Journal of Chromatography A, 881 (2000) 543555

www.elsevier.com/ locate/ chroma

Review
Chromatographic determination of carotenoids in foods
*
Jordi Oliver , Andreu Palou
` Laboratori de Biologia Molecular, Nutricio i Biotecnologia, Departament de Biologia Fonamental i Ciencies de la Salut,
Universitat de les Illes Balears, Crta. Valldemossa km 7.5, 07071 Palma de Mallorca, Spain
Abstract
In recent years, there has been particular emphasis on obtaining more accurate data on the types and concentrations of
carotenoids in foods for various health and nutrition activities. The analysis of carotenoids is complicated because of the
diversity and the presence of cistrans isomeric forms of this group of compounds. In addition, a wide variety of food
products of vegetal and animal origin, vegetables and animal samples contain carotenoids, and a great range of carotenoids
can be found in these samples. The characteristic conjugated double bond system of carotenoids produces the main problem
associated with work and manipulation on carotenoids, that is their particular instability, especially towards light, heat,
oxygen and acids. For this reason, several precautions are necessary when handling carotenoids. Another problem associated
with analysis of carotenoids is the difculty in obtaining standard compounds. High-performance liquid chromatographic
methods for the determination of carotenoids in foods are reviewed. The sample extraction and treatment, carotenoid
purication and standard manipulation are briey commented on. We present a critical assessment of chromatographic
methods developed for the determination of carotenoids in foods. Finally, some methods for carotenoid ester separation are
reviewed. 2000 Elsevier Science B.V. All rights reserved.
Keywords: Food analysis; Reviews; Carotenoids
Contents
1. Introduction ............................................................................................................................................................................ 544
2. Carotenoids ............................................................................................................................................................................ 545
2.1. Structure ........................................................................................................................................................................ 545
2.2. Extraction and sample treatment ...................................................................................................................................... 545
2.2.1. Extraction........................................................................................................................................................... 545
2.2.2. Saponication ..................................................................................................................................................... 547
2.3. Purication..................................................................................................................................................................... 547
3. Chromatographic determination of carotenoids .......................................................................................................................... 548
3.1. Determination of carotenoids by high-performance liquid chromatography.......................................................................... 549
3.2. Determination of carotenoid esters by high-performance liquid chromatography.................................................................. 552
4. Nomenclature ......................................................................................................................................................................... 552
Acknowledgements...................................................................................................................................................................... 553
References .................................................................................................................................................................................. 553
*Corresponding author. Fax: 134-971-173-184.
E-mail address: dbfjoo4@ps.uib.es (J. Oliver)
0021-9673/ 00/ $ see front matter 2000 Elsevier Science B.V. All rights reserved.
PI I : S0021- 9673( 00) 00329- 0
544 J. Oliver, A. Palou / J. Chromatogr. A 881 (2000) 543555
1. Introduction studies, there was exclusive focusing on provitamin
A carotenes or b-carotene. In general, food com-
The carotenoids are one of the most important position databases reported the vitamin A,
group of natural pigments, because of their wide provitamin A or b-carotene content of foods; at
distribution, structural diversity and numerous func- most, total carotenoid content was reported [20,21].
tions. Although the classical source of carotenoids is A list of individual non-provitamin A carotenoid
plants, they are also found in animals and micro- content of some foods, both raw and cooked, has
organisms [1,2]. The structure of the carotenoids been recently published [2224].
confers very special and remarkable properties which Other biological functions have been ascribed to
are the basis of their varied functions and actions in carotenoids [4,14]: stimulants of the immune re-
all kinds of living organisms. The carotenoids are sponse at different levels [2527], enhancers of gap-
essential for photosynthesis and for life in an oxygen junction communication [28,29], and quenchers of
atmosphere [3]. In addition to the provitamin A free radicals [30]. A role in the control of the cell
activity of some carotenoids, these pigments have growth and differentiation has been proposed
recently been implicated in the prevention of or through the modulation of the expression of tran-
protection against serious human health disorders scriptor factors and nuclear binding proteins [31
such as cancer, heart disease, macular degeneration 33]. There is strong in vitro evidence that carot-
and cataracts [2,49]. Carotenoids have also been enoids are powerful growth inhibitory agents in
successfully used for many years in the treatment of several human and murine tumor cell lines but not in
individuals suffering from photosensitivity disease, normal cells [14]. Fucoxanthin retarded the growth
for example, erithropoietic protoporphyria [10,11]. of human neuroblastoma GOTO cell lines [34],
Numerous observational studies has found that lycopene was capable of inhibiting the growth of
increased intake of carotenoid-rich foods, fruits and mammary and lung cancer cells [35], and crocin and
vegetables, have a protective effect against several crocetin inhibited the growth of HeLa cells [36].
