids are frequently near the detection limit. They suggested that the dissolution could be improved by using ultrasonic agitation; in addition, it would be advisable to use the initial mobile-phase composition to dissolve the extracts, in order to focus the sample at the head of the column. 2.2. Supercritical uid extraction (SFE) SFE has been used as an alternative method to traditional liquid extraction for isolating carotenoids from food samples, since this technique shows several advantages. For example, more speed in extraction, the evaporation step is not required, and carbon dioxide is non-toxic, its cost is low, it is non-ammable, and environmentally acceptable (Marsili and Callahan, 1993; Vagi et al., 2002). In addition, carbon dioxide has a low critical temperature (31 1C) making it ideal for extraction of thermally labile compounds (Vagi et al., 2002; Careri et al., 2001). Nevertheless, CO2 presents a low polarity and makes the extraction of polar compounds very difcult. This limitation can be solved by adding an organic modier such as MeOH or EtOH, in order to increase its solvation power (Careri et al., 2001). Regarding sample treatment, some researchers have reported that the removal of water from vegetable materials facilitates the SFE and homogenized samples are extracted much more rapidly due to the reduction of the particles size (Marsili and Callahan,

ARTICLE IN PRESS
100 s, A. Rodrguez-Bernaldo de Quiro H.S. Costa / Journal of Food Composition and Analysis 19 (2006) 97111

1993). The addition of an amount of Hydromatrix, in order to absorb the liquid portion from the sample is often used (Marsili and Callahan, 1993; Mathiasson et al., 2002). Gomez-Prieto et al. (2003) extracted all-trans-lycopene from tomato using CO2 without modier. They studied the effect of the carbon dioxide uid density on the yield of the extraction by carrying out several extractions at different densities (0.25, 0.35, 0.45, 0.55, 0.60, 0.70, 0.75, 0.80, 0.85 and 0.90 g/mL). The best results were found for 0.90 g/mL. A comparison between soxhlet extraction using n-hexane and EtOH as solvents and SFE to determine chlorophylls and carotenoids in marjoram (Origanum marojana L.) was described by Vagi et al. (2002). Three different temperatures (40, 50 and 60 1C) and three different pressures (100, 250 and 400 bar) were tested. The authors observed that the optimum conditions for extraction were 50 1C and 450 bar. Additionally, the amount of b-carotene extracted with EtOH was half of the amount obtained with supercritical uids. Ferreira de Franc a et al. (1999) applied SFE to determine lipids and carotenoids from buriti fruit (Mauritia exuosa). The effect of pressure and temperature was studied by performing a factorial experiment with replication for temperatures of 39.9 and 54.9 1C and pressures of 200 and 300 bar. The optimum conditions for this analysis were 200 bar and 39.9 1C. In another study, carried out by Baysal et al. (2000) several extraction conditions, such as temperature of the extractor (35, 45, 55 and 65 1C), pressure of the extraction uid (200, 250 and 300 bar), addition of cosolvent (5%, 10% and 15% EtOH), extraction time (1, 2 and 3 h) and CO2 ow rate (2, 4 and 8 kg/h) were optimized for the determination of lycopene and b-carotene, from tomato paste waste. The results revealed that the highest temperature used for the extraction gave the maximal extraction yield; however, the authors pointed out that, temperatures higher than 65 1C would give better extraction yield but this might cause an increase in carotene degradation. No signicant differences in lycopene and b-carotene extraction yields were observed, if the pressure was changed from 200 to 300 bar. Other authors have indicated that extraction pressures up to 400 bar can give higher yields (Vagi et al., 2002). Regarding extraction time and ow rate, the highest carotenoid yield was obtained with an extraction time of 2 h. It was observed that 1 h was not enough for extracting carotenoids and with 3 h the degradation process increased. The optimum ow rate for both lycopene and b-carotene was 4 kg/h. The addition of 5% EtOH as cosolvent improved the recoveries of both carotenoids. More recently, different extraction temperatures and pressures were assayed by Rozzi et al. (2002) to determine lycopene from tomato processing by-pro-

ducts. The results indicated that maximum recovery was achieved at 86 1C and 345 bar. Barth et al. (1995) compared classical solvent extraction and SFE for determining carotenoids from carrot (Daucus carota L.) tissue. They observed that total provitamin A activity was 7% greater with SFE than in the samples extracted with solvents. A method for selective separation of b-carotene isomers from the algae Dunaliella bardawil was investigated by Gamlieli-Bonshtein et al. (2002). The separation of the isomers was achieved due to their different dissolution rate in supercritical CO2. Several CO2 modiers (water, EtOH, methylene chloride, hexane) have been tested in order to improve the extraction efciency of a- and b-carotene from different vegetables (Marsili and Callahan, 1993). The addition of hexane did not signicantly increase the bcarotene solubility in supercritical CO2. With methylene chloride, the solubility of b-carotene increased but it induced the degradation of b-carotene. Finally, although b-carotene was less soluble in EtOH than in hexane, when EtOH was added as modier, the solubility of b-carotene in CO2 increased signicantly (Marsili and Callahan, 1993). Mendes et al. (2003) reported a supercritical carbon dioxide extraction of compounds with pharmaceutical importance from microalgae. Canthaxanthin and astaxanthin were extracted from Chlorella vulgaris. Several conditions of pressure and temperature were compared and the best results were achieved at 275 and 350 bar and 55 1C. The b-carotene produced by Dunaliella salina was a mixture of cis and trans isomers, being the cis isomer much more soluble than the trans isomer in supercritical CO2 extraction. The optimum conditions to extract the two isomers were achieved at 300 bar and 40 1C. A comparison of traditional extraction with ultrasound, microwave, sub- and SFE for unsaturated fatty acid (UFA), polyunsaturated fatty acid (PUFA) and carotene from rose hip seeds (Rosa canina L.) was presented by Szentmihalyi et al. (2002). Subcritical uid extraction appeared to be the best method for extraction of carotene while SFE was suitable for UFA and PUFA. Lopez et al. (2004) proposed the use of the SFE technique coupled to a continuous ow manifold including UV detector as a screening system to extract asthaxanthin from craysh. In Table 1 some examples of SFE conditions in carotenoid analysis are presented.

3. Saponication Prior to HPLC analysis, the saponication procedure has been often used as a step to simplify the separation by removing substances, such as chlorophylls and lipids,

ARTICLE IN PRESS
s, A. Rodrguez-Bernaldo de Quiro H.S. Costa / Journal of Food Composition and Analysis 19 (2006) 97111 Table 1 Examples of supercritical uid extraction conditions employed in carotenoid analysis Sample Analyte Supercritical uid SFE conditions (temperature, pressure, ow rate) T 40 1C Pressure: 281 bar Flow rate: 4 mL/min T 86 1C Pressure: 345 bar Flow rate: 2.5 mL/min Dry carotene beadlets: T 40 1C Pressure: 310 bar Flow rate: 2 mL/min Liver samples: T 80 1C Pressure: 310 bar Flow rate: 2 mL/min T 40 1C Pressure: 448 bar Flow rate: 0.51 mL/min T 55 1C lycopene T 65 1C b-carotene Pressure: 300 bar Flow Rate: 4 kg/h T 40 1C Pressure 342 bar Flow rate: 1.5 mL/min Reference 101

Tomato

All-trans-lycopene

CO2 without modier

Gomez-Prieto et al. (2003)

Tomato seeds and skins

Lycopene, b-carotene, acarotene, a-tocopherol, gtocopherol, and dtocopherol Vitamin A and b-carotene

CO2 without modier

Rozzi et al. (2002)

Vitamin supplements and calf liver tissue (dry carotene beadlets, liver samples)

CO2 without modier

Burri et al. (1997)

Algae Dunaliella bardawil

Geometrical isomers of bCarotene Lycopene and b-carotene

CO2 without modier

Gamlieli-Bonshtein et al. (2002) Baysal et al. (2000)

Tomato paste waste

CO2 and 5% EtOH as modier

Vegetables (carrots, collard greens, turnip greens, kale, mustard greens, broccoli owerets, zucchini, and yellow squash) Spirulina Pacica algae

a- and b-carotene

CO2 and EtOH as modier

Marsili and Callahan (1993)

b-carotene, bcryptoxanthin and zeaxanthin

CO2 and 15% EtOH as modier

T 80 1C (zeaxanthin) T 76 1C (bcryptoxanthin) T 60 1C (b-carotene) Pressure: 350 bar Flow rate: 2 mL/min T 40 1C Pressure: 606 bar Flow rate: 1 mL/min

Careri et al. (2001)

