JOURNAL OF FOOD COMPOSITION AND ANALYSIS
Journal of Food Composition and Analysis 19(2006)97–111
Study Review
Analysis of carotenoids in vegetable and plasma samples:A review
Ana Rodr?guez-Bernaldo de Quiro
s,Helena S.Costa ?Centro de Seguranc -a Alimentar e Nutric -a ?o,Instituto Nacional de Sau
′de Dr.Ricardo Jorge,Av.Padre Cruz,1649-016Lisboa,Portugal Received 29June 2004;received in revised form 11April 2005;accepted 15April 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 liquid–liquid 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 2005Elsevier 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 com-pounds 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
system,which in?uences 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 600carotenoids 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 modi?ed by food processing (Chen et al.,1995).In addition,carotenoid content in fruits and vegetables depends on several factors such as,genetic variety,maturity,post-harvest storage,processing and preparation.
However,the great interest in studying these com-pounds 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 enhan-cers of the immune response.Furthermore,some of them are involved in the cell communication and
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Abbreviations:ACN,acetonitrile;APCI,atmospheric pressure ionization interfaces;BHA,butylated hydroxyanisole;BHT,buty-lated 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 ultra-violet-visible
?Corresponding author.Tel.:+351217519267;fax:+351217526400.
E-mail address:helena.costa@insa.min-saude.pt (H.S.Costa).
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 degenera-tive diseases such as certain types of cancer,cardiovas-cular diseases(Machlin,1995)and age-related macular degeneration(Bone et al.,2000).In this study,associa-tions 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 separa-tion 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 chroma-tography for the determination of carotenoids with provitamin A activity in beet leaves,as well as in green leafy vegetables and other food samples(Almeida-Muradian 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 suf?cient for identi?cation 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(Azevedo-Meleiro and Rodriguez-Amaya,2004;Englberger et al.,2003a b;Heinonen et al.,1989;O tles and Atli,2000; Ha gg 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)(Mele ndez-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) (Ma rkus 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:hex-ane(1:1,v/v)and ethyl acetate(100%),to compare the extraction ef?ciency of carotenoids in tomato juice. They concluded that the best extraction ef?ciency 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 monoesteri?ed 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.moscha-ta).Rozzi et al.(2002)reported a method to determine lycopene,a-and b-carotene,a-,g-and d-tocopherol
A.Rodr?′guez-Bernaldo de Quiro′s,H.S.Costa/Journal of Food Composition and Analysis19(2006)97–111 98
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 hydroxyto-luene(BHT)to the solvent.On the contrary,Quacken-bush 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,pyr-ogallol has been recommended(Sharpless et al.,1999; Nierenberg and Nann,1992;Ha gg et al.,1994;Granelli and Helmersson,1996).
It is also advisable to add magnesium or calcium carbonate to the sample–extracting 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 above401C,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 puri?cation 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 range1:4to1:1(v/v), and best results were achieved with the proportion1:1 (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 (2:1v/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:liquid–liquid extraction using n-hexane as extracting solvent and solid-phase extraction using an ISOLUTE C18cartridge 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 liquid–liquid 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 concen-trations 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;Va gi et al.,2002).In addition,carbon dioxide has a low critical temperature(311C)making it ideal for extraction of thermally labile compounds(Va gi et al., 2002;Careri et al.,2001).Nevertheless,CO2presents a low polarity and makes the extraction of polar compounds very dif?cult.This limitation can be solved by adding an organic modi?er 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,
A.Rodr?′guez-Bernaldo de Quiro′s,H.S.Costa/Journal of Food Composition and Analysis19(2006)97–11199
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).