human chronic diseases [6,7,12,13]. However, more Nowadays the physiological and metabolic func-
recently, intervention trials have not been conclusive tions of the carotenoids are still not well understood
in the evaluation of the b-carotene supplementation [37]. The greatest problem is the structural diversity
effect on cardiovascular disease and cancers [13,14]. of the carotenoids, more than 700 have been char-
In well-nourished populations, b-carotene supple- acterized. Moreover, individual carotenoids are pres-
mentation is of little or no value in preventing these ent naturally in different cistrans isomeric forms,
human health disorders [15,16]; on the contrary, in which may affect their biochemistry [38]. It is
smokers, b-carotene supplementation increases necessary to have good, reliable data on carotenoid
rather than decreases lung cancer incidence [17,18]. composition of foods, thus, in this way, nutritional
In a recent study, a strong proliferative response in studies may obtain more realistic and valuable
lung tissue was observed in ferrets (animals that conclusions. In this aspect, the analytical methodolo-
metabolize b-carotene in much the same way as gies of carotenoids are the pathways to achieve these
humans) supplemented with high-dose of b-carotene, objectives. Chromatographic methods have been the
and this response was enhanced by exposure to traditional methods to separate and quantify the
tobacco smoke [19]. It was concluded that dimin- carotenoids, but the physical, chemical and biologi-
ished retinoid signaling could be a mechanism to cal features of this group of compounds have not
enhance lung tumorigenesis after high-dose b- facilitated the development of simple, easy-to-apply
carotene supplementation and exposure to tobacco methods. For example, the AOAC ofcial method
smoke [19]. [39] for the determination of carotenes and xantho-
In epidemiological studies we can nd associa- phylls in plants is still based on an open-column
tions between dietary components and the incidence separation and does not attempt to separate the
of various chronic diseases but do not dene specic individual carotenoids.
causeeffect relationships. Unmeasured variables are The continuing development of chromatographic
always present in this kind of study. Also in many methods is parallel to the evolution of the technique
J. Oliver, A. Palou / J. Chromatogr. A 881 (2000) 543555 545
and the hardware. The use of photodiode array transformation of the carotenoids that are present in
detection (DAD), which is capable of measuring the sample, and then the change in the carotenoid
simultaneously at a wide range of wavelengths, is composition of the sample. For this reason, several
widespread. The software associated with the DAD precautions are necessary when handling carot-
systems enables the manipulation of the wavelength enoids, and have been extensively revised in some
spectra of any chromatographic peak. other reviews [4043]. For example, the use of
In the present paper, the chromatographic methods antioxidants, laboratory experiments should be car-
for the determination of carotenoids in foods are ried out in dim lighting, in order to avoid as far as
reviewed. The methods for the separation of carot- possible the contact with direct sunlight, evaporation
enoid esters are discussed in a separate section. Prior should be performed by rotary evaporation and/ or
to these discussions, however, sample extraction and under a stream of nitrogen, and samples should be
treatment, carotenoid purication and standard ma- stored in the dark, under nitrogen or argon, at about
nipulation are briey commented on. 2208C.
The use of antioxidants is one of the most
common strategies to prevent oxidation during the
2. Carotenoids extraction and sample treatment, especially when the
samples are saponied to obtain free carotenoids.
2.1. Structure Ethoxyquin, pyrogallol, ascorbic acid and sodium
ascorbate are examples of antioxidants used [44], but
Structurally, the carotenoids are polyisoprenoid butylated hydroxytoluene (BHT) is the most exten-
compounds, which are synthesized by tail-to-tail sively used antioxidant. Normally BHT is used at
linkage of two C geranylgeranyl molecules. All the 0.01% or 0.1% in the extraction solution.
20
carotenoids are produced by variations of the parent
C skeleton. We can distinguish between the hydro-
40
carbon carotenoids, named carotenes made only of C 2.2.1. Extraction
and H, and the oxidized carotenoids, named xantho- There is no standard extraction procedure for
phylls (or oxycarotenoids) that present some O-sub- carotenoids because of the wide variety of food
stituent groups such as hydroxy, keto and epoxy products and animal samples containing these com-
groups. The spectrophotometric features of the carot- pounds, and the great range of carotenoids that can
enoids are produced by the conjugated double bond be found in these samples.
system. At the extremes of the molecule, the carot- The extraction procedures applied to food products
enoids present lineal or cyclic groups, cyclohexane begin with an extraction with methanol (MeOH) or a
and cyclopentane. The combination of these end- mixture of MeOH and other more apolar dissolvents.
groups with the addition of oxygen-containing func- Hart and Scott [23], for example, used a MeOH
tional groups and changes in the hydrogenation level tetrahydrofuran (THF) (1:1, v/ v) solution in the
permit the majority of the structures of the carot- carotenoid analysis of a wide variety of vegetables
enoids. Fig. 1 shows the chemical structure of and fruits, both raw and cooked. MeOH and diethyl
several common carotenoids. ether [45], MeOH and chloroform [46], MeOH and
hexane [47], and MeOH and acetonehexane [48,49]
2.2. Extraction and sample treatment have also been reported. Other groups prefer the use
of acetone alone [50] or in combination with light
The characteristic conjugated double bond system petroleum [51]. In a water convolvulvus (Ipomoea
of carotenoids produces the main problems associ- aquatica) and carrot carotenoid analysis, Chen and
ated with work and manipulation on carotenoids, that co-workers [52,53] used a more complex mixture
is their particular instability, especially towards light, extracting solution, hexaneacetoneMeOHtoluene
oxygen and heat. Acid and alkaline conditions can (10:7:6:7, v/ v). After the initial extraction of the
also be detrimental to certain carotenoids. Any of samples, the extract still contains some polar lipid
these factors may produce the degradation and/ or the contaminants, which can be removed by partitioning
546 J. Oliver, A. Palou / J. Chromatogr. A 881 (2000) 543555
Fig. 1. Chemical structures of several all-trans carotenoids.