Carrots (Daucus carota L. var. Caro Pride)

a- and b-carotene

CO2 and 5% chloroform as modier

Chandra and Nair (1997)

which could interfere with the chromatographic detection. Moreover, with the saponication process, valuable information about the nature and distribution of the carotenoids present in the sample can be obtained by evaluating their chromatographic prole before and after alkali treatment (Khachik et al., 1992c). However, a loss of total carotenoid content during saponication has been reported in the literature (Granado et al., 1992; Khachik et al., 1986). Fernandez et al. (2000) have compared alkali saponication and enzymatic hydrolysis on the total carotenoid concentration of Costa Rican crude palm oil. Findings showed greater concentration of carotenoids using enzymatic hydrolysis. The most sensitive compounds to alkaline treatments are

xanthophylls, particularly the epoxycarotenoids (Khachik et al., 1986). Therefore, when the sample to be analyzed has these pigments in its composition, this purication step should be avoided. As a general rule, for samples with low fat content, milder conditions in saponication step must be applied, and for high-fat content samples severe conditions should be employed (Khachik et al., 1992c). Saponication however, should be employed to evaluate the presence of carotenol esters that may otherwise be undetected. Table 2 summarizes different saponication conditions in various samples. After the saponication phase, carotenoids are extracted with ethyl ether (Hadden et al., 1999) diethyl ether (Goodner et al., 2001), n-hexane (Howard et al.,

ARTICLE IN PRESS
102 s, A. Rodrguez-Bernaldo de Quiro H.S. Costa / Journal of Food Composition and Analysis 19 (2006) 97111 Table 2 Different saponication conditions employed in carotenoid analysis Sample Juice of red Navel Orange (Cara cara) Analyte Neoxanthin (a,b), neochrome, violaxanthin, luteoxanthin, antheraxanthin, mutatoxanthin, lutein, isolutein, zeaxanthin, a- and bcryptoxanthin, phytoene, phytouene, a-, b- and g-carotene, lycopene Lutein, zeaxanthin, lutein 5,6-epoxide, antheraxanthin, b-cryptoxanthin Capsorubin, violaxanthin, capsanthin, anteraxanthin, lutein+zeaxanthin, cantaxanthin, b-cryptoxanthin, bcarotene All-transcis isomers of zeaxanthin, all-transcis isomers of lutein, lutein esters Lutein, zeaxanthin, b-cryptoxanthin, lycopene, trans cis a- and b-carotene, retinol, retinylpalmitate, d-, g- and atocopherol Lutein, zeaxanthin, lycopene, bcryptoxanthin, a-, b- and g-carotene Saponication conditions 10% methanolic KOH sol. (Overnight, room T, darkness) Reference Lee (2001)

Ultrafrozen orange juice Fatty foods (fat-cured crude sausage Sobrassada)

10% methanolic KOH sol. (1 h, room T, darkness) 10% methanolic KOH sol. containg 0.01% BHA (5 min, 50 1C)

Melendez-Mart nez et al. (2003) Oliver et al. (1998)

Marigold (Tagetes erecta) ower extract Standard Reference Material 2383 (Baby Food Composite)

15% methanolic KOH sol. (1 h, darkness) 40% methanolic KOH sol. (30 min, room T)

Hadden et al. (1999)

Sharpless et al. (1999)

Raw and cooked Spanish vegetables (lettuce, artichokes, Brussel sprouts, green beans, asparagus (green), beet, green peppers, spinach, tomato, red peppers, carrots, red cabbage, cucumber, squash, potato, onion, cabbage, cauliower) Edible wild vegetable Stinging Nettle (Urtica dioica L.) Virgin olive oil Corn Fresh and processed vegetables (broccoli, carrots and green beans) Sweetpotato (Ipomoea batatas, L.) Milk samples

Saturated methanolic KOH sol. (Under nitrogen atmosphere, 30 min, darkness)

Granado et al. (1992)

Lutein, lutein isomers, b-carotene, bcarotene isomers, neoxanthin, violaxanthin, lycopene a-tocopherol and b-carotene Lutein, zeaxanthin, and bcryptoxanthin Trans-b-carotene

Methanolic KOH sol. (room T)

Guil-Guerrero et al. (2003) Gimeno et al. (2000) Moros et al. (2002) Howard et al. (1999)

76% ethanolic KOH sol. (Under nitrogen atmosphere, 30 min, 70 1C) 80% ethanolic KOH sol. (In a water bath at boiling point, 10 min) 100% ethanolic KOH sol. (30 min, 70 1C) 10% ethanol:water (50:50, v/v) for 1 h, 80 1C 60% aqueous KOH sol. containing pyrogallol as antioxidant (30 min, 30 1C) 60% aqueous KOH sol. containing pyrogallol as antioxidant (under nitrogen atmosphere, 30 min, 70 1C) 80% aqueous KOH sol. (15 min, 70 1C)

a-carotene, b-carotene b-carotene

Huang et al. (1999) Granelli and Helmersson (1996) Ye et al. (2000)

Fortied foods (fortied breakfast cereal, peanut butter and margarine) Kale (Brassica oleracea var. Acephala cv. Vates)

All-rac-alpha-tocopheryl acetate, retinyl palmitate, b-carotene Lutein, b-carotene, retinol, phylloquinone

Kurilich et al. (2003)

1999), methylene chloride (Melendez-Mart nez et al., 2003), petroleum ether (Hart and Scott, 1995), or with mixtures, such as n-hexane:diethyl ether (70:30, v/v) (Heinonen et al., 1989; Otles and Atli, 2000), diethyl ether:petroleum ether (1:1, v/v) (Sharpless et al., 1999), hexane:ethyl acetate (85:15, v/v) (Gimeno et al., 2000), n-hexane:toluene (10:8, v/v) (Kurilich et al., 2003) and then, the extract is washed until KOH is eliminated. Khachik et al. (1986) observed an important loss in the xanthophylls content of raw broccoli after applying

a treatment with 30% methanolic potassium hydroxide under nitrogen atmosphere at room temperature during 3 h. On the contrary, the loss of carotenes was not signicant. Ye et al. (2000) reported that direct solvent extraction method presents an alternative technique to saponication for analysis of vitamins and beta-carotene in various fortied foods. Granado et al. (1992) pointed out in their paper, a loss in the concentration of xanthophylls related to the

ARTICLE IN PRESS
s, A. Rodrguez-Bernaldo de Quiro H.S. Costa / Journal of Food Composition and Analysis 19 (2006) 97111 103

saponication step. This loss in concentration can be partially corrected when the quantication is made on the basis of a calibration curve, which is also subject to the process of saponication. Hart and Scott (1995) for example, determined the carotenoid content in vegetables and fruits commonly consumed in United Kingdom. The following saponication procedure of carotenoid extracts was only applied for fruits and certain vegetables, like peppers: methanolic potassium hydroxide (10%) under nitrogen, in the dark for 1 h at room temperature. Oliver et al. (1998) investigated the carotenoid prole of fat-cured crude sausage (Sobrassada) before and after the saponication procedure, and have reported that the alkaline treatment of the carotenoid extracts hydrolyses all carotenoid esters. This treatment allowed the identication of other simple carotenoids, such as cryptocapsin, which were not detected in the nonsaponied extract. Several researchers have observed better carotenoid extraction when high temperatures are employed in the saponication step, but at the expense of xanthophylls recovery (Khachik et al., 1986; Scott, 1992). The traditional liquid extraction procedures described in the literature are cumbersome, labour intensive, timeconsuming, and involve multiple-step and the use of large amounts of volatile organic solvents with known health hazards and negative environmental impact. Fish et al. (2002) have developed a quantitative assay for lycopene that utilizes reduced volumes of organic solvents. Therefore, the interest in developing new methods for carotenoid extraction is increasing.

4. Chromatographic analysis of different samples Numerous methods for determining carotenoids in vegetables samples, as well as in human serum and plasma have been reported in the literature. Some of the examples recently published are shown in the present paper. Lin and Chen (2003) developed a method to determine the various carotenoids present in tomato juice, including all-trans-lutein, all-trans-b-carotene, alltrans-lycopene and their 13 cis-isomers. The separation was achieved by using a C30 column (250 mm 4.6 mm i.d., 5 mm particle size) from YMC and a gradient of two eluents, (A) ACN:1 butanol (70:30, v/v) and (B) methylene chloride. The analysis was completed within 55 min at a ow rate of 2 mL/min and the wavelength was set at 476 nm. A high-performance liquid chromatographic method to determine chlorophylls, carotenoids and their derivatives in some fresh and processed vegetables have been described (Gokmen et al., 2002). A C8 MikroPak