Go mez-Prieto et al.(2003)extracted all-trans-lyco-pene from tomato using CO2without modi?er.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, 0.60,0.70,0.75,0.80,0.85and0.90g/mL).The best results were found for0.90g/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 Va gi et al.(2002).Three different temperatures(40,50and601C)and three different pressures(100,250and400bar)were tested. The authors observed that the optimum conditions for extraction were501C and450bar.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 tempera-ture was studied by performing a factorial experiment with replication for temperatures of39.9and54.91C and pressures of200and300bar.The optimum conditions for this analysis were200bar and39.91C. In another study,carried out by Baysal et al.(2000) several extraction conditions,such as temperature of the extractor(35,45,55and651C),pressure of the extraction?uid(200,250and300bar),addition of cosolvent(5%,10%and15%EtOH),extraction time (1,2and3h)and CO2?ow rate(2,4and8kg/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 651C would give better extraction yield but this might cause an increase in carotene degradation.No signi?-cant differences in lycopene and b-carotene extraction yields were observed,if the pressure was changed from 200to300bar.Other authors have indicated that extraction pressures up to400bar can give higher yields (Va gi et al.,2002).Regarding extraction time and?ow rate,the highest carotenoid yield was obtained with an extraction time of2h.It was observed that1h was not enough for extracting carotenoids and with3h the degradation process increased.The optimum?ow rate for both lycopene and b-carotene was4kg/h.The addition of5%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 at861C and345bar.
Barth et al.(1995)compared classical solvent extrac-tion and SFE for determining carotenoids from carrot (Daucus carota L.)tissue.They observed that total provitamin A activity was7%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 investi-gated by Gamlieli-Bonshtein et al.(2002).The separa-tion of the isomers was achieved due to their different dissolution rate in supercritical CO2.
Several CO2modi?ers(water,EtOH,methylene chloride,hexane)have been tested in order to improve the extraction ef?ciency of a-and b-carotene from different vegetables(Marsili and Callahan,1993).The addition of hexane did not signi?cantly increase the b-carotene 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 modi?er,the solubility of b-carotene in CO2increased signi?cantly (Marsili and Callahan,1993).
Mendes et al.(2003)reported a supercritical carbon dioxide extraction of compounds with pharmaceutical importance from microalgae.Canthaxanthin and astax-anthin were extracted from Chlorella vulgaris.Several conditions of pressure and temperature were compared and the best results were achieved at275and350bar and551C.
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 CO2extraction.The optimum conditions to extract the two isomers were achieved at300bar and401C.
A comparison of traditional extraction with ultra-sound,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. Lo pez 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 cray?sh.
In Table1some examples of SFE conditions in carotenoid analysis are presented.
3.Saponi?cation
Prior to HPLC analysis,the saponi?cation procedure has been often used as a step to simplify the separation by removing substances,such as chlorophylls and lipids,
A.Rodr?′guez-Bernaldo de Quiro′s,H.S.Costa/Journal of Food Composition and Analysis19(2006)97–111 100
which could interfere with the chromatographic detec-tion.Moreover,with the saponi?cation process,valu-able information about the nature and distribution of the carotenoids present in the sample can be obtained by evaluating their chromatographic pro?le before and after alkali treatment(Khachik et al.,1992c).However, a loss of total carotenoid content during saponi?cation has been reported in the literature(Granado et al.,1992; Khachik et al.,1986).Fernandez et al.(2000)have compared alkali saponi?cation and enzymatic hydro-lysis on the total carotenoid concentration of Costa Rican crude palm oil.Findings showed greater concen-tration of carotenoids using enzymatic hydrolysis.The most sensitive compounds to alkaline treatments are xanthophylls,particularly the epoxycarotenoids(Kha-chik et al.,1986).Therefore,when the sample to be analyzed has these pigments in its composition,this puri?cation step should be avoided.As a general rule, for samples with low fat content,milder conditions in saponi?cation step must be applied,and for high-fat content samples severe conditions should be employed (Khachik et al.,1992c).Saponi?cation however,should be employed to evaluate the presence of carotenol esters that may otherwise be undetected.Table2summarizes different saponi?cation conditions in various samples. After the saponi?cation phase,carotenoids are extracted with ethyl ether(Hadden et al.,1999)diethyl ether(Goodner et al.,2001),n-hexane(Howard et al.,
Table1
Examples of supercritical?uid extraction conditions employed in carotenoid analysis
Sample Analyte Supercritical?uid SFE conditions
(temperature,pressure,
?ow rate)
Reference
Tomato All-trans-lycopene CO2without modi?er T?401C Go mez-Prieto et al.(2003)
Pressure:281bar
Flow rate:4mL/min
Tomato seeds and skins Lycopene,b-carotene,a-
carotene,a-tocopherol,g-
tocopherol,and d-
tocopherol CO2without modi?er T?861C Rozzi et al.(2002)
Pressure:345bar
Flow rate:2.5mL/min
Vitamin supplements and calf liver tissue(dry carotene beadlets,liver samples)Vitamin A and b-carotene CO2without modi?er Dry carotene beadlets:Burri et al.(1997)
T?401C
Pressure:310bar
Flow rate:2mL/min
Liver samples:
T?801C
Pressure:310bar
Flow rate:2mL/min
Algae Dunaliella bardawil Geometrical isomers of b-
Carotene CO2without modi?er T?401C Gamlieli-Bonshtein et al.