the extract against water or aqueous salt solutions investigated static and dynamic extractions using
[41]. CO with different modiers at 1% (MeOH, EtOH
2
The extraction procedure in other samples, serum and 2-propanol) at different pressures and tempera-
or plasma, is more constant. A rst step with ethanol tures. Extraction yields vary depending on the sam-
(EtOH) to precipitate proteins is usually used, with a ple composition with higher yields (96%) in high fat
second step of hexane extraction [49,54] or butanol food [57]. In sweet potatoes, extraction yield varies
ethyl acetate (1:1, v/ v) extraction [55]. depending on the moisture content of the sample,
Supercritical uid extraction (SFE) technique is a dehydration method and particle size has been
rapid, selective and easier to automate method for described [58], with better recoveries in a freeze
sample preparation prior to their characterization by dried sample.
other analytical methods (for review see Ref. [56]). SFE methods for b-carotene from leaves and
Carotenoids have become a focus of many studies in vegetables [59] and from vitamin supplements [60]
analytical-scale SFE [56]. Tonucci and Beecher [57] have been described. Both methods use SFE-grade
J. Oliver, A. Palou / J. Chromatogr. A 881 (2000) 543555 547
CO without a modier at 31 mPa with a 1-min these samples and the fact that, in general, the
2
static extraction followed by a 40-min dynamic simultaneous determination of retinoids and carot-
extraction at a 2 ml / min ow-rate at 408C. Com- enoids is required. Saponication is required because
pared with a liquidliquid extraction method of the high fat content (|45%) and carotenoids are
(MeOHchloroform, 4:1, v/ v) the recoveries of b- contained in the lipid matrix [65]. An enzymatic and
carotene were similar or somewhat higher for the bile salts treatment is necessary in milk samples, also
SFE protocol [60]. with EtOH protein precipitation and nally hexane
extraction [54,66]. Another milk saponication meth-
2.2.2. Saponication od with classical alkaline solution treatment has been
After the sample extraction, traditionally, the described [67,68], but it must be noted that retinoids
second step in the protocol of carotenoid determi- are even more unstable compounds than carotenoids.
nation is the alkaline saponication. Carotenoids in
vegetables and fruits are predominantly esteried by 2.3. Purication
fatty acids [42]. In addition, the degree of esterica-
tion can be different in function of the number of One of the main problems associated with analysis
hydroxyls present in the xanthophylls. Monohydrox- of carotenoids is the unavailability of appropriate
y-carotenoids, such as b-cryptoxanthin, could be standard compounds. The diversity, the inherent
found free or esteried by one fatty acid (monoester); instability and the presence of isomers of this group
dihydroxycarotenoids, such as lutein and zeaxanthin, of compounds means that most of them are not
could be found free or esteried by one (monoester) available commercially. The more common and
or two (diester) fatty acids. Thus, the identication of widespread carotenoids (b-carotene, a-carotene, lu-
peaks in high-performance liquid chromatography tein, zeaxanthin, lycopene, b-cryptoxanthin, etc.),
(HPLC) of these carotenoid esters can be difcult even some more rare carotenoids (capsanthin, cap-
[61]. In order to simplify the separation, most sorubin, etc.), may be purchased commercially.
methods for quantitative carotenoid analysis have a However, the purity of some of these commercial
previous phase of extract saponication [53]. carotenoids is not sufcient for a chromatographic
Normally, the saponication is carried out in a standard, and thus require some additional purica-
KOH solution: aqueous, ethanolic or methanolic tion steps.
solution, and the concentration may vary from 10 to Traditionally the carotenoid standards were a self-
60% (w/ v) [62]. The conditions under which the made preparation. From fruits and vegetables and
saponication is carried out may be at ambient with a purication process, each research obtained
temperature overnight or under heating which re- standard preparations of the most important carot-
duces the time of the reaction [44]. After the enoids of their samples. Phase separation, thin-layer
saponication, the sample is extracted with diethyl chromatography (TLC) and liquid chromatography
ether or hexane then the extract is washed several (LC) methods are used to obtain pure carotenoid
times with water until all the KOH is removed. standards, also more often nowadays a preparative
The degradation and loss of total carotenoid HPLC method has been described for this purpose.