(150 mm 4.6 mm i.d., 5 mm particle size) stainless steel column and a gradient mixture of MeOH:water as the mobile phase at a ow rate of 0.75 mL/min was used. The chromatograms were recorded simultaneously at 432, 450, 470, 652 and 666 nm using a DAD. A quaternary mixture of MeOH:ACN:methylene chloride:water (50:30:15:5, v/v/v/v) containing 0.1% BHT and 0.1% TEA as antioxidant and modier, respectively, and a C18 Kromasil (250 mm 4.6 mm i.d., 5 mm packing) were used by Melendez-Mart nez et al. (2003) to analyze the carotenoid prole in ultrafrozen orange juices. A more specic wavelength of 486 nm was selected and a ow rate of 2.5 mL/min. Carotenoids were identied by comparison with the spectra of the standards obtained with a DAD from 350 to 800 nm. Another method to determine chlorophyll and carotenoid pigments, in six cultivars of processed green peas (Pisum sativum, L.), was proposed by Edelenbos et al. (2001). A binary solvent gradient consisting of solvent (A) MeOH:water (80:20, v/v) and solvent (B) 100% ethyl acetate at a ow rate of 1 mL/min was used as mobile phase. The separations were performed on a LiChrospher 100 RP-18 column (5 mm packing, 244 mm 4 mm i.d.) and the temperature was maintained at 30 1C. Chromatograms were recorded at 440 nm with a DAD and absorption spectra of carotenoids and chlorophylls were recorded between 300 and 600 nm. More recently, Gomez-Prieto et al. (2003) achieved an optimal separation of phytoene, phytouene, b-carotene and lycopene, as well as all-trans-lycopene isomers from tomato samples. A linear gradient of two eluents (A) MeOH:water (96:4, v/v) and eluent (B) methyl-tert-butyl ether (MTBE) was used and ow rate was set at 1 mL/ min. A Develosil UG C30 (250 mm 4.6 mm i.d.) column maintained at 20 1C was employed and b-apocarotenal was used as internal standard. In order to determine, in the same injection, different carotenoids four wavelengths (285, 347, 450 and 472 nm) were selected. Vagi et al. (2002) developed an isocratic and reversed-phase high-performance liquid chromatographic method to determine chlorophylls and carotenoids in marjoram (Origanum majorana L.). In this study, a Nucleosil 5 C18 stainless steel column (250 mm 4 mm i.d.) and a ACN:MeOH:isopropyl alcohol (39:43:18, v/v/v) mobile phase at a ow rate of 0.9 mL/min was reported. Chromatograms were recorded at a wavelength of 430 nm. Analysis of xanthophylls in corn using a gradient system of two eluents (A) MeOH:MTBE:water (81:15:4, v/v/v) and (B) MeOH:MTBE (9:91, v/v) and a C30 column (4.6 mm 250 mm) was described by Moros et al. (2002). The ow rate was 1 mL/min and the chromatogram was monitored at 450 and 445 nm using a DAD.

ARTICLE IN PRESS
104 s, A. Rodrguez-Bernaldo de Quiro H.S. Costa / Journal of Food Composition and Analysis 19 (2006) 97111

Two normal-phase columns and a reversed-phase column were used to analyze the carotenoid in marigold ower (Tagetes erecta) extract (Hadden et al., 1999). A Zorbax SIL 5 mm column (250 mm 4.6 mm i.d.) and hexane:ethyl acetate (75:25, v/v) as mobile phase at a ow rate of 2 mL/min was used to separate all-translutein, cis-lutein isomers, as well as all-trans-zeaxanthin. The other normal-phase system included a b Cyclobond 5 mm column (250 mm 4.6 mm i.d.) with n-hexane:ethyl acetate (87:13, v/v) as mobile phase and a ow rate of 2.0 mL/min was employed to identify all-trans and cis isomers of lutein and all-trans-zeaxanthin. With the reversed-phase chromatography, a more complete separation of cis-lutein isomers was achieved. The analysis was performed using a YMC S3-SIL200 3 mm silica C30 column with a mobile phase consisting of 3% MTBE in MeOH at a ow rate of 1 mL/min. All-translutein, 9- and 90 -cis-lutein isomers, 13- and 130 -cis-lutein isomers and all-trans-zeaxanthin were identied. In all cases, the carotenoids were detected at 450 nm. In a recent study (Lee et al., 2001), more than 25 carotenoids from a sweet orange (Earlygold) sample were separated within 40 min using a ternary gradient elution consisting of: eluent (A) ACN:MeOH (75:25, v/ v), eluent (B) 100% MTBE and eluent (C) water containing 0.01% BHT and 0.05% TEA at a ow rate of 1 mL/min. The separation was performed on a C30 column (150 mm 4.6 mm i.d., 3 mm) from YMC maintained at 25 1C. In another experiment described by this author, the same conditions were applied to identify 29 carotenoids in juice from another type of orange, Red Navel orange (Cara cara). Carotenoids were detected at 450 nm (Lee, 2001). A method was developed to differentiate citrus of orange, mandarin and hybrid on the basis of their carotenoid prole. A gradient elution system, which was composed by MeOH, water and MTBE and, a C30 YMC column (25 cm 4.6 mm i.d., 5 mm) was used for the separation. The analysis was completed in 50 min, at a ow rate of 1 mL/min. In order to maximize absorbance in the red/orange region of the visible spectrum a wavelength of 486 nm was selected (Goodner et al., 2001). Previously, Mouly et al. (1999) employed another mobile phase that consisted of MeOH, MTBE and water to determine the carotenoid prole of Valencia orange juice. Carotenoids were identied using a DAD at 350, 430 and 486 nm. The separation was performed on a YMC C30 stainless steel column (25 cm 4.6 mm i.d., 5 mm). Many methods have also been reported for the analysis of carotenoids in biological samples. Thus, Lyan et al. (2001) described a method in which 13 identied carotenoids and 9 unknown carotenoids were separated from human plasma samples using a Nucleosil C18 column (15 cm 4.6 mm, 3 mm) and a Vydac C18

TP54 column (25 cm 4.6 mm, 5 mm) in series and a mixture of ACN:MeOH (containing 50 mM ammonium acetate):DCM:water (70:15:10:5, v/v/v/v) as mobile phase at a ow rate of 2 mL/min. More recently, another research group (Gueguen et al., 2002) proposed an isocratic method to determine ve carotenoids (lutein, zeaxanthin, b-cryptoxanthin, lycopene and a- and b-carotene), a-tocopherol and retinol from human serum. MeOH:ACN:THF (75:20:5, v/v/v) containing 0.01% BHT (w/v) was used as mobile phase, and a Nucleosil 100 C18 (25 cm 3 mm i.d., 5 mm) as stationary phase. Column temperature was set at 35 1C and a ow rate of 0.6 mL/min. Chromatograms were monitored at 290, 325 and 450 nm for the identication of a-tocopherol, retinol and carotenoids, respectively. Another isocratic reversed-phase liquid chromatographic method has been reported in the literature (Tzouganaki et al., 2002) to determine lycopene in plasma. The analysis was performed on a Nova Pak C18 column (3.9 mm 15 cm, 5 mm particle size) and the mobile phase consisted of MeOH:ACN:methylene chloride (55:30:15, %,v/v) at a ow rate of 1 mL/min. The detection was done at 472 nm.

4.1. High-performance liquid chromatography Carotenoid analysis of food and biological samples are mainly performed by HPLC. Reversed-phase separations (Rozzi et al., 2002; Edelenbos et al., 2001; Moros et al., 2002; Melendez-Mart nez et al., 2003; Murkovic et al., 2002; Hentschel et al., 2002; Lee, 2001; Lee et al., 2001; Lin and Chen, 2003; Deli et al., 2001; Iwase, 2002; Goodner et al., 2001; Vagi et al., 2002; Careri et al., 2001; Mathiasson et al., 2002; Gomez-Prieto et al., 2003; Gueguen et al., 2002; Lyan et al., 2001; Bako et al., 2002; Azevedo-Meleiro and Rodriguez-Amaya, 2004) have been widely used in the determination of this kind of compounds, although several normal-phase methods (Hollman et al., 1993; Casal et al., 2001; Englberger et al., 2003a) have also been reported in the literature. Both isocratic (Melendez-Mart nez et al., 2003; Mathiasson et al., 2002; Gamlieli-Bonshtein et al., 2002; Gueguen et al., 2002) and gradient elution systems (Rozzi et al., 2002; Moros et al., 2002; Gokmen et al., 2002; Lin and Chen, 2003; Gomez-Prieto et al., 2003; Azevedo-Meleiro and Rodriguez-Amaya, 2004) have been employed. In general, with gradient solvent methods, the resolution achieved is better than with isocratic systems. However, the former presents some drawbacks, such as a higher total analysis time because of the necessity to reequilibrate the column after each injection which represents a serious problem for routine analysis (Melendez-Mart nez et al., 2003).