(2002)
Pressure:448bar
Flow rate:0.5–1mL/min
Tomato paste waste Lycopene and b-carotene CO2and5%EtOH as
modi?er T?551C lycopene Baysal et al.(2000) T?651C b-carotene
Pressure:300bar
Flow Rate:4kg/h
Vegetables(carrots,collard greens,turnip greens,kale, mustard greens,broccoli ?owerets,zucchini,and yellow squash)a-and b-carotene CO2and EtOH as
modi?er
T?401C Marsili and Callahan
(1993)
Pressure342bar
Flow rate:1.5mL/min
Spirulina Paci?ca algae b-carotene,b-
cryptoxanthin and
zeaxanthin CO2and15%EtOH as
modi?er
T?801C(zeaxanthin)Careri et al.(2001)
T?761C(b-
cryptoxanthin)
T?601C(b-carotene)
Pressure:350bar
Flow rate:2mL/min
Carrots(Daucus carota L. var.Caro Pride)a-and b-carotene CO2and5%
chloroform as modi?er
T?401C Chandra and Nair(1997)
Pressure:606bar
Flow rate:1mL/min
A.Rodr?′guez-Bernaldo de Quiro′s,H.S.Costa/Journal of Food Composition and Analysis19(2006)97–111101
1999),methylene chloride(Mele ndez-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;O tles 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 with30%methanolic potassium hydroxide under nitrogen atmosphere at room temperature during 3h.On the contrary,the loss of carotenes was not signi?cant.
Ye et al.(2000)reported that direct solvent extraction method presents an alternative technique to saponi?ca-tion for analysis of vitamins and beta-carotene in various forti?ed foods.
Granado et al.(1992)pointed out in their paper,a loss in the concentration of xanthophylls related to the
Table2
Different saponi?cation conditions employed in carotenoid analysis
Sample Analyte Saponi?cation conditions Reference
Juice of red Navel Orange(Cara cara)Neoxanthin(a,b),neochrome,
violaxanthin,luteoxanthin,
antheraxanthin,mutatoxanthin,
lutein,isolutein,zeaxanthin,a-and b-
cryptoxanthin,phytoene,phyto?uene,
a-,b-and g-carotene,lycopene
10%methanolic KOH sol.
(Overnight,room T,darkness)
Lee(2001)
Ultrafrozen orange juice Lutein,zeaxanthin,lutein5,6-epoxide,
antheraxanthin,b-cryptoxanthin 10%methanolic KOH sol.(1h,room
T,darkness)
Mele ndez-Mart?nez et
al.(2003)
Fatty foods(fat-cured crude sausage‘‘Sobrassada’’)Capsorubin,violaxanthin,capsanthin,
anteraxanthin,lutein+zeaxanthin,
cantaxanthin,b-cryptoxanthin,b-
carotene
10%methanolic KOH sol.containg
0.01%BHA(5min,501C)
Oliver et al.(1998)
Marigold(Tagetes erecta)?ower extract All-trans–cis isomers of zeaxanthin,
all-trans–cis isomers of lutein,lutein
esters
15%methanolic KOH sol.(1h,
darkness)
Hadden et al.(1999)
Standard Reference Material 2383(Baby Food Composite)Lutein,zeaxanthin,b-cryptoxanthin,
lycopene,trans–cis a-and b-carotene,
retinol,retinylpalmitate,d-,g-and a-
tocopherol
40%methanolic KOH sol.(30min,
room T)
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,cauli?ower)Lutein,zeaxanthin,lycopene,b-
cryptoxanthin,a-,b-and g-carotene
Saturated methanolic KOH sol.