content [63] or individual carotenoids [24,46,50,64] Normally, to obtain a pure enough carotenoid
during the saponication have been described. How- solution almost two or three steps of purication are
ever, this loss depends on the conditions of the required. The best sources of each individual carot-
saponication [50,64] and on the sample composi- enoid are used, for example, neoxanthin from green
tion [46]. In recent years, many chromatographic cabbage, violaxanthin from spinach, antheraxanthin
methods for the simultaneous determination of free from potatoes, a-cryptoxanthin from carrot leaves,
and esteried carotenoids in fruit samples have been capsanthin and capsorubin from paprika, etc. The
described (see above), thus avoiding the saponica- spectrophotometric features of carotenoids are used
tion step. to identify and quantify the individual carotenoid that
The carotenoid extraction of milk and breast milk has been puried. There are many bibliographic
is more problematic because of the composition of references with spectral data of many carotenoids in
548 J. Oliver, A. Palou / J. Chromatogr. A 881 (2000) 543555
different solvents [40,43,69]. The purity degree of ment with a calibration curve of authentic standards
standard solution must be ensured by spectrophotom- at different concentrations.
etry and HPLC, for both commercial and self- Like HPLC for carotenoid determination, prepara-
puried standards. tive HPLC is more often cited for carotenoid puri-
Until the development of HPLC methods, open cation. In contrast with analytical HPLC, the pre-
column separations were used to quantify carot- parative HPLC methods are characterized by the use
enoids. The AOAC ofcial method for the determi- of columns with a bigger particle size (10 or 7 mm)
nation of carotenes and xanthophylls still uses an of stationary phase, bigger diameter column and/ or
open column method [39]. In an interlaboratory bigger ow-rate. For example, Wingerath et al. [75]
study of procedures for the analysis of vitamins in used a 7 mm C reversed-phase column and
18
foods [70] three laboratories out of 10 use an open MeOHMeCNDCMn-hexane (20:40:20:20, v/ v)
column method. as solvent system at a ow-rate of 4 ml / min to
Alumina, silica gel, MgO, MgCO , Ca(OH) , separate carotenol fatty acid esters of fruit juice.
3 2
CaCO , Celite, etc., have been used to purify Yuang and Chen [76] used an Ultrasphere C (5
3 18
carotenoids [41,43] with different solvent systems. mm) semi-preparative column with MeOHwater
Different proportions of light petroleum, diethyl DCM (90:8:2, v/ v) as mobile phase at a ow-rate of
ether, toluene and EtOH, alone or in combination, 3 ml / min to separate and purify trans-astaxanthin
have been described to purify most groups of from algae.
carotenoids by TLC on silica gel [43]. After the During the preparation and handling of standard
separation on TLC the bands are scraped off the carotenoids, special precautions are necessary to
layer and eluted from the silica gel with a suitable avoid the degradation or isomerization of puried
solvent; for the pigment identication and quantica- pigments. A stock solution must be prepared and
tion the wavelength spectra is recorded. stored in the dark, at 2208C, under nitrogen atmos-
The last development in the TLC method applied phere and using solvents with antioxidants. To
to the separation of carotenoids is reversed-phase prepare a standard solution, an appropriate aliquot of
TLC which consists of a separation on C plates. the stock solution is diluted with suitable solvent and
18
This method has been applied to the separation of the concentration of the solution must be calculated
various carotenoid standards using different propor- by spectrophotometry. The purity of the standard
tions of light petroleumacetonitrile (MeCN) solution should be conrmed by HPLC.
MeOH [71] achieving better separations than nor-
mal-phase TLC [44]. Chen and Chen [72] used a
solvent system of hexaneacetonechloroform 3. Chromatographic determination of
MeOH (70:25:10:5) to separate xanthophylls and carotenoids
b-carotene on silica gel to conrm the identication
of a HPLC method of water convolvulus. A prepara- The classical open column chromatographic meth-
tive TLC using 510% acetone in light petroleum on ods have been replaced by the new HPLC method,
silica gel plates for separation of xanthophyll esters because of the rapidity, non-destructiveness and ease
of fruits has been described [73]. in automating these techniques. The development of
More recently, a high-performance TLC (HPTLC) the HPLC apparatus is parallel with the development
method has been described to detect lycopene added of the laboratory hardware. The new HPLC chro-
to alcoholic and non-alcoholic beverages [74]. TLC matographs are more compact, more precise, more
was carried out on high-performance silica gel plates automated and their use is simpler. Most HPLC
(Whatman LHPKDF) and developed with dichloro- chromatographs have special software to control all
methane (DCM)MeOH (1:1, v/ v). A direct quanti- the apparatus parameters, to obtain chromatograms
cation procedure was applied successfully. The in and, at the same time, data integration and result
situ spectra of each band were recorded with a calculation automatically.
densitometer (370700 nm) and the maximum ab- The spectrophotometric HPLC detectors, which
sorption wavelength was used to quantify the pig- are used to detect carotenoids, have shown develop-
J. Oliver, A. Palou / J. Chromatogr. A 881 (2000) 543555 549
ments similar to chromatographs. The new spectro- ent kinds of MS detectors have been proposed as
photometric detectors have a high sensibility and suitable for carotenoid LCMS, namely, LCelectro-
accuracy, are capable of selecting any wavelength spray ionization (ESI) MS [80,81] and LCatmos-
and in some cases are programmable, during the pheric pressure chemical ionization (APCI) MS [82].