ARTICLE IN PRESS
s, A. Rodrguez-Bernaldo de Quiro H.S. Costa / Journal of Food Composition and Analysis 19 (2006) 97111 105

4.1.1. Mobile phase Acetonitrile (ACN), MeOH or mixtures of these solvents are the major constituents of the mobile phases used in the analysis of carotenoids. To optimize the separation of some carotenoids (for example geometrical isomers), the mobile phase is often modied by the addition of small amounts of other organic solvents (van den Berg et al., 2000); for example, DCM (Heinonen et al., 1989; Murkovic et al., 2002; Olmedilla et al., 1997; BakO et al., 2002), water (Edelenbos et al., 2001; Gokmen et al., 2002; Lee, 2001; Goodner et al., 2001; Mathiasson et al., 2002), hexane (Ye et al., 2000; Barua, 2001; Khachik et al., 1992a; Casal et al., 2001; Chandrika et al., 2003), acetone (Englberger et al., 2003a), chloroform (Huck et al., 2000; Schmitz et al., 1989), THF (Hulshof et al., 1997; Baysal et al., 2000; Gueguen et al., 2002; Talwar et al., 1998; Nierenberg and Nann, 1992; Burri et al., 1997; Englberger et al., 2003b), propanol (Rozzi et al., 2002; Markus et al., 1999; Hentschel et al., 2002; Vagi, et al., 2002), ethyl acetate (Edelenbos et al., 2001; Hadden et al., 1999; Epler et al., 1993; Nierenberg, 1985), methylene chloride (Khachik et al., 1986; Marsili and Callahan, 1993; Melendez-Mart nez et al., 2003; Barth et al., 1995; Tzouganaki et al., 2002) or various ethers (Mouly et al., 1999; Moros et al., 2002; Gomez-Prieto et al., 2003; Lacker et al., 1999). MeOH has been recommended in several reports (Epler et al., 1992, 1993) because it provides better recoveries than ACN or ACN-based solvents. In the same way, THF provided slightly higher recoveries than ethyl acetate (Huck et al., 2000; Epler et al., 1993). The use of chloroform must be avoided, as far as possible because of high toxicity; in addition, chlorinated solvents are associated with carotenoid losses (Huck et al., 2000). Antioxidants, such as ascorbic acid (Talwar et al., 1998) or BHT (Huck et al., 2000; Melendez-Mart nez et al., 2003; Murkovic et al., 2002; Hart and Scott, 1995; Lee, 2001; Lee et al., 2001; Deli et al., 2001; Gueguen et al., 2002; Lyan et al., 2001) are often added to mobile phases to stabilize them. Gueguen et al. (2002) observed a decrease of carotenoids with time when ascorbic acid was added to the mobile phase. On the contrary, in standard solutions, BHT protected efciently the carotenoids from the degradation process. Carotenoids are susceptible to oxidation and may undergo on-column degradation. Different studies (Huck et al., 2000; Hart and Scott, 1995; Epler et al., 1993) have revealed that the addition of solvent modiers, for example triethylamine (TEA) or ammonium acetate, to the mobile phase reduces losses or degradation. TEA behaves as a strong modier, and it reduces the retention time. It was tested, however, that concentrations around 0.05% did not change signicantly the elution times, and a good separation could be achieved (Hart and Scott, 1995).

Epler et al. (1993) described a method for determining the six major carotenoids in human serum (lutein, zeaxanthin, b-cryptoxanthin, lycopene, a- and b-carotene), as well as retinoids and tocopherols using several different columns and a gradient that included ACN, MeOH and ethyl acetate. It was reported that when 0.1% TEA was added to the solvents, the recovery was around 89% and only when 0.05 M ammonium acetate was added to MeOH, the recovery increased up to 94%. Therefore, it was suggested that the two modiers have different functions and only with the addition of both, TEA and ammonium acetate, the recovery could be improved. On the contrary, Hart and Scott (1995) did not observe signicant improvements on recovery when 0.05 M ammonium acetate was added to a mobile phase containing 0.05% TEA. However, due to the positive effect found by Epler et al. (1993), these authors added both ammonium acetate and TEA to the mobile phase. They pointed out that the specic actions of these compounds are unknown, though it is suggested that the modiers provide sufcient buffer capacity (considering that acidity was the critical factor for the recovery) to the mobile phase or, prevents reactions with free metal ions, so that with their use the recoveries achieved would be higher. Huck et al. (2000) investigated the selectivity factors for lutein/zeaxanthin and zeaxanthin/b-carotene for the evaluation of different mobile phase systems. A new quaternary mobile phase ACN:MeOH (containing 0.05% TEA and 0.05 M ammonium acetate):chloroform:n-heptane (75:25:2.5:2.5, v/v/v/v) was developed in order to improve the recoveries and separation of carotenoids. 4.1.2. Column Reversed-phase HPLC is widely used to separate carotenoids from different samples. C8 (Gokmen et al., 2002) and mainly C18 columns (Huck et al., 2000; Heinonen et al., 1989; Otles and Atli, 2000; Hagg et al., 1994; Hulshof et al., 1997; Granado et al., 1992; Khachik et al., 1986, 1992a; Gandul-Rojas et al., 1999; Howard et al., 1999; Markus et al., 1999; Sharpless et al., 1999; Scott and Hart, 1993; Murkovic et al., 2002; Taungbodhitham et al., 1998; Deli et al., 2001; Olmedilla et al., 1997; Howard et al., 2000; Riso and Porrini, 1997; Lunetta et al., 2002; Kiss et al., 2000; Darko et al., 2000; Englberger et al., 2003b; Huang et al., 1999; Assunc ao and Mercadante, 2003; Azevedo- Meleiro and Rodriguez-Amaya, 2004) have often been selected by researchers to perform the analysis. Epler et al. (1992) compared 65 reversed-phase liquid chromatographic columns to determine the selectivity and recovery of a mixture of the following carotenoids: lutein, zeaxanthin, b-cryptoxanthin, echinenone, lycopene, and a- and b-carotene using ACN and MeOH modied with THF or ethyl acetate as mobile phase.

ARTICLE IN PRESS
106 s, A. Rodrguez-Bernaldo de Quiro H.S. Costa / Journal of Food Composition and Analysis 19 (2006) 97111

Stationary phases were mainly C18, and were classied as monomeric, intermediate or polymeric. They found that the separation of lutein and zeaxanthin was only achieved by employing polymeric and some intermediate columns, but these columns provided lower recoveries than monomeric C18 columns. The authors also observed that the pore size of the column could affect the selectivity of carotenoids with different size, like zeaxanthin and b-carotene, but it did not affect if the size was very similar (for example a- and b-carotene). It has been reported in the literature (Khachik et al., 1992c) that C18 columns show a poor resolution in the separation of geometric (cis trans) isomers of carotenoids. Sander et al. (1994) developed a polymeric C30 liquid chromatographic column, specially designed for the separation of carotenoid isomers. Since then, numerous analytical methods have employed polymeric C30 columns and have been reported in the literature (Mouly et al., 1999; Moros et al., 2002; Hentschel et al., 2002; Kurilich et al., 2003; Lee, 2001; Lee et al., 2001; Gomez-Prieto et al., 2003; Dachtler et al., 1998; Lacker et al., 1999). Recently, a comprehensive review has described the application of C30 columns in the analysis of carotenoids, retinoids and other nutrients in natural samples (Sander et al., 2000). Dachtler et al. (1998) achieved a high resolution of lutein and zeaxanthin stereoisomers from bovine retine using a C30 stationary phase. Satisfactory separations of all trans-lycopene from tomato employing a C30 column were obtained by Gomez-Prieto et al. (2003). Marsili and Callahan (1993) tested three commercial C18 reversed-phase columns; Waters Novo-Pak column (4 mm particle size, 3.9 mm 150 mm), Supelcosil column (5 mm particle size, 4.6 mm 250 mm) and Vydac 201TP column (5 mm particle size, 4.6 mm 250 mm) in order to investigate the most suitable column to separate a-carotene from b-carotene. The results indicated that the best resolution was achieved with Vydac 201TP column and it was selected to determine b-carotene in vegetables. Khachik et al. (1992b) proposed an isocratic method to determine carotenoids, their oxidation product, as well as vitamins A and E from human plasma. A C18 and a silica-based nitrile-bonded column were used to separate polar carotenoids. The main xanthophylls in corn (lutein, zeaxanthin and b-cryptoxanthin) have been perfectly resolved, in less than 25 min, using a C30 column (Moros et al., 2002). 4.1.2.1. Column temperature. Another important factor that should be taken into account to achieve a satisfactory separation of carotenoids is the column temperature. Several authors (Huck et al., 2000; Scott and Hart, 1993) have reported that changes in ambient temperature cause signicant changes in the chromato-