(Under nitrogen atmosphere,30min,
darkness)
Granado et al.(1992)
Edible wild vegetable Stinging Nettle(Urtica dioica L.)Lutein,lutein isomers,b-carotene,b-
carotene isomers,neoxanthin,
violaxanthin,lycopene
Methanolic KOH sol.(room T)Guil-Guerrero et al.
(2003)
Virgin olive oil a-tocopherol and b-carotene76%ethanolic KOH sol.(Under
nitrogen atmosphere,30min,701C)
Gimeno et al.(2000)
Corn Lutein,zeaxanthin,and b-
cryptoxanthin 80%ethanolic KOH sol.(In a water
bath at boiling point,10min)
Moros et al.(2002)
Fresh and processed vegetables (broccoli,carrots and green beans)Trans-b-carotene100%ethanolic KOH sol.(30min,
701C)
Howard et al.(1999)
Sweetpotato(Ipomoea batatas, L.)a-carotene,b-carotene10%ethanol:water(50:50,v/v)for
1h,801C
Huang et al.(1999)
Milk samples b-carotene60%aqueous KOH sol.containing
pyrogallol as antioxidant(30min,
301C)Granelli and Helmersson(1996)
Forti?ed foods(forti?ed breakfast cereal,peanut butter and margarine)All-rac-alpha-tocopheryl acetate,
retinyl palmitate,b-carotene
60%aqueous KOH sol.containing
pyrogallol as antioxidant(under
nitrogen atmosphere,30min,701C)
Ye et al.(2000)
Kale(Brassica oleracea var. Acephala cv.Vates)Lutein,b-carotene,retinol,
phylloquinone
80%aqueous KOH sol.(15min,
701C)
Kurilich et al.(2003)
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saponi?cation step.This loss in concentration can be partially corrected when the quanti?cation is made on the basis of a calibration curve,which is also subject to the process of saponi?cation.
Hart and Scott(1995)for example,determined the carotenoid content in vegetables and fruits commonly consumed in United Kingdom.The following saponi?-cation procedure of carotenoid extracts was only applied for fruits and certain vegetables,like peppers:metha-nolic potassium hydroxide(10%)under nitrogen,in the dark for1h at room temperature.
Oliver et al.(1998)investigated the carotenoid pro?le of fat-cured crude sausage(‘‘Sobrassada’’)before and after the saponi?cation procedure,and have reported that the alkaline treatment of the carotenoid extracts hydrolyses all carotenoid esters.This treatment allowed the identi?cation of other simple carotenoids,such as cryptocapsin,which were not detected in the non-saponi?ed extract.
Several researchers have observed better carotenoid extraction when high temperatures are employed in the saponi?cation 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,time-consuming,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,all-trans-lycopene and their13cis-isomers.The separation was achieved by using a C30column(250mm?4.6mm i.d.,5m m particle size)from YMC and a gradient of two eluents,(A)ACN:1butanol(70:30,v/v)and(B) methylene chloride.The analysis was completed within 55min at a?ow rate of2mL/min and the wavelength was set at476nm.
A high-performance liquid chromatographic method to determine chlorophylls,carotenoids and their deri-vatives in some fresh and processed vegetables have been described(Gokmen et al.,2002).A C8MikroPak (150mm?4.6mm i.d.,5m m particle size)stainless steel column and a gradient mixture of MeOH:water as the mobile phase at a?ow rate of0.75mL/min was used. The chromatograms were recorded simultaneously at 432,450,470,652and666nm using a DAD.