chromatographic separation the wavelength can be Information on normal-phase HPLC and any other
changed. The more sophisticated detectors applied to chromatographic separations (TLC, LC, etc.) of
the HPLC separation of compounds with characteris- carotenoids has been reported in numerous previous
tic absorption spectra, such as carotenoids, are the reviews [38,41,44,83]. In the present report, we
DAD systems. These detection systems are capable summarize the latest published HPLC methods for
of recording the entire spectral range (from 190 to the determination and quantication of carotenoids in
800 nm) during analysis. Thus any chromatogram at foods, despite the previous commented reference,
selected wavelengths can be monitored, and the and all of them are reversed-phase separations. In a
absorption spectra of any peak chromatogram and at separate section, the simultaneous chromatographic
any moment during the chromatographic separation determinations of free carotenoids and carotenoid
can be recorded. esters published during the last years are reported.
All the properties of DAD make these detectors
suitable for carotenoid chromatographic separation. 3.1. Determination of carotenoids by high-
Nowadays, the published analytical carotenoid meth- performance liquid chromatography
ods use DAD more often. The wavelength range of
DAD in a carotenoid separation depends on the Granado et al. [22] analyzed the carotenoid com-
purpose of the method, for example, our group in a position in raw and cooked Spanish vegetables using
recent chromatographic method for carotenoid de- Spheri-5-RP-18 or Spheri-5-ODS columns, MeCN
termination uses a detection range of 350550 nm DCMMeOH (70:20:10, v/ v) as mobile phase and a
[46]. However, in HPLC analysis of serum samples ow-rate of 1.8 ml / min. For the separation of lutein
the DAD system was set at 250550 nm to allow the from zeaxanthin, the mobile phase was substituted
simultaneous determination of retinol, tocopherols with MeCNMeOH (85:15, v/ v). In this work, both
and carotenoids [77]. non-provitamin A (lutein, zeaxanthin and lycopene)
The majority of carotenoid separations reported in and provitamin A (b-cryptoxanthin, g-carotene, a-
the literature involve the use of reversed-phase carotene and b-carotene) carotenoids were reported,
HPLC, very few normal-phase HPLC have been whereas b-apo-89-carotenal and canthaxanthin were
reported in the last years. Recently, Khachik et al. not detected in any of the samples. Echinenone or
[54] used a normal-phase separation in an exhaustive retinyl palmitate were used as internal standard.
qualitative and quantitative analysis of carotenoids in Carotenoids were detected at 450 nm and retinyl
human milk and serum. However, normal-phase and palmitate at 325 nm with a programmable mul-
reversed-phase HPLC separations are described in tiwavelength detector.
this reference. In an interlaboratory study of methods A carotenoid analysis of carrot, spinach, tomatoes,
for the determination of fat-soluble vitamins, only corn (canned) and tangerines using a stainless steel
two normal-phase HPLC separations have been (25034.6 mm I.D.) column packed with Vydac 201
reported as opposed to ve reversed-phase sepa- TP, 5 mm particle size, and a mobile phase consisting
rations and three open-column chromatographic of MeOHTHF (95:5, v/ v) have been described
methods [70]. [84]. The ow-rate was at 1 ml / min, the chromato-
It is well known that traditional gas chromatog- gram was recorded at 450 nm and ethyl-b-apo-89-
raphy (GC) is not suitable for the analysis of carotenoate was used as internal standard. Lutein,
carotenoids, because of their inherent instability and zeaxanthin, a-carotene, b-carotene and lycopene
their low volatility [44], extensible to GCmass were separated and quantied with this method.
spectrometry (MS) [78]. A recent review on the Muller [24] used three different mobile phases for
latest innovations in LCMS applied to carotenoid the carotenoid separation and determination of fruit
analysis has been published [78,79], and two differ- and vegetable samples on Vydac 5 mm C analytical
18
550 J. Oliver, A. Palou / J. Chromatogr. A 881 (2000) 543555
column with a ow-rate of 1 ml / min and column ml / min of MeCNMeOHchloroformhexane
temperature of 208C. An isocratic mobile phase (75:12.5:7.5:7.5, v/ v). b-Apo-89-carotenal was used
system was a mixture of MeOHacetone (95:5, v/ v) as internal standard. For a similar purpose, the use of
(previously described in Ref. [65]), another suitable a 3 mm ODS-Hypersil column and a linear gradient
isocratic system consisted of MeOHMeCNam- of two eluents (eluent A: MeOHMeCNwater0.2
monium acetate (85:15:0.01, v/ v/ w); and, a gradient M TrisHCl buffer, 15:65:19:1, v/ v; eluent B:
separation using two solvents, MeCN2-propanol MeOHhexane, 7:1, v/ v) has been described.