graphic response of carotenoids; therefore, it is important to work at constant temperature. Scott and Hart (1993) studied the effect of column temperature in the separation of a mixture of carotenoids in a reference standard solution and in an extract of a dried food mixture. Five different temperatures were tested (15, 20, 22.5, 25 and 30 1C) and the results indicated that changes in the temperature affect both elution time and prole. The best separation was achieved at 2022.5 1C. They also pointed out that the optimal working temperature should be assessed not only for different stationary phases but also for the same stationary phase when the column was replaced. Likewise, Huck et al. (2000) optimized the column temperature in order to achieve an efcient separation of lutein, zeaxanthin, b-cryptoxanthin and b-carotene. The temperature was varied between 21 and 80 1C. The best selectivity was achieved at 21 1C and, at temperatures higher than 60 1C carotenoids degradation was signicant. 4.2. Liquid chromatography-mass spectrometry (LC-MS) As reported in several studies, LC-MS is an innovative and powerful analytical tool for the identication of carotenoids, since it provides information about the structure and in addition, it is a very sensitive technique. The LC-MS methods developed for carotenoid analysis include mainly atmospheric pressure ionization interfaces (APCI) (Huck et al., 2000; Murkovic et al., 2002; Kurilich et al., 2003; Khachik et al., 1992a, b; Lacker et al., 1999; Lienau et al., 2003; Weller and Breithaupt, 2003; Fang et al., 2003; van Breemen et al., 2002; Breithaupt et al., 2002; Hagiwara et al., 1998; Wang et al., 2000) or electrospray ionization interfaces (ESI) (Hadden et al., 1999; Careri et al., 1999). Lacker et al. (1999) developed a method for the identication of a mixture of carotenoids, including astaxanthin, cantaxanthin, zeaxanthin, echinenone and b-carotene, as well as cistrans isomers of b-carotene using LC-MS in the APCI mode. The analysis was performed on a 25 cm 4.6 mm column, packed with ProntoSil silica gel 3 mm, modied with triacontyltrichlorosilane C30 and a mobile phase of MeOH:MTBE (70:30, v/v) was employed. Mass spectra were achieved by scanning the mass range from m/z 200 to 700. The limit of detection for b-carotene determined in positiveion APCI-MS was 1 pmol. An HPLC-MS-MS (APCI) method for the identication of carotenoids in different vegetables was described by Huck et al. (2000). A Phenomenex Luna C18 (25 cm 2 mm, 5 mm) column was used as stationary phase and the mobile phase system consisted of ACN (0.1% BHT): MEOH (containing 0.05 M ammonium acetate and 0.05% TEA):CHCl3 (containing 0.1%

ARTICLE IN PRESS
s, A. Rodrguez-Bernaldo de Quiro H.S. Costa / Journal of Food Composition and Analysis 19 (2006) 97111 107

BHT):n-heptane (containing 0.1% BHT) (50:40:5:5, v/v/v/v) at a ow rate of 0.2 mL/min. Mass spectra were acquired over the scan range m/z 3002000. The detection limits obtained in positive-ion mode were in the range of nanogram. Several authors have used mass spectrometry to ensure correct peak identication and purity in a complex matrix. Thus, Murkovic et al. (2002) and Khachik et al. (1992b) both used LC-MS-APCI in positive-ion mode to conrm the carotenoids present in different varieties of pumpkins and to identify the structure of keto- and hydroxycarotenoids in human plasma, respectively. An HPLC-MS (ESI) was used by Hadden et al. (1999) to conrm the carotenoids present in marigold ower extract. Fang et al. (2003) proposed a method to determine total lycopene and its cis and all trans isomers in human plasma using LC-MS-MS in the negative-ion mode because in these conditions, a fragment of m/z 467 was formed from the molecular ion m/z 536 by elimination of a terminal isoprene group. Total lycopene was eluted as a single peak on C18 column and an isocratic mobile phase consisting of ACN:MTBE (95:5, v/v) was employed. To separate isomers, a C30 column and a gradient system composed by MeOH and MTBE was used. Another attractive application of LC-APCI-MS method, to detect stable isotopic labelled carotenoids was described by Kurilich et al. (2003). Careri et al. (1999) reported an interesting reversedphase liquid chromatography-electrospray mass spectrometry interfaced with TurboIonspray for the separation of b-carotene and xanthophylls. The separation was achieved on two ODS Hypersil columns connected in series (200 2.1 mm, 5 mm and 100 2.1 mm, 5 mm) and a mobile phase of ACN:MeOH (0.1 M ammonium acetate):DCM. The determinations were performed by operating the mass spectrometer in the positive-ion mode over m/z 500650. Breithaupt et al. (2002) used a LC-APCI-MS for the identication of 8 lutein monoesters produced from lutein diesters of marigold owers (Tagetes erecta L.) after an incomplete enzymatic saponication of lutein diester. Lutein diesters from several fruits were also identied. The separation was performed on a C30 YMC column (250 4.6 mm i.d.) and the mobile phase consisted of two eluents (A) MeOH:MTBE:water (81:15:4, v/v/v) and (B) MeOH:MTBE:water (6:90:4, v/v/v). Mass spectra were acquired over the scan range m/z 801200. The authors developed a method, using the same conditions, to evaluate the carotenoids in chicken plasma after feeding diets containing free and esteried lutein (Breithaupt et al., 2003). A method to separate and identify zeaxanthin esters of several plant extracts using LC-APCI-MS has been

described (Weller and Breithaupt, 2003). For separation, a C30 YMC column (250 4.6 mm i.d., 5 mm) maintained at 35 1C was used. Mass spectra of zeaxanthin esters were achieved by scanning the mass range from m/z 400 to 1200. The limit of detection for zeaxanthin diesters was estimated to be 0.4 mg/mL. Carotenoids present in mango, all-trans-b-carotene, all-trans- and cis-b-cryptoxanthin, all-trans-zeaxanhin, luteoxanthin isomers, all-trans- and cis-violaxanthin and all-trans and cis-neoxanthin, were identied using a mass spectrometer coupled to a liquid chromatograph in an experiment carried out by Mercadante et al. (1997).

Acknowledgements A. Rodr guez-Bernaldo de Quiros would like to thank Xunta de Galicia for providing a Grant from the programme stages in research centres and to Dr. M. Antonia Calhau who welcomed her at Centro de Seguranc a Alimentar e Nutric ao, Instituto Nacional - de Saude Dr. Ricardo Jorge during this period of time. References
Almeida, L.B., Penteado, M.V.C., 1988. Carotenoids and pro-vitamin A value of white eshed Brazilian sweet potatoes (Ipomoea batatas Lam.). Journal of Food Composition and Analysis 1, 341352. Almeida-Muradian, L.B., Magri, L.M., Rios, M.D.G., 1997. Chemical characterization of Brazilian beet leaves: provitamin A value with and without the saponication step for raw and cooked samples. Bollettino Chimico Farmaceutico. 136, 537542. Almeida-Muradian, L.B., Rios, M.D.G., Sasaki, R., 1998. Determination of provitamin A of green leafy vegetables by high performance liquid chromatography and open column chromatography. Bolletino Chimico Farmaceutico. 137, 290294. Assuncao, R.B., Mercadante, A.Z., 2003. Carotenoids and ascorbic - acid composition from commercial products of cashew apple (Anacardium occidentale L.). Journal of Food Composition and Analysis 16, 647657. Azevedo-Meleiro, C.H., Rodriguez-Amaya, D.B., 2004. Conrmation of the identity of the carotenoids of tropical fruits by HPLC-DAD and HPLC-MS. Journal of Food Composition and Analysis 17, 385396. Bako, E., Deli, J., Toth, G., 2002. HPLC study on the carotenoid composition of Calendula products. Journal of Biochemical and Biophysical Methods. 53, 241250. Barth, M.M., Zhou, C., Kute, K.M., Rosenthal, G.A., 1995. Determination of optimum conditions for supercritical uid extraction of carotenoids from carrot (Daucus carota L.) Tissue. Journal of Agricultural and Food Chemistry 43, 28762878. Barua, A.B., 2001. Improved normal-phase and reversed-phase gradient high-performance liquid chromatography procedures for the analysis of retinoids and carotenoids in human serum, plant and animal tissues. Journal of Chromatography A 936, 7182. Barua, A.B., Olson, J.A., 1998. Reversed-phase gradient highperformance liquid chromatographic procedure for simultaneous analysis of very polar to nonpolar retinoids, carotenoids and tocopherols in animal and plant samples. Journal of Chromatography B 707, 6979.