A quaternary mixture of MeOH:ACN:methylene chloride:water(50:30:15:5,v/v/v/v)containing0.1% BHT and0.1%TEA as antioxidant and modi?er, respectively,and a C18Kromasil(250mm?4.6mm i.d., 5m m packing)were used by Mele ndez-Mart?nez et al. (2003)to analyze the carotenoid pro?le in ultrafrozen orange juices.A more speci?c wavelength of486nm was selected and a?ow rate of 2.5mL/min.Carotenoids were identi?ed by comparison with the spectra of the standards obtained with a DAD from350to800nm. Another method to determine chlorophyll and car-otenoid 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 of1mL/min was used as mobile phase.The separations were performed on a LiChrospher100RP-18column(5m m packing, 244mm?4mm i.d.)and the temperature was main-tained at301C.Chromatograms were recorded at 440nm with a DAD and absorption spectra of carotenoids and chlorophylls were recorded between 300and600nm.
More recently,Go mez-Prieto et al.(2003)achieved an optimal separation of phytoene,phyto?uene,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 at1mL/ min.A Develosil UG C30(250mm?4.6mm i.d.) column maintained at201C was employed and b-apo-carotenal was used as internal standard.In order to determine,in the same injection,different carotenoids four wavelengths(285,347,450and472nm)were selected.
Va gi et al.(2002)developed an isocratic and re-versed-phase high-performance liquid chromatographic method to determine chlorophylls and carotenoids in marjoram(Origanum majorana L.).In this study,a Nucleosil5C18stainless steel column(250mm?4mm i.d.)and a ACN:MeOH:isopropyl alcohol(39:43:18, v/v/v)mobile phase at a?ow rate of0.9mL/min was reported.Chromatograms were recorded at a wave-length of430nm.
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(4.6mm?250mm)was described by Moros et al.(2002).The?ow rate was1mL/min and the chromatogram was monitored at450and445nm using a DAD.
A.Rodr?′guez-Bernaldo de Quiro′s,H.S.Costa/Journal of Food Composition and Analysis19(2006)97–111103
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 SIL5m m column(250mm?4.6mm i.d.)and hexane:ethyl acetate(75:25,v/v)as mobile phase at a ?ow rate of2mL/min was used to separate all-trans-lutein,cis-lutein isomers,as well as all-trans-zeaxanthin. The other normal-phase system included a b Cyclobond 5m m column(250mm?4.6mm i.d.)with n-hexa-ne:ethyl acetate(87:13,v/v)as mobile phase and a?ow rate of2.0mL/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-SIL2003m m silica C30column with a mobile phase consisting of3% MTBE in MeOH at a?ow rate of1mL/min.All-trans-lutein,9-and90-cis-lutein isomers,13-and130-cis-lutein isomers and all-trans-zeaxanthin were identi?ed.In all cases,the carotenoids were detected at450nm.
In a recent study(Lee et al.,2001),more than25 carotenoids from a sweet orange(Earlygold)sample were separated within40min using a ternary gradient elution consisting of:eluent(A)ACN:MeOH(75:25,v/ v),eluent(B)100%MTBE and eluent(C)water containing0.01%BHT and0.05%TEA at a?ow rate of1mL/min.The separation was performed on a C30 column(150mm?4.6mm i.d.,3m m)from YMC main-tained at251C.In another experiment described by this author,the same conditions were applied to identify29 carotenoids in juice from another type of orange,Red Navel orange(Cara cara).Carotenoids were detected at 450nm(Lee,2001).
A method was developed to differentiate citrus of orange,mandarin and hybrid on the basis of their carotenoid pro?le.A gradient elution system,which was composed by MeOH,water and MTBE and,a C30 YMC column(25cm?4.6mm i.d.,5m m)was used for the separation.The analysis was completed in50min,at a?ow rate of1mL/min.In order to maximize absorbance in the red/orange region of the visible spectrum a wavelength of486nm 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 pro?le of Valencia orange juice.Carotenoids were identi?ed using a DAD at350,430and486nm.The separation was performed on a YMC C30stainless steel column(25cm?4.6mm i.d.,5m m).