(40:60, v/ v) and water (previously described [50]). Mnguez-Mosquera and Hornero-Mendez [88] de-
The samples were saponied and losses were occa- veloped a reversed-phase method to quantify red
sionally observed especially for xanthophylls. peppers, paprika and oleoresin carotenoids. The
Lycopene was the most unstable xanthophyll with separation was carried out on a Spherisorb ODS C ,
18
saponication treatment. 5 mm particle size column using a non-linear gra-
Ben-Amotz and Fishler [85] reported extensive dient of acetone and water at a ow-rate of 1.5
data on common fruit and vegetable carotenoid ml / min. Spectral data of carotenoids identied were
composition. In this study, a Vydac 201 TP54 C 5 reported in the mobile phase.
18
mm column maintained at 308C and MeOHMeCN Weissenberg et al. [45] optimized the separation of
(9:1, v/ v) mobile phase at a ow-rate of 1 ml / min is red pepper pigments with a non-aqueous ternary
described. b-Apo-89-carotenal or echinenone was mobile phase. Different proportions of MeCN, 2-
used as internal standard. Some carotenoid isomer propanol and ethyl acetate at 0.8 ml / min as ow-rate
content has been reported with special emphasis on were assessed in two C columns with different
18
9-cis-b-carotene. length (25 and 12.5 cm, LiChrospher 100 RP-18, 5
A reversed-phase ion-pair HPLC applied to the mm and Superspher RP-18, 4 mm, respectively). The
separation of carotenoids and chlorophylls in olive best separation of capsorubin and capsanthin was
has been described [86]. Separation was carried out achieved in a 12.5 cm column length and MeCN2-
on a Spherisorb ODS-2 5 mm particle size column propanolethyl acetate (80:10:10, v/ v) as a mobile
and with a non-linear gradient of eluent A (water phase; however, canthaxanthin and lutein ran to-
solution PMeOH, 1:1:8, v/ v/ v) and eluent B gether. Peak distortion was observed with increased
(acetoneMeOH, 1:1, v/ v) at a ow-rate of 2 ml / MeCN proportion of the solvent.
min. The ion-pair reagent (solution P) is 0.05 M Previously, Craft and Wise [65] optimized an
tetrabutylammonium acetate and 1 M ammonium isocratic HPLC separation of carotenoids in a poly-
acetate in water. Eighteen pigments, including chlo- meric C column. Nine solvent modiers were
18
rophylls and carotenoids, were separated in 30 min. investigated using a MeOH-based mobile phase.
Spectral data of carotenoids identied in the eluent Column temperature was also investigated. 3 to 5%
was reported. THF in MeOH and a column temperature of 208C
A non-aqueous reversed-phase HPLC using an result in good separation of a mixture of seven
isocratic mobile phase consisting of MeCNDCM carotenoids (lutein, zeaxanthin, b-cryptoxanthin,
MeOH (65:25:10, v/ v/ v) has been described [87]. echinenone, a-carotene, b-carotene and lycopene).
An Analytichem C column was used, ow-rate was Multiple peak formation and peak distortion have
18
1 ml / min and the chromatographic separation was been described associated with the analysis of carot-
monitored using DAD. The peak spectrum was used enoids by HPLC [89,90]. These problems have been
to identify some carotenoids of red grapefruit. DAD associated with the solubility of the carotenoids in
was used for spectral characterization of the sepa- the HPLC eluent, the nature of the injection solvent
rated pigments, some spectral information was re- [89,90] and also with the reaction between carot-
ported. enoids and metal surfaces [90]. Particularly,
Another simultaneous determination of chloro- lycopene seems a very unstable compound in stain-
phylls and carotenoids has been proposed by Chen less steel columns with metal frits, so recoveries
and Chen [72]. The column was packed with Ul- lower than 60% have been described [84,90]. The
tremex C , 5 mm particle size and ow-rate was 1 addition of antioxidants (BHT) in the stock solvent
18
J. Oliver, A. Palou / J. Chromatogr. A 881 (2000) 543555 551
solution or to store the stock solution dried has been Interest in the carotenoid isomer analysis has
shown to improve lycopene recovery [90]. Scott [90] grown in the last years, numerous chromatographic
also reported a between-column variation using methods specially focused on carotenoid isomers
columns containing different batches of packing have been described (see Ref. [38] for review). The
material (Vydac TP20154): changes in peak re- traditional reversed-phase chromatographic columns
sponses, elution times and differences in the peak seldom have an adequate ability to separate the
prole separation were shown. A progressively cistrans isomers of a particular carotenoid. A better
worse result has been reported on C NovaPak performance for this purpose has been attributed to
18
columns that previously were useful for carotenoid polymeric ODS columns [92].