ARTICLE IN PRESS
108 s, A. Rodrguez-Bernaldo de Quiro H.S. Costa / Journal of Food Composition and Analysis 19 (2006) 97111 Englberger, L., Schierle, J., Marks, G.C., Fitzgerald, M.H., 2003a. Micronesian banana, taro, and other foods: newly recognized sources of provitamin A and other carotenoids. Journal of Food Composition and Analysis 16, 319. Englberger, L., Aalbersberg, W., Ravi, P., Bonnin, E., Marks, G.C., Fitzgerald, M.H., Elymore, J., 2003b. Further analyses on Micronesian banana, taro, breadfruit and other foods for provitamin A carotenoids and minerals. Journal of Food Composition and Analysis 16, 219236. Epler, K.S., Sander, L.C., Ziegler, R.G., Wise, S.A., Craft, N.E., 1992. Evaluation of reversed-phase liquid chromatographic columns for recovery and selectivity of selected carotenoids. Journal of Chromatography 595, 89101. Epler, K.S., Ziegler, R.G., Craft, N.E., 1993. Liquid chromatographic method for the determination of carotenoids, retinoids and tocopherols in human serum and in food. Journal of Chromatography 619, 3748. Fang, L., Pajkovic, N., Wang, Y., Gu, C., van Breemen, R.B., 2003. Quantitative analysis of lycopene isomers in human plasma using high-performance liquid chromatography-tandem mass spectrometry. Analytical Chemistry 75, 812817. Fernandez, R.X.E., Shier, N.W., Watkins, B.A., 2000. Effect of alkali saponication, enzymatic hydrolysis and storage time on the total crotenoid concentration of Costa Rican crude palm oil. Journal of Food Composition and Analysis 13, 179187. Ferreira de Franca, L., Reber, G., Meireles, M.A.A., Machado, N.T., Brunner, G., 1999. Supercritical extraction of carotenoids and lipids from buriti (Mauritia exuosa), a fruit from the Amazon region. Journal of Supercritical Fluids 14, 247256. Ferruzzi, M.G., Sander, L.C., Rock, C.L., Schwartz, S.J., 1998. Carotenoid determination in biological microsamples using liquid chromatography with a coulometric electrochemical array detecor. Analytical Biochemistry 256, 7481. Fish, W.W., Perkins-Veazie, P., Collins, J.K., 2002. A quantitative assay for lycopene that utilizes reduced volumes of organic solvents. Journal of Food Composition and Analysis 15, 309317. Fisher, J.F., Rouseff, R.L., 1986. Solid-phase extraction and HPLC determination of b-cryptoxanthin and a- and b-carotene in orange juice. Journal of Agricultural and Food Chemistry 34, 985989. Gamlieli-Bonshtein, I., Korin, E., Cohen, S., 2002. Selective separation of cistrans geometrical isomers of b-Carotene via CO2 supercritical uid extraction. Biotechnology and Bioengineering 80, 169174. Gandul-Rojas, B., Cepero, M.R.-L., M nguez-Mosquera, M.I., 1999. Chlorophyll and carotenoid patterns in olive fruits, Olea europaea Cv. Arbequina. Journal of Agricultural and Food Chemistry 47, 22072212. Gimeno, E., Calero, E., Castellote, A.I., Lamuela-Raventos, R.M., de la Torre, M.C., Lopez-Sabater, M.C., 2000. Simultaneous determination of a-tocopherol and b-carotene in olive oil by reversedphase high-performance liquid chromatography. Journal of Chromatography A 881, 255259. Gimeno, E., Castellote, A.I., Lamuela-Raventos, R.M., de la Torre Boronat, M.C., Lopez-Sabater, M.C., 2001. Rapid high-performance liquid chromatographic method for the simultaneous determination of retinol, alpha-tocopherol and beta-carotene in human plasma and low-density lipoproteins. Journal of Chromatography B 758, 315322. Gokmen, V., Bahceci, S., Acar, J., 2002. Liquid chromatographic method for the determination of chlorophylls, carotenoids, and their derivatives in fresh and processed vegetables. Journal of Liquid Chromatography and Related Technologies 25, 12011213. Gomez-Prieto, M.S., Caja, M.M., Herraiz, M., Santa-Mar a, G., 2003. Supercritical uid extraction of all-trans-Lycopene from tomato. Journal of Agricultural and Food Chemistry 51, 37. Barua, A.B., Kostic, D., Olson, J.A., 1993. New simplied procedures for the extraction and simultaneous high-performance liquid chromatographic analysis of retinol, tocopherols and carotenoids in human serum. Journal of Chromatography 617, 257264. Baysal, T., Ersus, S., Starmans, D.A.J., 2000. Supercritical CO2 extraction of beta-carotene and lycopene from tomato paste waste. Journal of Agricultural and Food Chemistry 48, 55075511. Bone, R.A., Landrum, J.T., Dixon, Z., Chen, Y., Llerena, C.M., 2000. Lutein and zeaxanthin in the eyes, serum and diet of human subjects. Experimental Eye Research 71, 239245. Breithaupt, D.E., Wirt, U., Bamedi, A., 2002. Differentiation between lutein monoester regioisomers and detection of lutein diesters from marigold owers (Tagetes erecta L.) and several fruits by liquid chromatography-mass spectrometry. Journal of Agricultural and Food Chemistry 50, 6670. Breithaupt, D.E., Weller, P., Grashorn, M.A., 2003. Quantication of carotenoids in chicken plasma after feeding free or esteried lutein and capsanthin using high-performance liquid chromatography and liquid chromatography-mass spectrometry analysis. Poultry Science 82, 395401. Burri, B.J., Neidlinger, T.R., Lo, A.O., Kwan, C., Wong, M.R., 1997. Supercritical uid extraction and reversed-phase liquid chromatography methods for vitamin A and beta-carotene heterogeneous distribution of vitamin A in the liver. Journal of Chromatography A 762, 201206. Careri, M., Elviri, L., Mangia, A., 1999. Liquid chromatographyelectrospray mass spectrometry of b-carotene and xanthophylls: validation of the analytical method. Journal of Chromatography A 854, 233244. Careri, M., Furlattini, L., Mangia, A., Musci, M., Anklam, E., Theobald, A., von Holst, C., 2001. Supercritical uid extraction for liquid chromatographic determination of carotenoids in Spirulina Pacica algae: a chemometric approach. Journal of Chromatography A 912, 6171. Carotenature (The carotenoids page), 2000. Retrieved September 4, 2002 from the World Wide Web: http://www.carotenature.com. Casal, S., Macedo, B., Oliveira, M.B.P.P., 2001. Simultaneous determination of retinol, b-carotene and a-tocopherol in adipose tissue by high-performance liquid chromatography. Journal of Chromatogrphy B 763, 18. Chandra, A., Nair, M.G., 1997. Supercritical uid carbon dioxide extraction of a- and b-carotene from carrot (Daucus carota L.). Phytochemical Analysis 8, 244246. Chandrika, U.G., Jansz, E.R., Wickramasinghe, S.M.D.N., Warnasuriya, N.D., 2003. Carotenoids in yellow- and red-esh papaya (Carica papaya L.). Journal of the Science of Food and Agriculture 83, 12791282. Chen, B.H., Peng, H.Y., Chen, H.E., 1995. Changes of carotenoids, color and vitamin A contents during processing of carrot juice. Journal of Agricultural and Food Chemistry 43, 19121918. Dachtler, M., Kohler, K., Albert, K., 1998. Reversed-phase highperformance liquid chromatographic identication of lutein and zeaxanthin stereoisomers in bovine retina using a C30 bonded phase. Journal of Chromatography B 720, 211216. Darko, E., Schoefs, B., Lemoine, Y., 2000. Improved liquid chromatographic method for the analysis of photosynthetic pigments of higher plants. Journal of Chromatography A 876, 111116. Deli, J., Molnar, P., Matus, Z., Toth, G., 2001. Carotenoid composition in the fruits of red paprika (Capsicum annuum var. lycopersiciforme rubrum) during ripening; biosynthesis of carotenoids in red paprika. Journal of Agricultural and Food Chemistry 49, 15171523. Edelenbos, M., Christensen, L.P., Grevsen, K., 2001. HPLC determination of chlorophyll and carotenoid pigments in processed green pea cultivars (Pisum sativum L.). Journal of Agricultural and Food Chemistry 49, 47684774.