Many methods have also been reported for the analysis of carotenoids in biological samples.Thus, Lyan et al.(2001)described a method in which13 identi?ed carotenoids and9unknown carotenoids were separated from human plasma samples using a Nucleosil C18column(15cm?4.6mm,3m m)and a Vydac C18TP54column(25cm?4.6mm,5m m)in series and a mixture of ACN:MeOH(containing50mM ammonium acetate):DCM:water(70:15:10:5,v/v/v/v)as mobile phase at a?ow rate of2mL/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)containing0.01%BHT(w/v)was used as mobile phase,and a Nucleosil100C18(25cm?3mm i.d.,5m m) as stationary phase.Column temperature was set at 351C and a?ow rate of0.6mL/min.Chromatograms were monitored at290,325and450nm for the identi?cation of a-tocopherol,retinol and carotenoids, respectively.
Another isocratic reversed-phase liquid chromato-graphic 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(3.9mm?15cm,5m m particle size)and the mobile phase consisted of MeOH:ACN:methylene chloride(55:30:15,%,v/v)at a?ow rate of1mL/min. The detection was done at472nm.
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;Mele ndez-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;Va gi et al., 2002;Careri et al.,2001;Mathiasson et al.,2002; Go mez-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(Mele n-dez-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; Go mez-Prieto et al.,2003;Azevedo-Meleiro and Ro-driguez-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 re-equilibrate the column after each injection which represents a serious problem for routine analysis (Mele ndez-Mart?nez et al.,2003).
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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 geome-trical isomers),the mobile phase is often modi?ed 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;Va gi,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; Mele ndez-Mart?nez et al.,2003;Barth et al.,1995; Tzouganaki et al.,2002)or various ethers(Mouly et al., 1999;Moros et al.,2002;Go mez-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; Mele ndez-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 ef?ciently the carotenoids from the degrada-tion 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 modi?ers,for example triethylamine(TEA)or ammo-nium acetate,to the mobile phase reduces losses or degradation.TEA behaves as a strong modi?er,and it reduces the retention time.It was tested,however,that concentrations around0.05%did not change signi?-cantly 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-caro-tene),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 around89%and only when0.05M ammonium acetate was added to MeOH,the recovery increased up to94%. Therefore,it was suggested that the two modi?ers 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 signi?cant improvements on recovery when 0.05M ammonium acetate was added to a mobile phase containing0.05%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 speci?c actions of these compounds are unknown,though it is suggested that the modi?ers provide suf?cient 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 and0.05M ammonium acetate):chloro-form: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 C18columns(Huck et al.,2000; Heinonen et al.,1989;O tles and Atli,2000;Ha gg 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;Ma rkus 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-a o and Mercadante,2003;Azevedo-Meleiro and Rodriguez-Amaya,2004)have often been selected by researchers to perform the analysis.
Epler et al.(1992)compared65reversed-phase liquid chromatographic columns to determine the selectivity and recovery of a mixture of the following carotenoids: lutein,zeaxanthin,b-cryptoxanthin,echinenone,lyco-pene,and a-and b-carotene using ACN and MeOH modi?ed with THF or ethyl acetate as mobile phase.
A.Rodr?′guez-Bernaldo de Quiro′s,H.S.Costa/Journal of Food Composition and Analysis19(2006)97–111105
Stationary phases were mainly C18,and were classi?ed as monomeric,intermediate or polymeric.They found that the separation of lutein and zeaxanthin was only achieved by employing polymeric and some intermedi-ate columns,but these columns provided lower recov-eries than monomeric C18columns.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 C18columns show a poor resolution in the separation of geometric(cis–trans)isomers of carote-noids.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 C30columns 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; Go mez-Prieto et al.,2003;Dachtler et al.,1998;Lacker et al.,1999).Recently,a comprehensive review has described the application of C30columns 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 C30stationary phase.Satisfactory separations of all trans-lycopene from tomato employing a C30column were obtained by Go mez-Prieto et al.(2003).
Marsili and Callahan(1993)tested three commercial C18reversed-phase columns;Waters Novo-Pak column (4m m particle size,3.9mm?150mm),Supelcosil col-umn(5m m particle size,4.6mm?250mm)and Vydac 201TP column(5m m particle size,4.6mm?250mm)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 Vydac201TP 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 than25min,using a C30column(Moros et al., 2002).
4.1.2.1.Column temperature.Another important fac-tor 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 signi?cant changes in the chromato-graphic response of carotenoids;therefore,it is im-portant to work at constant temperature.