separation [90]. Stahl et al. [95] achieved the separation of ve
Epler et al. [91] observed bad carotenoid sepa- isomers of b-carotene and seven isomers of lycopene
rations (no peaks were observed) on columns that in human serum and tissues. The separation was
previous carotenoid separations were acceptable after carried out on 5 mm particle size Suplex PKB 100
a minute exposition to triuoroacetic acid. The same column with either MeOHMeCN2-propanol
column after treatment with MeOHaqueous 0.01 M (54:44:2, v/ v) or MeOHMeCN2-propanolwater
ammonium acetate (50:50, v/ v) for 45 min was (10:40:40:10, v/ v). An eluent system of MeCN
restored to its initial good carotenoid-separating MeOHDCM (75:15:10, v/ v) or MeCNMeOH
state. What is more, ve columns which in a (90:10 or 5:95) has been described as a better mobile
previously published evaluating paper of the same phase for the separation of b-carotene isomers and
research group [92] had poor recovery (,48%), after MeOHDCM (99:1, v/ v) for the separation of a-
treatment with MeOHammonium acetate and using carotene isomers and for the simultaneous separation
a mobile phase with 0.05% TEA the columns of a- and b-carotene isomers [53].
become suitable for carotenoid separation (recovery The congurational isomers of the all-trans-, and
.87%) [91]. most of the congurational isomers of the 9-cis-,
A study of the column temperature effect on the 13-cis- and 15-cis-astaxanthin have been separated
carotenoid chromatographic determination has been on a chiral HPLC separation [96]. Recently, a
published [93]. The column used in this study was a polymeric C stationary phase was developed to
30
5 mm Spherisorb ODS 2 column and the mobile optimize carotenoid reversed-phase chromatography
phase consisted of MeCNMeOHDCM (75:20:5, [97]. This novel stationary phase has demonstrated
v/ v) containing 0.1% BHT at a 1.5 ml / min ow- superior resolution for carotenoid isomer separation
rate. The sample was a reference standard mixture of (lutein, zeaxanthin, b-cryptoxanthin, a-carotene, b-
lutein, zeaxanthin, b-cryptoxanthin, echinenone, carotene and lycopene) in comparison to traditional
lycopene, a-carotene and b-carotene. A reduction of C columns [9799].
18
about 1 min in elution time for every 18C rise in Sander et al. [97] achieved a good resolution and
temperature was shown, and the optimum tempera- identication of four isomers of b-carotene (all-
ture was at 2022.58C. trans, 9-cis, 13-cis and 15-cis) in a not fully opti-
Differences between monomeric and polymeric mized separation of a carotene and xanthophyll
octadecylsilica (ODS) reversed-phase columns in standard solution using C stationary phase column
30
chromatographic separation of carotenoids using and a gradient of 81:15:4 to 6:90:4 of MeOH
MeOH-based mobile phase have been described methyl-tert.-butyl ether (MTBE)water during 90
[92,94]. In general, the polymeric ODS stationary min. Both monomeric and polymeric C columns
18
phase has a better selectivity for carotenoids than were unable to separate b-carotene isomers in this
monomeric ODS stationary phase. MeOH-based standard solution [97]. Similar separation has been
solvents provided higher recoveries than MeCN- described in a canned sweet potatoes extract with a
based solvents [92]; the most appropriate solvent linear gradient from 85:15 to 10:90 of MeOH
system was MeOHTHF (90:10, v/ v) [94]. Lutein MTBE containing 1 mM ammonium acetate over 60
and zeaxanthin were difcult to separate in the min [80].
monomeric ODS phases [92]. A superior resolution of C stationary phase
30
552 J. Oliver, A. Palou / J. Chromatogr. A 881 (2000) 543555
respect to traditional C columns have been de- matographic methods are characterized by a re-
18
scribed to separate several standard solutions of versed-phase HPLC on C columns. Most of the
18
individual carotenoids and xanthophylls (b-carotene, methods listed use DAD to facilitate the identica-
a-carotene, zeaxanthin, lutein, b-cryptoxanthin and tion of the carotenoid esters comparing peak spectra
lycopene) using different proportions of MeOH along the chromatogram. Seven isocratic [107109]
MTBE as mobile phase [98,99]. and eight gradient [46,48,50,51,75,76,110,111] mo-
The electrochemical detection of carotenoids in bile phase systems have been described. In general,
chromatographic separations has been described, but both isocratic and gradient mobile phases are based
with utilization of conventional one or two channel on MeOH, acetone, 2-propanol and MeCN with
electrochemical cells [100103]. Recently, a HPLC some modiers (water, hexane, ethyl acetate or
carotenoid determination with a coulimetric electro- DCM).
chemical array detector equipped with eight channels Independently of the mobile phase composition,
in series has been described [49]. The potential there are similar patterns of chromatographic sepa-
settings were from 100 to 520 mV in 60 mV ration. For example, in paprika samples (fruits,
increments. The chromatographic conditions were 5 powder or food with high content of paprika)
mm polymeric C as stationary phase and a gradient [46,48,107,108,110,111] the chromatogram could be
30
with different concentrations of MeOHMTBEam- divided into four zones: the rst zone corresponds to
monium acetate (eluent A, 95:3:2, eluent B, 25:73:2). free carotenoids, the second zone to monoesteried
The electrochemical detectors have shown to be 100- pigments, the third zone to a-carotene, b-carotene
to 1000-fold more sensitive than conventional spec- and their isomers, and nally the fourth zone to
trophotometric detectors [49,102]. In addition to the diesteried carotenoids. The fatty acid moiety of
high sensitivity, the electrochemical array detector carotenoid esters has been identied in some refer-
signal was characteristic for each individual carot- ences [48,73,75,109,110,112,113], and there are a
enoid (lutein, zeaxanthin, b-cryptoxanthin, a- great variety of saturated fatty acids, from decanoic
carotene and b-carotene), even for cistrans isomers acid to oleic acid which have been reported. The
of b-carotene (all-trans, 9-cis, 13-cis and 15-cis) degree of esterication varied between carotenoids,