ARTICLE IN PRESS
s, A. Rodrguez-Bernaldo de Quiro H.S. Costa / Journal of Food Composition and Analysis 19 (2006) 97111 Goodner, K.L., Rouseff, R.L., Hofsommer, H.J., 2001. Orange, mandarin and hybrid classication using multivariate statistics based on carotenoid proles. Journal of Agricultural and Food Chemistry 49, 11461150. Granado, F., Olmedilla, B., Blanco, I., Rojas-Hidalgo, E., 1992. Carotenoid composition in raw and cooked Spanish vegetables. Journal of Agricultural and Food Chemistry 40, 21352140. Granelli, K., Helmersson, S., 1996. Rapid high-performance liquid chromatographic method for determination of beta-carotene in milk. Journal of Chromatography A 721, 355358. Gueguen, S., Herbeth, B., Siest, G., Leroy, P., 2002. An isocratic liquid chromatographic method with diode-array detection for the simultaneous determination of alpha-tocopherol, retinol, and ve carotenoids in human serum. Journal of Chromatographic Science 40, 6976. Guil-Guerrero, J.L., Rebolloso-Fuentes, M.M., Isasa, M.E.T., 2003. Fatty acids and carotenoids from Stinging Nettle (Urtica dioica L.). Journal of Food Composition and Analysis 16, 111119. Hadden, W.L., Watkins, R.H., Levy, L.W., Regalado, E., Rivadeneira, D.M., van Breemen, R.B., Schwartz, S.J., 1999. Carotenoid composition of marigold (Tagetes erecta) ower extract used as nutritional supplement. Journal of Agricultural and Food Chemistry 47, 41894194. Hagg, M., Ylikoski, S., Kumpulainen, J., 1994. Vitamin C and a- and b-carotene contents in vegetables consumed in Finland during 19881989 and 19921993. Journal of Food Composition and Analysis 7, 252259. Hagiwara, T., Yasuno, T., Funayama, K., Suzuki, S., 1998. Determination of lycopene, alpha-carotene and beta-carotene in serum by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry with selected-ion monitoring. Journal of Chromatography B 708, 6773. Hart, D.J., Scott, K.J., 1995. Development and evaluation of an HPLC method for the analysis of carotenoids in foods, and the measurement of the carotenoid content of vegetables and fruits commonly consumed in the UK. Food Chemistry 54, 101111. Hegazi, M.M., Perez-Ruzafa, A., Almela, L., Candela, M.-E., 1998. Separation and identication of chlorophylls and carotenoids from Caulerpa prolifera, Jania rubens and Padina pavonica by reversedphase high-performance liquid chromatography. Journal of Chromatography A 829, 153159. Heinonen, M.I., Ollilainen, V., Linkola, E.K., Varo, P.T., Koivistoinen, P.E., 1989. Carotenoids in Finnish foods: vegetables, fruits and berries. Journal of Agricultural and Food Chemistry 37, 655659. Hentschel, V., Kranl, K., Hollmann, J., Lindhauer, M.G., Bohm, V., Bitsch, R., 2002. Spectrophotometric determination of yellow pigment content and evaluation of carotenoids by high-performance liquid chromatography in durum wheat grain. Journal of Agricultural and Food Chemistry 50, 66636668. Hollman, P.C.H., Slangen, J.H., Wagstaffe, P.J., Faure, U., Southgate, D.A.T., Finglas, P.M., 1993. Intercomparison of methods for the determination of vitamins in foods. Part 1. Fat-soluble vitamins. Analyst 118, 475480. Howard, L.A., Wong, A.D., Perry, A.K., Klein, B.P., 1999. bCarotene and ascorbic acid retention in fresh and processed vegetables. Journal of Food Science 64, 929936. Howard, L.R., Talcott, S.T., Brenes, C.H., Villalon, B., 2000. Changes in phytochemical and antioxidant activity of selected pepper cultivars (Capsicum species) as inuenced by maturity. Journal of Agricultural and Food Chemistry 48, 17131720. Hsieh, Y.-P.C., Karel, M., 1983. Rapid extraction and determination of a- and b-carotenes in foods. Journal of Chromatography 259, 515518. Huang, A.S., Tanudjaja, L., Lum, D., 1999. Content of alpha-, beta-carotene, and dietary ber in 18 sweetpotato varieties grown 109 in Hawaii. Journal of Food Composition and Analysis 12, 147151. Huck, C.W., Popp, M., Scherz, H., Bonn, G.K., 2000. Development and evaluation of a new method for the determination of the carotenoid content in selected vegetables by HPLC and HPLCMS-MS. Journal of Chromatographic Science 38, 441449. Hulshof, P.J.M., Xu, C., van de Bovenkamp, P., Muhilal, West, C.E., 1997. Application of a validated method for the determination of provitamin A carotenoids in Indonesian Foods of different maturity and origin. Journal of Agricultural and Food Chemistry 45, 11741179. Iwase, H., 2002. Simultaneous sample preparation for high-performance liquid chromatographic determination of Vitamin A and bcarotene in emulsied nutritional supplements after solid-phase extraction. Analytica Chimica Acta 463, 2129. Khachik, F., Beecher, G.R., Whittaker, N.F., 1986. Separation, identication and quantication of the major carotenoid and chlorophyll constituents in extracts of several green vegetables by liquid chromatography. Journal of Agricultural and Food Chemistry 34, 603616. Khachik, F., Goli, M.B., Beecher, G.R., Holden, J., Lusby, W.R., Tenorio, M.D., Barrera, M.R., 1992a. Effect of food preparation on qualitative and quantitative distribution of major carotenoid constituents of tomatoes and several green vegetables. Journal of Agricultural and Food Chemistry 40, 390398. Khachik, F., Beecher, G.R., Goli, M.B., Lusby, W.R., Smith Jr., J.C., 1992b. Separation and identication of carotenoids and their oxidation products in the extracts of human plasma. Analytical Chemistry 64, 21112122. Khachik, F., Beecher, G.R., Goli, M.B., Lusby, W.R., 1992c. Separation and quantitation of carotenoids in foods. Methods in Enzymology 213, 347359. Kiss, G.A.C., Forgacs, E., Cserhati, T., Mota, T., Morais, H., Ramos, A., 2000. Optimisation of the microwave-assisted extraction of pigments from paprika (Capsicum annuum L.) powders. Journal of Chromatography A 889, 4149. Krinskey, N.I., 1994. The biological properties of carotenoids. Pure and Applied Chemistry 66, 10031010. Kurilich, A.C., Britz, S.J., Clevidence, B.A., Novotny, J.A., 2003. Isotopic labeling and LC-APCI-MS quantication for investigating absorption of carotenoids and phylloquinone from kale (Brassica oleracea). Journal of Agricultural and Food Chemistry 51, 48774883. Lacker, T., Strohschein, S., Albert, K., 1999. Separation and identication of various carotenoids by C30 reversed-phase highperformance liquid chromatography coupled to UV and atmospheric pressure chemical ionization mass spectrometric detection. Journal of Chromatography A 854, 3744. Lee, H.S., 2001. Characterization of carotenoids in juice of red navel orange (Cara cara). Journal of Agricultural and Food Chemistry 49, 25632568. Lee, B.L., Chua, S.C., Ong, H.Y., Ong, C.N., 1992. High-performance liquid chromatographic method for routine determination of vitamins A and E and beta-carotene in plasma. Journal of Chromatography 581, 4147. Lee, H.S., Castle, W.S., Coates, G.A., 2001. High-performance liquid chromatography for the characterization of carotenoids in the new sweet orange (Earlygold) grown in Florida, USA. Journal of Chromatography A 913, 371377. Lienau, A., Glaser, T., Tang, G., Dolnikowski, G.G., Grusak, M.A., Albert, K., 2003. Bioavailability of lutein in humans from intrinsically labeled vegetables determined by LC-APCI-MS. Journal of Nutritional Biochemistry 14, 663670. Lin, C.H., Chen, B.H., 2003. Determination of carotenoids in tomato juice by liquid chromatography. Journal of Chromatography A 1012, 103109.