Scott and Hart(1993)studied the effect of column temperature in the separation of a mixture of carote-noids in a reference standard solution and in an extract of a dried food mixture.Five different temperatures were tested(15,20,22.5,25and301C)and the results indicated that changes in the temperature affect both elution time and pro?le.The best separation was achieved at20–22.51C.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 ef?cient separation of lutein,zeaxanthin,b-cryptoxanthin and b-carotene. The temperature was varied between21and801C.The best selectivity was achieved at211C and,at tempera-tures higher than601C carotenoids degradation was signi?cant.
4.2.Liquid chromatography-mass spectrometry
(LC-MS)
As reported in several studies,LC-MS is an innova-tive and powerful analytical tool for the identi?cation 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 inter-faces(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 identi?cation of a mixture of carotenoids,including astaxanthin,cantaxanthin,zeaxanthin,echinenone and b-carotene,as well as cis–trans isomers of b-carotene using LC-MS in the APCI mode.The analysis was performed on a25cm?4.6mm column,packed with ProntoSil silica gel3m m,modi?ed with triacontyltri-chlorosilane C30and a mobile phase of MeOH:MTBE (70:30,v/v)was employed.Mass spectra were achieved by scanning the mass range from m/z200to700.The limit of detection for b-carotene determined in positive-ion APCI-MS was1pmol.
An HPLC-MS-MS(APCI)method for the identi?ca-tion of carotenoids in different vegetables was described by Huck et al.(2000).A Phenomenex Luna C18 (25cm?2mm,5m m)column was used as stationary phase and the mobile phase system consisted of ACN (0.1%BHT):MEOH(containing0.05M ammonium acetate and0.05%TEA):CHCl3(containing0.1%
A.Rodr?′guez-Bernaldo de Quiro′s,H.S.Costa/Journal of Food Composition and Analysis19(2006)97–111 106
BHT):n-heptane(containing0.1%BHT)(50:40:5:5, v/v/v/v)at a?ow rate of0.2mL/min.Mass spectra were acquired over the scan range m/z300–2000.The detection limits obtained in positive-ion mode were in the range of nanogram.
Several authors have used mass spectrometry to ensure correct peak identi?cation 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 con?rm 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 con?rm 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/z467was formed from the molecular ion m/z536by elimination of a terminal isoprene group.Total lycopene was eluted as a single peak on C18column and an isocratic mobile phase consisting of ACN:MTBE(95:5,v/v)was employed.To separate isomers,a C30column 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 reversed-phase liquid chromatography-electrospray mass spectro-metry interfaced with TurboIonspray for the separation of b-carotene and xanthophylls.The separation was achieved on two ODS Hypersil columns connected in series(200?2.1mm,5m m and100?2.1mm,5m m)and a mobile phase of ACN:MeOH(0.1M ammonium acetate):DCM.The determinations were performed by operating the mass spectrometer in the positive-ion mode over m/z500–650.
Breithaupt et al.(2002)used a LC-APCI-MS for the identi?cation of8lutein monoesters produced from lutein diesters of marigold?owers(Tagetes erecta L.) after an incomplete enzymatic saponi?cation of lutein diester.Lutein diesters from several fruits were also identi?ed.The separation was performed on a C30YMC column(250?4.6mm 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/z80–1200.The authors developed a method,using the same conditions,to evaluate the carotenoids in chicken plasma after feeding diets containing free and esteri?ed 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 C30YMC column(250?4.6mm i.d.,5m m)main-tained at351C was used.Mass spectra of zeaxanthin esters were achieved by scanning the mass range from m/z400to1200.The limit of detection for zeaxanthin diesters was estimated to be0.4m g/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 identi?ed using a mass spectrometer coupled to a liquid chroma-tograph in an experiment carried out by Mercadante et al.(1997).
Acknowledgements
A.Rodr?guez-Bernaldo de Quiro s would like to thank Xunta de Galicia for providing a Grant from the programme‘‘stages in research centres’’and to Dr.M. Anto nia Calhau who welcomed her at Centro de Seguranc-a Alimentar e Nutric-a o,Instituto Nacional de Sau de Dr.Ricardo Jorge during this period of time. References
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