[49]. for example in red pepper fruits, zeaxanthin is
predominantly found in the monoesteried form and
3.2. Determination of carotenoid esters by high- capsorubin in the diesteried form [114]. Goda et al.
performance liquid chromatography [113] have shown in paprika a preferential position
of esterication group of the capsanthin hydroxyl
Hydroxycarotenoids in vegetables are predomi- group pertaining to the cyclopentane versus the
nantly esteried by fatty acids [42]. The carotenoid cyclohexane group.
composition of fruits is not constant, depends on
variety, fruit ripening, environmental conditions, etc.
Similarly, in some fruits, during ripening the esteri- 4. Nomenclature
cation degree of carotenoids increases [104,105]. In
addition, a high stability against possible thermo-, BHT Butylated hydroxytoluene
photo- and enzymatic oxidation reactions has been DCM Dichloromethane
related to the esterication degree [106,107]. In EtOH Ethanol
recent years, the methods for the carotenoid de- GC Gas chromatography
termination without saponication have increased. HPLC High-performance liquid chromatog-
The use of DAD has facilitated the identication of raphy
the esteried forms of carotenoids. HPTLC High-performance thin-layer chromatog-
In Table 1, recent chromatographic methods to raphy
separate carotenoid esters in fruits and foods are LC Liquid chromatography
summarized. Because of the wide range of apolarity MeCN Acetonitrile
of the carotenoids and carotenoid esters, the chro- MeOH Methanol
J. Oliver, A. Palou / J. Chromatogr. A 881 (2000) 543555 553
Table 1
HPLC methods for carotenoid ester separations
Column Eluent Detection Samples Ref.
ODS Resolve C Linear gradient of MeOH and ethyl acetate 470 nm Red bell peppers [48]
18
Flow-rate: 1.8 ml / min
5 mm Zorbax C Gradient of acetonewater (75:25, v/ v) and 460 nm Paprika [110]
18
acetoneMeOH (75:25, v/ v). Flow-rate: 1 ml / min
10 mm Chromsil C Acetonewater (90:10, v/ v), acetonewaterhexane 290 nm, 350 nm Paprika fruits [106]
18
(85:10:5, v/ v) or MeCN2-propanolwater (39:57:5:4, v/ v). and 438 nm and powder
Flow-rate: 1 ml / min
5 mm ODS RP-18e and Non-linear gradient of MeCN2-propanol (40:60, v/ v) DAD (462 nm) Pepper fruits and [50]
5 mm ODS RP-8 serially and water. Flow-rate: 0.8 ml / min. powder paprika
connected
6 mm LiChrosorb C MeCN2-propanolMEOHwater (39:52:5:4, v/ v). Flow-rate: DAD Spice red pepper, tomato, [107]
18
0.91.5 ml / min carrot, orange and mandarin
5 mm Suplex pKB 100 or Linear gradient of MeOHMeCNDCMhexane at DAD Tangerine and orange [75]
5 mm LiChrospher C 10:85:2.5:2.5 (v/ v) and 10:45:22.5:22.5 (v/ v). Flow- concentrates
18
rate: 0.7 ml / min
YMC-Pack ODS-A. MeOHethyl acetate (62:38 or 88:12, v/ v) or MeOH 470 nm Banana peel [108]
Column temperature: 308C waterethyl acetate (80:10:10). Flow-rate: 1 ml / min
5 mm Spherisorb ODS1 Non-linear gradient of MeOHMeCN (50:50, v/ v), water DAD (450 nm) Paprika extract [109]
and ethyl acetate. Flow-rate: 1.5 ml / min
5 mm Ultrasphere C Linear gradient of DCMMeOHMeCNwater at DAD Alga (Haematococcus [76]
18
Column temperature: 258C 5:85:5.5:4.5 (v/ v) and 22:28:45.5:4.5 (v/ v). Flow-rate: 1 ml / min pluvialis)
5 mm Spherisorb C ODS2 Non-linear gradient of acetonewater at 100:50 to DAD (max plot) Powder paprika and fat [46]
18
100:5. Flow-rate: 1 ml / min cured crude sausage
(Sobrassada)
5 mm Spherisorb C ODS-2 Non-linear gradient of different proportions of acetone, DAD Aqueous paprika extract [51]
18
Column temperature: 408C MeOH and water. Flow-rate: 1 ml / min and orange juice
MS Mass spectrometry tura, Pesca i Alimentacio of the Balearic Island
MTBE Methyl-tert.-butyl ether Government.
ODS Octadecylsilica
SFE Supercritical uid extraction
THF Tetrahydrofuran
References
TLC Thin-layer chromatography
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