ARTICLE IN PRESS
110 s, A. Rodrguez-Bernaldo de Quiro H.S. Costa / Journal of Food Composition and Analysis 19 (2006) 97111 Olmedilla, B., Granado, F., Rojas-Hidalgo, E., Blanco, I., 1990. A rapid separation of ten carotenoids, three retinoids, alphatocopherol and d-alpha-tocopherol acetate by high performance liquid chromatography and its application to serum and vegetable samples. Journal of Liquid Chromatography 13, 14551483. Olmedilla, B., Granado, F., Blanco, I., Rojas-Hidalgo, E., 1992. Determination of nine carotenoids, retinol, retynil palmitate and atocopherol in control human serum using two internal standards. Food Chemistry 45, 205213. Olmedilla, B., Granado, F., Gil-Martinez, E., Blanco, I., RojasHidalgo, E., 1997. Reference values for retinol, tocopherol, and main carotenoids in serum of control and insulin-dependent diabetic Spanish subjects. Clinical Chemistry 43, 10661071. Otles, S., Atli, Y., 2000. Analysis of carotenoids in tomato paste by HPLC and OCC. American Laboratory 32, 2224. Pfander, H., 1987. Key to Carotenoids. Birkhauser Verlag, Basel. Quackenbush, F.W., Smallidge, R.L., 1986. Nonaqueous reversedphase liquid chromatographic system for separation and quantitation of provitamins A. Journal of the Association of Ofcial Analytical Chemists 69, 767772. Riso, P., Porrini, M., 1997. Determination of carotenoids in vegetable foods and plasma. International Journal for Vitamin and Nutrition Research 67, 4754. Rodrigues, P., Morais, H., Mota, T., Olivera, S., Forgacs, E., Cserhati, T., 1998. Use of HPLC and multivariate methods for the evaluation of the stability of colour pigments of paprika (Capsicum annuum) powder. Analytica Chimica Acta 372, 411416. Rodriguez-Amaya, D.B., 1996. Assessment of the provitamin A contents of foodsthe Brazilian experience. Journal of Food Composition and Analysis 9, 196230. Rodriguez-Amaya, D.B., Kimura, M., Godoy, H.T., Arima, H.K., 1988. Assessment of provitamin A determination by open column chromatography/visible absorption spectrophotometry. Journal of Chromatographic Science 26, 624629. Rozzi, N.L., Singh, R.K., Vierling, R.A., Watkins, B.A., 2002. Supercritical uid extraction of lycopene from tomato processing byproducts. Journal of Agricultural and Food Chemistry 50, 26382643. Sander, L.C., Sharpless, K.E., Craft, N.E., Wise, S.A., 1994. Development of engineered stationary phases for the separation of carotenoid isomers. Analytical Chemistry 66, 16671674. Sander, L.C., Sharpless, K.E., Pursch, M., 2000. C30 stationary phases for the analysis of food by liquid chromatography. Journal of Chromatography A 880, 189202. Schmitz, H.H., Artz, W.E., Poor, C.L., Dietz, J.M., Erdman Jr., J.W., 1989. High-performance liquid chromatography and capillary supercritical-uid chromatography separation of vegetable carotenoids and carotenoid isomers. Journal of Chromatography 479, 261268. Scott, K.J., 1992. Observations on some of the problems associated with the analysis of carotenoids in foods by HPLC. Food Chemistry 45, 357364. Scott, K.J., Hart, D.J., 1993. Further observations on problems associated with the analysis of carotenoids by HPLC-2: Column temperature. Food Chemistry 47, 403405. Sharpless, K.E., Arce-Osuna, M., Thomas, J.B., Gill, L.M., 1999. Value assignment of retinal, retinyl palmitate, tocopherol and carotenoid concentrations in standard reference material 2383 (Baby Food Composite). Journal of AOAC International 82, 288296. Su, Q., Rowley, K.G., Balazs, N.D.H., 2002. Carotenoids: separation methods applicable to biological samples. Journal of Chromatography B 781, 393418. Lopez, M., Arce, L., Garrido, J., Rios, A., Valcarcel, M., 2004. Selective extraction of astaxanthin from crustaceans by use of supercritical carbon dioxide. Talanta 64, 726731. Lunetta, J.M., Zulim, R.A., Dueker, S.R., Lin, Y., Flaig, V., Schneider, P.D., Wolfe, B.M., Clifford, A.J., 2002. Method for the simultaneous determination of retinol and beta-carotene concentrations in human tissues and plasma. Analytical Biochemistry 304, 100109. Lyan, B., Aza s-Braesco, V., Cardinault, N., Tyssandier, V., Borel, P., Alexandre-Gouabau, M.C., Grolier, P., 2001. Simple method for clinical determination of 13 carotenoids in human plasma using an isocratic high-performance liquid chromatographic method. Journal of Chromatography B 751, 297303. Machlin, L.J., 1995. Critical assessment of epidemiological data concerning the impact of antioxidant nutrients on cancer and cardiovascular disease. Critical Reviews in Food Science and Nutrition 35, 4150. Markus, F., Daood, H.G., Kapitany, J., Biacs, P.A., 1999. Change in the carotenoid and antioxidant content of spice red pepper (Paprika) as a function of ripening and some technological factors. Journal of Agricultural and Food Chemistry 47, 100107. Marsili, R., Callahan, D., 1993. Comparison of a liquid solvent extraction technique and supercritical uid extraction for the determination of a- and b-carotene in vegetables. Journal of Chromatogrphic Sciences 31, 422428. Mathiasson, L., Turner, C., Berg, H., Dahlberg, L., Theobald, A., Anklam, E., Ginn, R., Sharman, M., Ulberth, F., Gabernig, R., 2002. Development of methods for the determination of vitamins A, E and beta-carotene in processed foods based on supercritical uid extraction: a collaborative study. Food Additives and Contaminants 19, 632646. Melendez-Mart nez, A.J., Vicario, I.M., Heredia, F.J., 2003. A routine high-performance liquid chromatography method for carotenoid determination in ultrafrozen orange juices. Journal of Agricultural and Food Chemistry 51, 42194224. Mendes, R.L., Nobre, B.P., Cardoso, M.T., Pereira, A.P., Palavra, A.F., 2003. Supercritical carbon dioxide extraction of compounds with pharmaceutical importance from microalgae. Inorganica Chimica Acta 356, 328334. Mercadante, A.Z., 1999. Chromatographic separation of carotenoids. Archivos Latinoamericanos de Nutricion 49, 52S57S. Mercadante, A.Z., Rodriguez-Amaya, D.B., Britton, G., 1997. HPLC and mass spectrometric analysis of carotenoids from mango. Journal of Agricultural and Food Chemistry 45, 120123. Moros, E.E., Darnoko, D., Cheryan, M., Perkins, E.G., Jerrell, J., 2002. Analysis of xanthophylls in corn by HPLC. Journal of Agricultural and Food Chemistry 50, 57875790. Mouly, P.P., Gaydou, E.M., Corsetti, J., 1999. Determination of the geographical origin of valencia orange juice using carotenoid liquid chromatographic proles. Journal of Chromatography A 844, 149159. Murkovic, M., Mulleder, U., Neunteu, H., 2002. Carotenoid content in different varieties of pumpkins. Journal of Food Composition and Analysis 15, 633638. Nierenberg, D.W., 1985. Serum and plasma beta-carotene levels measured with an improved method of high-performance liquid chromatography. Journal of Chromatography 339, 273284. Nierenberg, D.W., Nann, S.L., 1992. A method for determining concentrations of retinol, tocopherol, and ve carotenoids in human plasma and tissue samples. American Journal of Clinical Nutrition 56, 417426. Oliver, J., Palou, A., Pons, A., 1998. Semi-quantication of carotenoids by high-performance liquid chromatography: saponicationinduced losses in fatty foods. Journal of Chromatography A 829, 393399.

ARTICLE IN PRESS
s, A. Rodrguez-Bernaldo de Quiro H.S. Costa / Journal of Food Composition and Analysis 19 (2006) 97111 Szentmihalyi, K., Vinkler, P., Lakatos, B., Illes, V., Then, M., 2002. Rose hip (Rosa canina L.) oil obtained from waste hip seeds by different extraction methods. Bioresource Technology 82, 195201. Talwar, D., Ha, T.K.K., Cooney, J., Brownlee, C., St JO0 Reilly, D., 1998. A routine method for the simultaneous measurement of retinol, a-tocopherol and ve carotenoids in human plasma by reverse phase HPLC. Clinica Chimica Acta 270, 85100. Taungbodhitham, A.K., Jones, G.P., Wahlqvist, M.L., Briggs, D.R., 1998. Evaluation of extraction method for the analysis of carotenoids in fruits and vegetables. Food Chemistry 63, 577584. Tzouganaki, Z.D., Atta-Politou, J., Koupparis, M.A., 2002. Development and validation of liquid chromatographic method for the determination of lycopene in plasma. Analytica Chimica Acta 467, 115123. Vagi, E., Simandi, B., Daood, H.G., Deak, A., Sawinsky, J., 2002. Recovery of pigments from Origanum majorana L. by extraction with supercritical carbon dioxide. Journal of Agricultural and Food Chemistry 50, 22972301. van Breemen, R.B., Xu, X., Viana, M.A., Chen, L., StacewiczSapuntzakis, M., Duncan, C., Bowen, P.E., Shari, R., 2002. Liquid chromatography-mass spectrometry of cis- and all-translycopene in human serum and prostate tissue after dietary 111 supplementation with tomato sauce. Journal of Agricultural and Food Chemistry 50, 22142219. van den Berg, H., Faulks, R., Granado, H.F., Hirschberg, J., Olmedilla, B., Sandmann, G., Southon, S., Stahl, W., 2000. The potential for the improvement of carotenoid levels in foods and the likely systemic effects. Journal of the Science of Food and Agriculture 80, 880912. Wang, Y., Xu, X., van Lieshout, M., West, C.E., Lugtenburg, J., Verhoeven, M.A., Creemers, A.F., Muhilal, van Breemen, R.B., 2000. A liquid chromatography-mass spectrometry method for the quantication of bioavailability and bioconversion of betacarotene to retinol in humans. Analytical Chemistry 72, 49995003. Weller, P., Breithaupt, D.E., 2003. Identication and quantication of zeaxanthin esters in plants using liquid chromatography-mass spectrometry. Journal of Agricultural and Food Chemistry 51, 70447049. Ye, L., Landen, W.O., Eitenmiller, R.R., 2000. Liquid chromatographic analysis of all-trans-retinyl palmitate, beta-carotene, and vitamin E in fortied foods and the extraction of encapsulated and nonencapsulated retinyl palmitate. Journal of Agricultural and Food Chemistry 48, 40034008.